Chimeric costimulatory proteins containing mutant intracellular domains with increased expression

Abstract

The present application relates to functionally optimized intracellular costimulatory domains, optionally in combination with cell-intrinsic immune checkpoint inhibitory or immune stimulatory receptors or portions thereof, that can be used in adoptive cell therapy for the treatment of human diseases and disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 163,171, filed March 19, 2021, and U.S. Provisional Application No. 63 / 133,494, filed January 4, 2021, the contents of each of which are incorporated by reference in their entirety herein. [Background technology]

[0002] Although adoptive cell therapy shows efficacy in cancer treatment, the efficacy of these therapies can be further improved by genetic engineering of T cells for better proliferation and persistence. T cells require functionally non-redundant costimulatory signals from the CD28 family and tumor necrosis factor receptor (TNFR) family along with antigen-induced TCR signaling to promote full-fledged activation and sustained proliferation. In the development of genetically engineered T cell therapeutics, it is necessary to introduce chimeric T cell costimulatory molecules that can be locally activated when T cells engage with pathological antigens and potently enhance T cell activation for increased therapeutic efficacy. Currently, second generation chimeric costimulatory molecules that incorporate one costimulatory signaling domain from either the CD28 family or the TNFR family of proteins have been widely adopted in CAR T cell therapy. Chimeric antigen receptors (CARs) that incorporate one costimulatory signaling domain increase T cell function through both activation and costimulatory signals, thus resulting in enhanced antitumor efficacy and T cell persistence. Considering that the ability of T cells to proliferate depends on the structural design, current second generation costimulatory proteins may not be optimal for inducing durable tumor remission. Therefore, efforts are desired to develop third generation chimeric molecules that combine two costimulatory signaling domains from CD28 and TNFR family members to exploit the non-overlapping functions of the two families of costimulatory molecules to further enhance T cell therapeutic capabilities. In addition, such third generation chimeric costimulatory molecules can be incorporated into TCR T therapy where T cell activation remains suboptimal due to insufficient costimulatory signals during activation of exogenously expressed TCRs by antigens. However, existing recombinant DNA strategies often suffer from reduced cell surface expression of chimeric proteins that combine two costimulatory signaling domains, preventing the realization of the functional potential of the chimeric proteins. The present application addresses such a need. Summary of the Invention [Problem to be solved by the invention]

[0003] This application discloses third generation chimeric T cell costimulatory molecules that incorporate two signaling domains from the CD28 and TNFR families and are expressed at levels significantly improved over previously reported for enhanced T cell function, as well as methods for making the costimulatory molecules. [Means for solving the problem]

[0004] Provided herein is a novel chimeric costimulatory intracellular domain. Provided herein is a chimeric costimulatory intracellular domain comprising (a) a first signaling domain based on an intracellular signaling domain of a CD28 family protein, and (b) at least a second signaling domain comprising a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

[0005] In some embodiments, the first signaling domain based on the intracellular signaling domain of a CD28 family protein is selected from CD28 protein, ICOS protein, or a combination thereof. In some embodiments, at least the second signaling domain is based on a variant of the intracellular signaling domain of a TNFR family protein selected from CD137 (4-1BB) and CD134 (OX-40).

[0006] The chimeric costimulatory intracellular domain provided herein comprises (a) a first signaling domain based on the intracellular signaling domain of CD28 protein, ICOS protein, or a combination thereof, and (b) at least a second signaling domain comprising a mutant CD137(4-1BB) intracellular domain or a mutant CD134(OX-40) intracellular domain. In some embodiments, the mutant CD137(4-1BB) intracellular domain or the mutant CD134(OX-40) intracellular domain comprises a deletion, insertion, or substitution of one or more amino acids in the membrane proximal portion of the CD137 or CD134 intracellular domain. Without being bound by theory, in some embodiments, one or more amino acids in the membrane proximal portion may be a ubiquitination site involved in the ubiquitination and degradation of the CD137 or CD134 protein.

[0007] Also disclosed herein are functionally optimized intracellular costimulatory domains for use in novel adoptive cell therapies, optionally in combination with cell-intrinsic immune checkpoint inhibitory or immune stimulatory receptors or portions thereof, developed to treat human diseases and disorders, including hematological and solid tumors. Also disclosed herein are functionally optimized intracellular costimulatory domains for use in combination with T cell receptors (TCRs), e.g., endogenous TCRs or high affinity TCRs targeting tumor-associated antigens. Optionally, the intracellular costimulatory domains are used in combination with a second component (e.g., a cell surface receptor or portion thereof) that directs immune cell migration or induces immune cell activation and / or proliferation to bind to target tissues or cells, such as PD-1 switch receptors (PD-1-based costimulatory molecules) that can increase T cell functionality in tumors, such as PD-L1 / PD-L2 expressing tumors. Also disclosed herein are therapies that utilize PD-1 checkpoint blockade in a cell-intrinsic manner, which simultaneously minimizes autoimmune side effects and provides increased functionality on tumors. The present application discloses recombinant T cell co-stimulatory receptors (RTCRs) based on T cell co-receptors or chimeric antigen receptors (CARs) that contain the functionally optimized intracellular co-stimulatory domains of the present application. The present application also discloses T cell co-receptors that contain functionally optimized intracellular co-stimulatory domains and PD-1 extracellular domains (i.e., PD-1 switch receptors or PD-1-based co-stimulatory molecules). The present application also discloses CD19 and B cell maturation Ag (BCMA)-based CARs that contain functionally optimized intracellular co-stimulatory domains that promote CD19 and B cell maturation Ag (BCMA) binding-mediated T cell activation, proliferation, and tumor killing. The RTCRs disclosed in the present application can be used for checkpoint target validation, safety screening, and development of preclinical animal models to evaluate the efficacy of the combination of the functionally optimized intracellular co-stimulatory domains of the present application with any TCR or CAR. Additional cell-intrinsic immune checkpoint inhibitors with effective TCRs are also developed.

[0008] The present disclosure provides a recombinant T cell costimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least a second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

[0009] The present disclosure provides a recombinant T cell costimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least second signaling domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

[0010] The present disclosure also provides a nucleic acid encoding the RTCR disclosed herein.The present disclosure also provides a vector comprising the nucleic acid disclosed herein.The present disclosure also provides a cell comprising the nucleic acid or vector disclosed herein.

[0011] The present disclosure also provides a modified T lymphocyte (T cell), comprising: (a) a modification in an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; and (b) a recombinant T cell costimulatory receptor (RTCR) as disclosed herein.

[0012] The present disclosure also provides a composition comprising the RTCR disclosed herein. The present disclosure also provides a composition comprising a nucleic acid encoding the RTCR disclosed herein. The present disclosure also provides a composition comprising a vector comprising a nucleic acid disclosed herein. The present disclosure also provides a composition comprising a cell disclosed herein. The present disclosure also provides a composition comprising a modified T cell disclosed herein. The present disclosure also provides a composition comprising a cell population, the population comprising a plurality of cells comprising a nucleic acid encoding an RTCR as disclosed herein, or a vector comprising a nucleic acid encoding an RTCR as disclosed herein.The present disclosure also provides a composition comprising a cell population, the population comprising a plurality of modified T cells as disclosed herein.

[0013] The present disclosure provides a method of producing a plurality of modified T cells, the method comprising: a) providing a plurality of primary T cells as disclosed herein; b) providing a composition comprising an RTCR as disclosed herein, a nucleic acid encoding an RTCR as disclosed herein, or a vector comprising a nucleic acid encoding an RTCR as disclosed herein; and c) introducing the composition of (b) into the plurality of primary T cells of (a) to produce a plurality of modified T cells under conditions in which the RTCR is stably expressed in the plurality of modified T cells. In some embodiments, the method of producing a plurality of modified T cells as disclosed herein further comprises modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the endogenous TCR. In some embodiments, the method of producing a plurality of modified T cells as disclosed herein further comprises modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the endogenous TCR. In some embodiments, the method of producing a plurality of modified T cells disclosed herein further comprises d) maintaining the plurality of modified T cells in a suitable cell culture medium; and e) either i) cryopreserving the plurality of modified T cells in a suitable cell freezing medium, or ii) preparing the plurality of modified T cells for administration to a subject suffering from a disease or disorder.

[0014] The disclosure also provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective number of cells comprising a nucleic acid encoding an RTCR disclosed herein or a vector comprising a nucleic acid encoding an RTCR disclosed herein, a therapeutically effective number of any one of the engineered T cells disclosed herein, a therapeutically effective amount of any one of the compositions disclosed herein, or a therapeutically effective number of engineered T cells produced by the methods disclosed herein.

[0015] The present disclosure also provides a chimeric costimulatory intracellular protein (CIP) comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least the second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

[0016] The present disclosure also provides a chimeric costimulatory intracellular protein (CIP) comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least the second signaling domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

[0017] Throughout this specification, the term "comprising" or variations such as "comprises" or "comprise" are understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0018] Throughout this specification, the terms "signal domain," "signaling domain," and "signal transduction domain" are used interchangeably, unless the context dictates otherwise.

[0019] While the present disclosure has been described in conjunction with the detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the present disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0020] The patent and scientific literature referred to herein establishes knowledge available to those of skill in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI deposits, as indicated by accession numbers, cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference. [Brief description of the drawings]

[0021] [Figure 1] Figure 1 shows an alignment of the intracellular tails of TNF receptor superfamily members used in the T cell costimulatory molecules of the present application. The membrane proximal polybasic region is shown in italics. Potential PI3K binding sites are bold and underlined. TRAF1 / 2 binding motif: major motif Px(Q / E)E and minor motif Px(Q / E)x are underlined. Potential ubiquitination sites are shown in bold.

[0022] [Figure 2A]1 shows the modular design of second and third generation costimulatory molecules. 2 shows the modular design of costimulatory molecules showing the signal peptide, extracellular domain, transmembrane domain, and intracellular signaling domain. 3 shows the structure and sequence of the first signaling domain: ICOS, CD28, and the ICOS intracellular domain with a portion of the CD28 domain inserted. 4 shows regions of the ICOS and CD28 intracellular domains with specific binding function and known binding partners. 5 shows the amino acid / nucleic acid sequences of the costimulatory molecules and intracellular domains as indicated.

[0023] [Figure 2B] 1 shows the modular design of second and third generation costimulatory molecules. 2 shows the modular design of costimulatory molecules showing the signal peptide, extracellular domain, transmembrane domain, and intracellular signaling domain. 3 shows the structure and sequence of the first signaling domain: ICOS, CD28, and the ICOS intracellular domain with a portion of the CD28 domain inserted. 4 shows regions of the ICOS and CD28 intracellular domains with specific binding function and known binding partners. 5 shows the amino acid / nucleic acid sequences of the costimulatory molecules and intracellular domains as indicated.

[0024] [Diagram 3] 1 shows a combination of an extracellular effector domain and an intracellular signaling domain of the present application.

[0025] [Figure 4A] Figure 1 shows that deletion of the N-terminal portion of the 4-1BB signaling domain, including the polybasic domain and lysine residues, rescues the expression of costimulatory molecules. Figure 2 shows the expression of human PD1 and huEGFRt on the surface of T lymphocytes after lentiviral transduction with the indicated constructs. Figure 3 shows normalized PD1 surface expression on huEGFRt expressing cells expressing different costimulatory molecules with wild-type or truncated 4-1BB domain or ICOS- or CD28-based chimeric intracellular domains containing the OX-40 domain, as indicated. The amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0026] [Figure 4B] Figure 1 shows that deletion of the N-terminal portion of the 4-1BB signaling domain, including the polybasic domain and lysine residues, rescues the expression of costimulatory molecules. Figure 2 shows the expression of human PD1 and huEGFRt on the surface of T lymphocytes after lentiviral transduction with the indicated constructs. Figure 3 shows normalized PD1 surface expression on huEGFRt expressing cells expressing different costimulatory molecules with wild-type or truncated 4-1BB domain or ICOS- or CD28-based chimeric intracellular domains containing the OX-40 domain, as indicated. The amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0027] [Figure 5A] Cytokine production and proliferation of T cells expressing different costimulatory molecules with ICOS-based chimeric intracellular domains containing wild-type or truncated 4-1BB or OX-40 domains. Antibody-mediated cross-linking of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (FIG. 5A), TNF (FIG. 5B), and IFNγ (FIG. 5C) were measured by bead-based multiplex assays in culture supernatants after 18 h of stimulation of T cells transduced with the indicated constructs with plate-bound anti-PD1 [2 μg / mL] and the indicated amount of plate-bound anti-CD3. The x-axis shows the different costimulatory molecules and the y-axis shows the amount of cytokine produced expressed as absorbance units (AU). Proliferation of T cells stimulated with the indicated plate-bound antibodies for 96 h is shown. Amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0028] [Figure 5B]Cytokine production and proliferation of T cells expressing different costimulatory molecules with ICOS-based chimeric intracellular domains containing wild-type or truncated 4-1BB or OX-40 domains. Antibody-mediated cross-linking of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (FIG. 5A), TNF (FIG. 5B), and IFNγ (FIG. 5C) were measured by bead-based multiplex assays in culture supernatants after 18 h of stimulation of T cells transduced with the indicated constructs with plate-bound anti-PD1 [2 μg / mL] and the indicated amount of plate-bound anti-CD3. The x-axis shows the different costimulatory molecules and the y-axis shows the amount of cytokine produced expressed as absorbance units (AU). Proliferation of T cells stimulated with the indicated plate-bound antibodies for 96 h is shown. Amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0029] [Figure 5C] Cytokine production and proliferation of T cells expressing different costimulatory molecules with ICOS-based chimeric intracellular domains containing wild-type or truncated 4-1BB or OX-40 domains. Antibody-mediated cross-linking of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (FIG. 5A), TNF (FIG. 5B), and IFNγ (FIG. 5C) were measured by bead-based multiplex assays in culture supernatants after 18 h of stimulation of T cells transduced with the indicated constructs with plate-bound anti-PD1 [2 μg / mL] and the indicated amount of plate-bound anti-CD3. The x-axis shows the different costimulatory molecules and the y-axis shows the amount of cytokine produced expressed as absorbance units (AU). Proliferation of T cells stimulated with the indicated plate-bound antibodies for 96 h is shown. Amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0030] [Figure 5D]Cytokine production and proliferation of T cells expressing different costimulatory molecules with ICOS-based chimeric intracellular domains containing wild-type or truncated 4-1BB or OX-40 domains. Antibody-mediated cross-linking of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (FIG. 5A), TNF (FIG. 5B), and IFNγ (FIG. 5C) were measured by bead-based multiplex assays in culture supernatants after 18 h of stimulation of T cells transduced with the indicated constructs with plate-bound anti-PD1 [2 μg / mL] and the indicated amount of plate-bound anti-CD3. The x-axis shows the different costimulatory molecules and the y-axis shows the amount of cytokine produced expressed as absorbance units (AU). Proliferation of T cells stimulated with the indicated plate-bound antibodies for 96 h is shown. Amino acid / nucleic acid sequences of the chimeric intracellular domains are as indicated.

[0031] [Figure 6A] 6 shows that PD-L1 engagement of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (upper panel), IFN (middle panel), and TNFγ (lower panel) measured by bead-based multiplex assay in culture supernatants after 18 h stimulation of T cells transduced with the indicated constructs are shown. The x-axis shows the amount of anti-CD3 antibody (μg / ml), and the y-axis shows cytokine production as a percentage of control. IL-2 (upper panel), IFNγ (middle panel), and TNF (lower panel) production by T cells transduced with the indicated costimulatory molecules containing the CD28 intracellular domain (FIG. 6B, upper, middle, and lower panels) and the ICOS intracellular domain (FIG. 6C, upper, middle, and lower panels), respectively, and stimulated with the indicated concentrations of soluble anti-CD3 antibody in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). The x-axis shows the amount of anti-CD3 antibody (μg / ml) and the y-axis shows cytokine production as a percentage of control. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0032] [Figure 6B]6 shows that PD-L1 engagement of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (upper panel), IFN (middle panel), and TNFγ (lower panel) measured by bead-based multiplex assay in culture supernatants after 18 h stimulation of T cells transduced with the indicated constructs are shown. The x-axis shows the amount of anti-CD3 antibody (μg / ml), and the y-axis shows cytokine production as a percentage of control. IL-2 (upper panel), IFNγ (middle panel), and TNF (lower panel) production by T cells transduced with the indicated costimulatory molecules containing the CD28 intracellular domain (FIG. 6B, upper, middle, and lower panels) and the ICOS intracellular domain (FIG. 6C, upper, middle, and lower panels), respectively, and stimulated with the indicated concentrations of soluble anti-CD3 antibody in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). The x-axis shows the amount of anti-CD3 antibody (μg / ml) and the y-axis shows cytokine production as a percentage of control. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0033] [Figure 6C]6 shows that PD-L1 engagement of costimulatory molecules increases T cell cytokine production in vitro. IL-2 (upper panel), IFN (middle panel), and TNFγ (lower panel) measured by bead-based multiplex assay in culture supernatants after 18 h stimulation of T cells transduced with the indicated constructs are shown. The x-axis shows the amount of anti-CD3 antibody (μg / ml), and the y-axis shows cytokine production as a percentage of control. IL-2 (upper panel), IFNγ (middle panel), and TNF (lower panel) production by T cells transduced with the indicated costimulatory molecules containing the CD28 intracellular domain (FIG. 6B, upper, middle, and lower panels) and the ICOS intracellular domain (FIG. 6C, upper, middle, and lower panels), respectively, and stimulated with the indicated concentrations of soluble anti-CD3 antibody in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). The x-axis shows the amount of anti-CD3 antibody (μg / ml) and the y-axis shows cytokine production as a percentage of control. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0034] [Figure 7A]Figure 5 shows that engagement of the costimulatory molecule PD-L1 increases T cell cytotoxicity and proliferation in vitro. T cells expressing PD-1 constructs and K562 cells were mixed and stimulated as in Figure 6. Shown are the number of remaining K562 cells (upper panel) and the number of T cells (lower panel) assessed by flow cytometry after 96 hours of stimulation with the indicated concentrations of soluble anti-CD3 antibodies in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). Shown is the proliferation of T cells expressing various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by "X") or HLA-A2 and PD-L1 (indicated by "*") and 0.3 μg / ml of anti-CD3 as measured by the shift in Cell Trace violet dilution shown on the x-axis. Graph showing the number of remaining target cells (K562) assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. Graph showing the number of T cells assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. The x-axis shows the amount of CD3 antibody (μg / ml) and the y-axis shows the number of cells. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0035] [Figure 7B]Figure 5 shows that engagement of the costimulatory molecule PD-L1 increases T cell cytotoxicity and proliferation in vitro. T cells expressing PD-1 constructs and K562 cells were mixed and stimulated as in Figure 6. Shown are the number of remaining K562 cells (upper panel) and the number of T cells (lower panel) assessed by flow cytometry after 96 hours of stimulation with the indicated concentrations of soluble anti-CD3 antibodies in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). Shown is the proliferation of T cells expressing various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by "X") or HLA-A2 and PD-L1 (indicated by "*") and 0.3 μg / ml of anti-CD3 as measured by the shift in Cell Trace violet dilution shown on the x-axis. Graph showing the number of remaining target cells (K562) assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. Graph showing the number of T cells assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. The x-axis shows the amount of CD3 antibody (μg / ml) and the y-axis shows the number of cells. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0036] [Figure 7C]Figure 5 shows that engagement of the costimulatory molecule PD-L1 increases T cell cytotoxicity and proliferation in vitro. T cells expressing PD-1 constructs and K562 cells were mixed and stimulated as in Figure 6. Shown are the number of remaining K562 cells (upper panel) and the number of T cells (lower panel) assessed by flow cytometry after 96 hours of stimulation with the indicated concentrations of soluble anti-CD3 antibodies in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). Shown is the proliferation of T cells expressing various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by "X") or HLA-A2 and PD-L1 (indicated by "*") and 0.3 μg / ml of anti-CD3 as measured by the shift in Cell Trace violet dilution shown on the x-axis. Graph showing the number of remaining target cells (K562) assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. Graph showing the number of T cells assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. The x-axis shows the amount of CD3 antibody (μg / ml) and the y-axis shows the number of cells. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0037] [Figure 7D]Figure 5 shows that engagement of the costimulatory molecule PD-L1 increases T cell cytotoxicity and proliferation in vitro. T cells expressing PD-1 constructs and K562 cells were mixed and stimulated as in Figure 6. Shown are the number of remaining K562 cells (upper panel) and the number of T cells (lower panel) assessed by flow cytometry after 96 hours of stimulation with the indicated concentrations of soluble anti-CD3 antibodies in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). Shown is the proliferation of T cells expressing various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by "X") or HLA-A2 and PD-L1 (indicated by "*") and 0.3 μg / ml of anti-CD3 as measured by the shift in Cell Trace violet dilution shown on the x-axis. Graph showing the number of remaining target cells (K562) assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. Graph showing the number of T cells assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. The x-axis shows the amount of CD3 antibody (μg / ml) and the y-axis shows the number of cells. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0038] [Figure 7E]Figure 5 shows that engagement of the costimulatory molecule PD-L1 increases T cell cytotoxicity and proliferation in vitro. T cells expressing PD-1 constructs and K562 cells were mixed and stimulated as in Figure 6. Shown are the number of remaining K562 cells (upper panel) and the number of T cells (lower panel) assessed by flow cytometry after 96 hours of stimulation with the indicated concentrations of soluble anti-CD3 antibodies in the presence of K562 cells expressing HLA-A2 (left panel) or HLA-A2 and PD-L1 (right panel). Shown is the proliferation of T cells expressing various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by "X") or HLA-A2 and PD-L1 (indicated by "*") and 0.3 μg / ml of anti-CD3 as measured by the shift in Cell Trace violet dilution shown on the x-axis. Graph showing the number of remaining target cells (K562) assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. Graph showing the number of T cells assessed by flow cytometry 96 hours after stimulation with T cells expressing costimulatory molecules in the presence of increasing amounts of anti-CD3 antibody (μg / ml) as indicated. The x-axis shows the amount of CD3 antibody (μg / ml) and the y-axis shows the number of cells. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0039] [Figure 8] Figure 1 shows that engagement of costimulatory molecules increases T cell proliferation in vitro. T cells were stimulated for 96 hours on plate-bound antibodies with 2 μg / mL anti-PD1 and anti-CD3 [mg / mL] concentrations (indicated by "*") or anti-CD3 alone (indicated by "X") as shown on the y-axis, and proliferation of T cells expressing various PD1 constructs was measured by shifts in crystal violet tracings as shown on the x-axis. Amino acid / nucleic acid sequences of the costimulatory molecules are as shown.

[0040] [Figure 9A]9A-9C show the effect of polybasic and lysine residue mutations on expression or function of costimulatory molecules incorporating ICOS and 4-1BB signaling domains. A series of flow cytometry plots showing proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-based costimulatory molecules (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (lines unlabeled). A graph showing PD-1 expression (expressed as fold increase from endogenous levels) from the FACS plots of FIG. 9A. A graph showing cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the graphs in the bottom row. Graphs show proliferation of T cells after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0041] [Figure 9B]9A-9C show the effect of polybasic and lysine residue mutations on expression or function of costimulatory molecules incorporating ICOS and 4-1BB signaling domains. A series of flow cytometry plots showing proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-based costimulatory molecules (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (lines unlabeled). A graph showing PD-1 expression (expressed as fold increase from endogenous levels) from the FACS plots of FIG. 9A. A graph showing cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the graphs in the bottom row. Graphs show proliferation of T cells after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0042] [Figure 9C]9A-9C show the effect of polybasic and lysine residue mutations on expression or function of costimulatory molecules incorporating ICOS and 4-1BB signaling domains. A series of flow cytometry plots showing proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-based costimulatory molecules (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (lines unlabeled). A graph showing PD-1 expression (expressed as fold increase from endogenous levels) from the FACS plots of FIG. 9A. A graph showing cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the graphs in the bottom row. Graphs show proliferation of T cells after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0043] [Figure 9D]9A-9C show the effect of polybasic and lysine residue mutations on expression or function of costimulatory molecules incorporating ICOS and 4-1BB signaling domains. A series of flow cytometry plots showing proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-based costimulatory molecules (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (lines unlabeled). A graph showing PD-1 expression (expressed as fold increase from endogenous levels) from the FACS plots of FIG. 9A. A graph showing cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the graphs in the bottom row. Graphs show proliferation of T cells after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0044] [Figure 10A]Figure 10 shows the expression and function of costimulatory molecules incorporating ICOS and OX-40 signaling domains. A series of flow cytometry plots showing the proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-switched receptors (PD1-based costimulatory molecules) (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (not shown). Figure 10B shows PD-1 expression (fold of endogenous expression) from the FACS plots of Figure 10A. Figure 10C shows cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the bottom row of graphs. Graphs show T cell proliferation after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). The differences between T cell proliferation in the presence or absence of PD-L1 on target cells are shown in the far right graph. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0045] [Figure 10B]Figure 10 shows the expression and function of costimulatory molecules incorporating ICOS and OX-40 signaling domains. A series of flow cytometry plots showing the proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-switched receptors (PD1-based costimulatory molecules) (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (not shown). Figure 10B shows PD-1 expression (fold of endogenous expression) from the FACS plots of Figure 10A. Figure 10C shows cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the bottom row of graphs. Graphs show T cell proliferation after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). The differences between T cell proliferation in the presence or absence of PD-L1 on target cells are shown in the far right graph. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0046] [Figure 10C]Figure 10 shows the expression and function of costimulatory molecules incorporating ICOS and OX-40 signaling domains. A series of flow cytometry plots showing the proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-switched receptors (PD1-based costimulatory molecules) (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (not shown). Figure 10B shows PD-1 expression (fold of endogenous expression) from the FACS plots of Figure 10A. Figure 10C shows cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the bottom row of graphs. Graphs show T cell proliferation after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). The differences between T cell proliferation in the presence or absence of PD-L1 on target cells are shown in the far right graph. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0047] [Figure 10D]Figure 10 shows the expression and function of costimulatory molecules incorporating ICOS and OX-40 signaling domains. A series of flow cytometry plots showing the proliferation of T cells expressing either wild-type PD1 receptor (indicated by "*") or different PD1-switched receptors (PD1-based costimulatory molecules) (indicated by "X") as indicated by the labels on top of each plot. T cells expressing endogenous PD-1 were used as controls (not shown). Figure 10B shows PD-1 expression (fold of endogenous expression) from the FACS plots of Figure 10A. Figure 10C shows cytokine production (IL-2, left panel; IFNy, center panel; and TNF, right panel) (y-axis) by T cells expressing different costimulatory molecules in response to K562 cells (top row) and K562-PDL1 expressing cells (middle row) when stimulated with the indicated concentrations of anti-CD3 (x-axis) as indicated. The differences in levels of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells are shown in the bottom row of graphs. Graphs show T cell proliferation after 96 hours of culture with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph) when stimulated with the indicated concentrations of anti-CD3 (x-axis). The differences between T cell proliferation in the presence or absence of PD-L1 on target cells are shown in the far right graph. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0048] [Figure 11A] Figure 11 shows that engagement of costimulatory molecules increases T cell conjugation with PD-L1 expressing cells. Flow cytometry gating strategy of 30 min conjugation of CFSE labeled T cells with CTV labeled K562 targets. Quantification of results from the two experiments shown in Figure 11A is shown and normalized to control conjugation. Amino acid / nucleic acid sequences of costimulatory molecules with and without signaling peptides are as indicated.

[0049] [Figure 11B]Figure 11 shows that engagement of costimulatory molecules increases T cell conjugation with PD-L1 expressing cells. Flow cytometry gating strategy of 30 min conjugation of CFSE labeled T cells with CTV labeled K562 targets. Quantification of results from the two experiments shown in Figure 11A is shown and normalized to control conjugation. Amino acid / nucleic acid sequences of costimulatory molecules with and without signaling peptides are as indicated.

[0050] [Figure 12A] Figure 1 shows increased T cell proliferation and function upon engagement of costimulatory molecules with PD-L1 expressing cells. Flow cytometry plots showing surface expression of PD-1 and TCR β chain with (lower middle and right plots) and without (upper middle and right plots) costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated, in T cells expressing either wild-type HLA-A2 / NY-ESO-1 specific TCR or mutant NY-ESO TCR, when co-cultured with K562 cells, 72 hours after lentiviral transfection. Graphs showing IL-2 (top graph) and IFNγ (bottom graph) producing T cells expressing NY-ESO-1 / PD1 based costimulatory molecule combinations as indicated, when co-cultured with HLA-A2 and antigen expressing A375 tumor cells, with T cell:A375 cell ratios shown on the x-axis. Graph showing dose-dependent killing of A375 cells by T cells expressing the indicated wild-type NY-ESO TCR or mutant high affinity (HA) NY-ESO TCR with / without costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated. The x-axis shows dose (T cell:A375 cell ratio) and percentage of total input A375 cells surviving. Amino acid / nucleic acid sequences of costimulatory molecules with and without signaling peptides are as indicated.

[0051] [Figure 12B]Figure 1 shows increased T cell proliferation and function upon engagement of costimulatory molecules with PD-L1 expressing cells. Flow cytometry plots showing surface expression of PD-1 and TCR β chain with (lower middle and right plots) and without (upper middle and right plots) costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated, in T cells expressing either wild-type HLA-A2 / NY-ESO-1 specific TCR or mutant NY-ESO TCR, when co-cultured with K562 cells, 72 hours after lentiviral transfection. Graphs showing IL-2 (top graph) and IFNγ (bottom graph) producing T cells expressing NY-ESO-1 / PD1 based costimulatory molecule combinations as indicated, when co-cultured with HLA-A2 and antigen expressing A375 tumor cells, with T cell:A375 cell ratios shown on the x-axis. Graph showing dose-dependent killing of A375 cells by T cells expressing the indicated wild-type NY-ESO TCR or mutant high affinity (HA) NY-ESO TCR with / without costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated. The x-axis shows dose (T cell:A375 cell ratio) and percentage of total input A375 cells surviving. Amino acid / nucleic acid sequences of costimulatory molecules with and without signaling peptides are as indicated.

[0052] [Figure 12C]Figure 1 shows increased T cell proliferation and function upon engagement of costimulatory molecules with PD-L1 expressing cells. Flow cytometry plots showing surface expression of PD-1 and TCR β chain with (lower middle and right plots) and without (upper middle and right plots) costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated, in T cells expressing either wild-type HLA-A2 / NY-ESO-1 specific TCR or mutant NY-ESO TCR, when co-cultured with K562 cells, 72 hours after lentiviral transfection. Graphs showing IL-2 (top graph) and IFNγ (bottom graph) producing T cells expressing NY-ESO-1 / PD1 based costimulatory molecule combinations as indicated, when co-cultured with HLA-A2 and antigen expressing A375 tumor cells, with T cell:A375 cell ratios shown on the x-axis. Graph showing dose-dependent killing of A375 cells by T cells expressing the indicated wild-type NY-ESO TCR or mutant high affinity (HA) NY-ESO TCR with / without costimulatory molecule constructs containing ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt) as indicated. The x-axis shows dose (T cell:A375 cell ratio) and percentage of total input A375 cells surviving. Amino acid / nucleic acid sequences of costimulatory molecules with and without signaling peptides are as indicated.

[0053] [Figure 13A] 13A-C show that third generation tail mutations increase surface expression of the CD-19 CAR receptor on transduced primary T cells. Histograms of CD-19Fc binding to untransduced T cells (marked by x) or T cells transduced with the indicated constructs (marked by *) are shown. MFI measurements of the histograms shown in FIG. 13A normalized to FMC63scFV_BB_Z are shown. Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0054] [Figure 13B]13A-C show that third generation tail mutations increase surface expression of the CD-19 CAR receptor on transduced primary T cells. Histograms of CD-19Fc binding to untransduced T cells (marked by x) or T cells transduced with the indicated constructs (marked by *) are shown. MFI measurements of the histograms shown in FIG. 13A normalized to FMC63scFV_BB_Z are shown. Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0055] [Figure 14A] Figure 1 shows that modified 3rd generation tails increase cytokine production and tumor killing. Figure 2 shows in vitro killing of CD19 positive cells by CAR-R transduced primary T cells. Figure 3 shows the number of remaining cells of a B cell line (Nalm6 cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). Figure 4 shows the number of remaining cells of a B cell line (Raji cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). The y-axis shows the number of remaining CD19 positive cells, which corresponds to the ratio of T cells to CD19 positive cells shown on the x-axis. Figure 1 shows 18-h IFNγ production, as indicated on the y-axis, by T cells expressing second and third generation, CD28-based receptors (left panel) and ICOS-based receptors (right panel), at the T cell:target cell ratios indicated on the x-axis, in response to 18-h incubation with CD19-positive B cells. Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0056] [Figure 14B]Figure 1 shows that modified 3rd generation tails increase cytokine production and tumor killing. Figure 2 shows in vitro killing of CD19 positive cells by CAR-R transduced primary T cells. Figure 3 shows the number of remaining cells of a B cell line (Nalm6 cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). Figure 4 shows the number of remaining cells of a B cell line (Raji cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). The y-axis shows the number of remaining CD19 positive cells, which corresponds to the ratio of T cells to CD19 positive cells shown on the x-axis. Figure 1 shows 18-h IFNγ production, as indicated on the y-axis, by T cells expressing second and third generation, CD28-based receptors (left panel) and ICOS-based receptors (right panel), at the T cell:target cell ratios indicated on the x-axis, in response to 18-h incubation with CD19-positive B cells. Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0057] [Figure 14C] Figure 1 shows that modified 3rd generation tails increase cytokine production and tumor killing. Figure 2 shows in vitro killing of CD19 positive cells by CAR-R transduced primary T cells. Figure 3 shows the number of remaining cells of a B cell line (Nalm6 cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). Figure 4 shows the number of remaining cells of a B cell line (Raji cells) after 96 hours of co-culture with CAR-T cells expressing 2nd and 3rd generation receptors based on CD28 (left panel) and ICOS (right panel). The y-axis shows the number of remaining CD19 positive cells, which corresponds to the ratio of T cells to CD19 positive cells shown on the x-axis. Figure 1 shows 18-h IFNγ production, as indicated on the y-axis, by T cells expressing second and third generation, CD28-based receptors (left panel) and ICOS-based receptors (right panel), at the T cell:target cell ratios indicated on the x-axis, in response to 18-h incubation with CD19-positive B cells. Amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0058] [Figure 15A] Figure 2 shows that modified third generation signaling domains increase CD19-CAR function in vitro compared to the original third generation signaling domain. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells, as shown on the x-axis. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) CD28-based second and third generation receptors (right panel) over repeated stimulation with RAJI B cells, as shown on the x-axis. Graphs showing cumulative target cell (Nalm6) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells as indicated on the x-axis. Graphs showing cumulative target cell (Raji) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Raji B cells as indicated on the x-axis. A series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated after time 0 or 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. FIG. 1 is a series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated, at time 0 or after 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0059] [Figure 15B]Figure 2 shows that modified third generation signaling domains increase CD19-CAR function in vitro compared to the original third generation signaling domain. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells, as shown on the x-axis. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) CD28-based second and third generation receptors (right panel) over repeated stimulation with RAJI B cells, as shown on the x-axis. Graphs showing cumulative target cell (Nalm6) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells as indicated on the x-axis. Graphs showing cumulative target cell (Raji) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Raji B cells as indicated on the x-axis. A series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated after time 0 or 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. FIG. 1 is a series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated, at time 0 or after 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0060] [Figure 15C]Figure 2 shows that modified third generation signaling domains increase CD19-CAR function in vitro compared to the original third generation signaling domain. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells, as shown on the x-axis. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) CD28-based second and third generation receptors (right panel) over repeated stimulation with RAJI B cells, as shown on the x-axis. Graphs showing cumulative target cell (Nalm6) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells as indicated on the x-axis. Graphs showing cumulative target cell (Raji) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Raji B cells as indicated on the x-axis. A series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated after time 0 or 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. FIG. 1 is a series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated, at time 0 or after 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0061] [Figure 15D]Figure 2 shows that modified third generation signaling domains increase CD19-CAR function in vitro compared to the original third generation signaling domain. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells, as shown on the x-axis. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) CD28-based second and third generation receptors (right panel) over repeated stimulation with RAJI B cells, as shown on the x-axis. Graphs showing cumulative target cell (Nalm6) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells as indicated on the x-axis. Graphs showing cumulative target cell (Raji) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Raji B cells as indicated on the x-axis. A series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated after time 0 or 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. FIG. 1 is a series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated, at time 0 or after 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0062] [Figure 15E]Figure 2 shows that modified third generation signaling domains increase CD19-CAR function in vitro compared to the original third generation signaling domain. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells, as shown on the x-axis. Graph showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) CD28-based second and third generation receptors (right panel) over repeated stimulation with RAJI B cells, as shown on the x-axis. Graphs showing cumulative target cell (Nalm6) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Nalm6 B cells as indicated on the x-axis. Graphs showing cumulative target cell (Raji) numbers (indicating target cell killing) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel) and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with Raji B cells as indicated on the x-axis. A series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated after time 0 or 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. FIG. 1 is a series of flow cytometry plots showing Tim3 and PD-1 expression on CAR-T cells as indicated, at time 0 or after 5 consecutive stimulations with RAJI B cell targets. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0063] [Figure 16A]FIG. 16B shows that modified third generation signaling domains increase BCMA-CAR function in vitro compared to the original third generation sequence. Flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by "X") or T cells transduced with the indicated BCMA CAR-T receptors including CD28-based and ICOS-based second and third generation costimulatory molecules (indicated by "*") as indicated. FIG. 16C shows BCMA-Fc binding (MFI) (x-axis) from transduced T cells of the FACS plots of FIG. 16A. FIG. 16D shows a series of graphs showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel), and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells. Graphs showing cumulative target cell numbers (indicating T cell killing) (y-axis) of T cells expressing a) CD28-based 2nd and 3rd generation receptors (left panel), and b) ICOS-based 2nd and 3rd generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells, as indicated on the x-axis. A series of graphs showing cytokine production (IL-2, left panel, TNF, middle panel, and IFNγ, right panel) (y-axis) using CAR-T cells transduced with the indicated BCMA CAR constructs containing CD28-based or ICOS-based costimulatory molecules, as indicated, incubated for 18 hours at effector to target ratios indicated on the x-axis. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0064] [Figure 16B]FIG. 16B shows that modified third generation signaling domains increase BCMA-CAR function in vitro compared to the original third generation sequence. Flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by "X") or T cells transduced with the indicated BCMA CAR-T receptors including CD28-based and ICOS-based second and third generation costimulatory molecules (indicated by "*") as indicated. FIG. 16C shows BCMA-Fc binding (MFI) (x-axis) from transduced T cells of the FACS plots of FIG. 16A. FIG. 16D shows a series of graphs showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel), and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells. Graphs showing cumulative target cell numbers (indicating T cell killing) (y-axis) of T cells expressing a) CD28-based 2nd and 3rd generation receptors (left panel), and b) ICOS-based 2nd and 3rd generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells, as indicated on the x-axis. A series of graphs showing cytokine production (IL-2, left panel, TNF, middle panel, and IFNγ, right panel) (y-axis) using CAR-T cells transduced with the indicated BCMA CAR constructs containing CD28-based or ICOS-based costimulatory molecules, as indicated, incubated for 18 hours at effector to target ratios indicated on the x-axis. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0065] [Figure 16C]FIG. 16B shows that modified third generation signaling domains increase BCMA-CAR function in vitro compared to the original third generation sequence. Flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by "X") or T cells transduced with the indicated BCMA CAR-T receptors including CD28-based and ICOS-based second and third generation costimulatory molecules (indicated by "*") as indicated. FIG. 16C shows BCMA-Fc binding (MFI) (x-axis) from transduced T cells of the FACS plots of FIG. 16A. FIG. 16D shows a series of graphs showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel), and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells. Graphs showing cumulative target cell numbers (indicating T cell killing) (y-axis) of T cells expressing a) CD28-based 2nd and 3rd generation receptors (left panel), and b) ICOS-based 2nd and 3rd generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells, as indicated on the x-axis. A series of graphs showing cytokine production (IL-2, left panel, TNF, middle panel, and IFNγ, right panel) (y-axis) using CAR-T cells transduced with the indicated BCMA CAR constructs containing CD28-based or ICOS-based costimulatory molecules, as indicated, incubated for 18 hours at effector to target ratios indicated on the x-axis. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0066] [Figure 16D]FIG. 16B shows that modified third generation signaling domains increase BCMA-CAR function in vitro compared to the original third generation sequence. Flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by "X") or T cells transduced with the indicated BCMA CAR-T receptors including CD28-based and ICOS-based second and third generation costimulatory molecules (indicated by "*") as indicated. FIG. 16C shows BCMA-Fc binding (MFI) (x-axis) from transduced T cells of the FACS plots of FIG. 16A. FIG. 16D shows a series of graphs showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel), and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells. Graphs showing cumulative target cell numbers (indicating T cell killing) (y-axis) of T cells expressing a) CD28-based 2nd and 3rd generation receptors (left panel), and b) ICOS-based 2nd and 3rd generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells, as indicated on the x-axis. A series of graphs showing cytokine production (IL-2, left panel, TNF, middle panel, and IFNγ, right panel) (y-axis) using CAR-T cells transduced with the indicated BCMA CAR constructs containing CD28-based or ICOS-based costimulatory molecules, as indicated, incubated for 18 hours at effector to target ratios indicated on the x-axis. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated.

[0067] [Figure 16E]FIG. 16B shows that modified third generation signaling domains increase BCMA-CAR function in vitro compared to the original third generation sequence. Flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by "X") or T cells transduced with the indicated BCMA CAR-T receptors including CD28-based and ICOS-based second and third generation costimulatory molecules (indicated by "*") as indicated. FIG. 16C shows BCMA-Fc binding (MFI) (x-axis) from transduced T cells of the FACS plots of FIG. 16A. FIG. 16D shows a series of graphs showing cumulative T cell numbers (indicating T cell proliferation) (y-axis) of T cells expressing a) CD28-based second and third generation receptors (left panel), and b) ICOS-based second and third generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells. Graphs showing cumulative target cell numbers (indicating T cell killing) (y-axis) of T cells expressing a) CD28-based 2nd and 3rd generation receptors (left panel), and b) ICOS-based 2nd and 3rd generation receptors (right panel) over repeated stimulation with RPMI-8226 multiple myeloma target cells, as indicated on the x-axis. A series of graphs showing cytokine production (IL-2, left panel, TNF, middle panel, and IFNγ, right panel) (y-axis) using CAR-T cells transduced with the indicated BCMA CAR constructs containing CD28-based or ICOS-based costimulatory molecules, as indicated, incubated for 18 hours at effector to target ratios indicated on the x-axis. The amino acid / nucleic acid sequences of the costimulatory molecules are as indicated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Provided herein is a novel chimeric costimulatory intracellular domain. Provided herein is a chimeric costimulatory intracellular domain comprising (a) a first signaling domain based on an intracellular signaling domain of a CD28 family protein, and (b) at least a second signaling domain comprising a mutant intracellular signaling domain of a TNFR family protein.

[0069] CD28 family proteins have a single extracellular immunoglobulin variable-like (IgV) domain followed by a short cytoplasmic tail. Members of the CD28 family proteins include CD28, CD28H, inducible costimulatory factor (ICOS), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152), programmed death-1 (PD-1), and B- and T-lymphocyte attenuator (BTLA). CD28, CD28H, and ICOS are costimulatory proteins expressed on T cells that promote activation, high levels of cytokine / chemokine expression, resistance to apoptosis, and proliferation of T cells.

[0070] Tumor necrosis factor receptor (TNFR) family proteins include TNFR1 (tumor necrosis factor receptor 1 / TNFRSF1A), TNFR2 (tumor necrosis factor receptor 2 / TNFRSF1B), lymphotoxin β receptor / TNFRSF3, OX40 / TNFRSF4, CD40 / TNFRSF5, Fas / TNFRSF6, decoy receptor 3 / TNFRSF6B, CD27 / TNFRSF7, CD30 / TNFRSF8, 4-1BB / TNFRSF9, DR4 (death receptor 4 / TNFRSF10A), DR5 (death receptor 5 / TNFRSF10A), and TNFRSF1 (death receptor 5 / TNFRSF10B). F10B), decoy receptor 1 / TNFRSF10C, decoy receptor 2 / TNFRSF10D, RANK (receptor activator of NF-κB / TNFRSF11A), OPG (osteoprotegerin / TNFRSF11B), DR3 (cell death receptor 3 / TNFRSF25), TWEAK receptor / TNFRSF12A, TACI / TNFRSF13B, BAFF-R (BAFF receptor / TNFRSF13C), HVEM (herpes virus entry mediator / TNFRSF14), nerve growth factor receptor / TNFRSF16, BCMA (B cell Tumor necrosis factor superfamily (TNFSF) ligands and TNF receptor superfamily (TNFRSF) receptors include cytoplasmic cytoplasmic antigen / TNFRSF17, GITR (glucocorticoid-inducible TNF receptor / TNFRSF18), TAJ (toxic and JNK inducer / TNFRSF19), RELT / TNFRSF19L, DR6 (cell death receptor 6 / TNFRSF21), TNFRSF22, TNFRSF23, ectodysplasin A2 isoform receptor / TNFRS27, and ectodysplasin 1-anhidrosis receptor. The interaction between TNFRSF provides costimulatory signals that control survival, proliferation, differentiation, and effector functions of immune cells. Depending on the specific intracellular signals induced by TNFRSF members, they can be classified into three groups-death domain (DD)-containing receptors, decoy receptors, and TNF receptor-associated factor (TRAF)-binding receptors. Some TNFRSFs, such as TNFR-1, Fas, DR3, DR4, DR5, and DR6, contain their own DD and / or interact with other cytoplasmic DD-containing adaptor molecules.Some other TNFRSFs, such as TNFR-2, CD27, CD30, CD40, glucocorticoid-inducible TNFR family-related gene (GITR), Fn1, lymphotoxin beta receptor (LTβR), OX40, receptor activator of NF-κB (RANK), and XEDAR, lack the DD and contain a motif with 4-6 amino acids called a TRAF interaction motif (TIM) that recruits TRAF proteins. TRAF proteins are adaptor molecules that activate multiple downstream signaling pathways, such as NF-κB, Janus kinase (JNK), ERK, p38MAPK, and PI3K, that aid in cell survival, proliferation, and cytokine production. In some embodiments, the first signaling domain based on the intracellular signaling domain of a CD28 family protein is selected from a CD28 protein, an ICOS protein, or a combination thereof. In some embodiments, at least the second signaling domain is based on a variant of the intracellular signaling domain of a TNFR family protein selected from CD137 (4-1BB) and CD134 (OX-40).

[0071] Provided herein is a novel chimeric costimulatory intracellular domain based on the third generation costimulatory domain of the present application. Reduced surface expression is a major obstacle in the development of chimeric costimulatory proteins for therapeutic purposes. The present disclosure provides a novel chimeric costimulatory intracellular domain generated through mutations in the third generation costimulatory domain of the present application, which is highly expressed and highly functional compared to current second and third generation chimeric receptors effective in inducing costimulation. The chimeric costimulatory intracellular domain provided herein comprises (a) a first signaling domain based on the intracellular signaling domain of CD28 protein, ICOS protein, or a combination thereof, and (b) at least a second signaling domain that is a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain. In some embodiments, the mutant CD137 (4-1BB) intracellular domain or the mutant CD134 (OX-40) intracellular domain comprises one or more amino acid deletions, insertions, or substitutions in the membrane proximal portion of the CD137 or CD134 intracellular domain. In some embodiments, one or more amino acids in the membrane proximal portion are ubiquitination sites involved in ubiquitination and degradation of CD137 or CD134 proteins. In some embodiments, the mutant CD137 (4-1BB) intracellular domain or mutant CD134 (OX-40) intracellular domain comprises a deletion, insertion, or substitution of one or more lysine residues in the membrane proximal portion of the CD137 or CD134 intracellular domain. In some embodiments, the lysine residues are ubiquitination sites involved in ubiquitination and degradation of CD137 or CD134 proteins. In some embodiments, the chimeric costimulatory intracellular domain provided herein further comprises a third signaling domain. In some embodiments, the third signaling domain may be based on the CD3 zeta signaling domain.

[0072] In some embodiments, the novel costimulatory intracellular domain of the present application can be combined or fused in-frame with the extracellular domain of any known costimulatory protein, a cell-intrinsic immune checkpoint inhibitor, a chimeric antigen receptor, an antibody or a portion thereof, a ligand or its receptor, a cytokine or its receptor, a chemokine or its receptor, or a complement receptor to form a functional recombinant T cell costimulatory receptor (RTCR). In some embodiments, the RTCR can be expressed in a cell in combination with another T cell receptor (TCR), a chimeric antigen receptor, or a costimulatory protein. The RTCR comprising the novel costimulatory intracellular domain disclosed herein, when co-expressed with the TCR in a T cell, significantly increases the cell surface expression of the RTCR and / or the cell proliferation, activation, persistence, cytokine production, and / or effector function of the T cell compared to second generation costimulatory receptors.

[0073] Highly effective adoptive cellular therapy including cell-intrinsic inhibitors of T cell exhaustion that target shared safe tumor-associated antigens and can tolerate suppressive tumor microenvironment are described in this application. Exemplary chimeric molecules expressing the extracellular domain of PD-1 and a functionally optimized chimeric intracellular costimulatory domain are disclosed herein. Engineered T cells expressing the chimeric molecules of the present disclosure are generated to demonstrate the efficacy of the chimeric molecules in enhancing T cell stimulation, activation and proliferation. Both molecules are expressed on the same T cell to generate TCR-T products that are highly reactive to tumor cells expressing both cognate MHC / peptide complexes and high levels of PD-L1 / / PD-L2.

[0074] To illustrate, a cell-intrinsic inhibitor of T cell exhaustion is developed by co-expression of a third generation chimeric PD-1 receptor in combination with a T cell receptor targeting a tumor-associated antigen or specific antigen to enhance the efficacy of T cell-mediated killing of tumor cells. The third generation chimeric receptor disclosed herein can be used in combination with any endogenous or engineered T cell receptor as well as a chimeric artificial receptor (CAR). The results herein show that the third generation costimulatory molecules disclosed herein generate T cells with high physiological avidity and sustained proliferative potential while overriding negative signaling by PD-1 and instead delivering costimulatory signals in a PD-L1-rich environment. The novel costimulatory molecules can be co-expressed with tumor-associated antigen (TAA)-specific TCRs and used to target PD-L1 / PD-L2 and TAA-expressing tumors. As this shows, the synergistic effect between TCR activation and costimulatory molecules significantly increases the therapeutic window and generates potentially more effective candidates for clinical studies. In the disclosure described below, the design of third generation chimeric proteins is systematically optimized to further validate the improved anti-cancer efficacy in vitro and to explore the anti-tumor efficacy in vivo.

[0075] Costimulatory molecules are generated that incorporate the extracellular domain of PD-1 with the intracellular domains of CD28, ICOS, CD134, and CD137, alone and in various combinations. These sequences are optimized for surface expression and functionality by incorporating key mutations / deletions within the signaling domains of the chimeric receptors, focusing on the junction between CD28 and the TNF receptor family signaling domain. The functionality of these receptors is tested based on surface expression, in vitro signaling, in vitro T cell conjugation, cytokine production, proliferation, and cytotoxicity using a combination of soluble and plate-bound antibody stimulation with K562 target cells expressing PD-L1 or A375 tumor cells.

[0076] The disclosure herein provides an approach in which TCR-T products co-express chimeric costimulatory molecules together with recombinant TAA-specific TCRs or endogenous TCRs. This approach can target tumor-associated antigens with simultaneous antagonism of checkpoint inhibition and delivery of costimulatory signals to the infused T cell product. This approach not only leads to a much improved product, but also helps to develop a universal function-enhancing platform for further TCR-T products.

[0077] A key technical challenge impeding the clinical adoption of third-generation CARs combining two domains has been the unusually low expression of the chimeric proteins at the cell surface and the associated reduced functionality (Zhao, 2015, Guedan, 2018). This was also true for costimulatory molecules, where the combination of CD28 and CD137 signaling domains resulted in low-expressed, non-functional receptors (Ankri, 2013). Disclosed herein are switch receptor / costimulatory molecules based on the ICOS / CD137 signaling domain and optimized for surface expression, a significant improvement over previous studies using the CD28 signaling domain alone, mediating both increased effector function and persistence of adoptively transferred cells. The results described herein show that the third-generation costimulatory molecules disclosed herein generate T cells with high physiological avidity and sustained proliferative potential, while overriding negative signaling by PD-1 and instead delivering costimulatory signals in a PD-L1-rich environment. The novel switch receptor / costimulatory molecules disclosed herein can be co-expressed with endogenous TCRs or TAA-specific TCRs and used to target PD-L1 / PD-L2 expressing tumors, demonstrating that synergy between TCR activation and costimulatory molecules significantly increases the therapeutic window for potentially more effective candidates for clinical investigation.

[0078] Tumor-associated and tumor-specific antigens allow for immunological targeting of tumors with relatively minimal risk of off-tumor, on-target side effects. Tumor cells can upregulate these antigens, which can then be targeted by human immune responses or ACT. The present disclosure combines third-generation CAR-based costimulatory molecules, which exhibit superior functionality over CD28-based receptors, with new high-affinity TCRs targeting TAAs to generate TCR-T products that resist the suppressive functions of the TME.

[0079] The present disclosure provides a recombinant T cell costimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least a second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

[0080] In some embodiments, the mutant intracellular signaling domain of a TNFR family protein is one of a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

[0081] The present disclosure provides a recombinant T cell costimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least second signaling domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

[0082] Unless otherwise indicated, the terms "co-stimulatory molecule", "costimulatory molecule", "costimulatory molecule", "co-stimulatory protein", "costimulatory protein", "costimulatory protein", "co-stimulatory receptor", "costimulatory receptor", "costimulatory receptor" and "switch receptor" are used interchangeably and refer to a recombinant T cell costimulatory receptor (RTCR) comprising a novel chimeric costimulatory intracellular domain of the present application. These terms may be used in combination with terms such as "recombinant T cell", "recombinant", "chimeric T cell", and "chimera" to refer to the RTCR of the present application.

[0083] As described herein, a "recombinant T cell costimulatory receptor" or "switch receptor" of the present disclosure is a "costimulatory molecule," "costimulatory receptor" or "costimulatory protein" generated by operably linking an extracellular domain to an intracellular chimeric intracellular protein of the present disclosure.

[0084] As described herein, "CD137" is a member of the tumor necrosis factor (TNF) receptor family, also referred to as 4-1BB, CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and is induced by lymphocyte activation (ILA). As described herein, the terms "CD137", "4-1BB", "4-1BB wt", "4-1BB wild type", "BB", "BB wt" and "BB wild type" are used interchangeably throughout, unless otherwise indicated, for example, when describing constructs or costimulatory molecules of the present application.

[0085] In some embodiments, the CD137 intracellular domain may be derived from a mammalian CD137. In some embodiments, the mammalian CD137 may be human CD137, mouse CD137, rat CD137, or monkey CD137. In some embodiments, the CD137 intracellular domain may be derived from human CD137, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the human CD137 amino acid sequences according to GenBank Accession Nos. U03397, AAA62478, NP_001552, Q07011, AAH06196, and XP_006710681. In some embodiments, the CD137 intracellular domain may be derived from mouse CD137, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the mouse CD137 amino acid sequences according to GenBank Accession Nos. NP_001070977.1, NP_001070976.1, NP_035742.1, NP_033430.1, P20334.1, XP_011248530.1, XP_011248530.1, ABI30213.1, BAE32724.1, and AAH28507.1. In some embodiments, the CD137 intracellular domain may be derived from rat CD137 or an isoform or variant thereof that comprises an amino acid sequence identical to any one of the rat CD137 amino acid sequences according to GenBank Accession Nos.: NP_852049.1, NP_001020944.1, BAD99404.1, XP_008762504.1, XP_006239534.1, EDL81196.1, AAH97483.1, EHB16663.1, EHB16663.1, KFO38282.1, XP_010618177.1, XP_029414155.1, XP_029414154.1, XP_021099219.1, and XP_012888584.1. In some embodiments, the CD137 intracellular domain may be derived from monkey CD137, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the monkey CD137 amino acid sequences having GenBank Accession Nos. ABY47575.1, ABI30212.1, ABY47577.1, ABY47576.1 and ABY47578.1.

[0086] In some embodiments, the CD137 intracellular domain described herein comprises the amino acid sequence starting at amino acid position 214 to the last amino acid at the C-terminus of the amino acid sequence of a human CD137 protein described herein. In some embodiments, the CD137 intracellular domain described herein comprises the amino acid sequence starting at amino acid position 215 to the last amino acid at the C-terminus of the amino acid sequence of a mouse CD137 protein described herein.

[0087] In some embodiments, the mutant CD137 intracellular domain described herein is derived from any one of the CD137 proteins described herein comprising one or more mutations, which may be additions / insertions, deletions / truncations or substitutions / replacements of one or more amino acids within the amino acid sequence of the CD137 protein. In some embodiments, the mutant CD137 intracellular domain described herein is any one of the CD137 intracellular domain sequences described herein comprising one or more mutations, which may be additions / insertions, deletions / truncations or substitutions / replacements of one or more amino acids within the amino acid sequence of the CD137 intracellular domain. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 intracellular domain described herein comprising a deletion or substitution of one or more amino acids within the amino acid sequence of the CD137 intracellular domain that may be targeted for ubiquitination. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein described herein comprising a deletion or substitution of one or more lysine residues within the amino acid sequence of the CD137 intracellular domain that may be targeted for ubiquitination. In some embodiments, a mutant CD137 intracellular domain described herein is a CD137 protein described herein that comprises a deletion or substitution of one, two, three or four lysine residues within the amino acid sequence of the CD137 intracellular domain that can be targeted for ubiquitination. In some embodiments, the lysine residues within the amino acid sequence of the CD137 intracellular domain described herein that can be deleted or replaced are at amino acid positions 214, 218, 219 and / or 225 of the CD137 intracellular domain.

[0088] In some embodiments, the mutant CD137 intracellular domain may be a truncated CD137 intracellular domain. The truncated CD137 intracellular domain described herein may be any one of the CD137 proteins described herein, wherein a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 100, 200 or more amino acids has been deleted from the N-terminus of the CD137 protein described herein. The truncated CD137 intracellular domain described herein may be any one of the CD137 intracellular domain sequences described herein, wherein a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids has been deleted from the N-terminus of the CD137 intracellular domain described herein. In some embodiments, the amino acids deleted from the N-terminus of the CD137 intracellular domain comprise one or more proximal polybasic amino acids of the CD137 intracellular domain.

[0089] In some embodiments, the mutant CD137 intracellular domain can be a truncated CD137 intracellular domain. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence from amino acid position 13 to amino acid position 42 of a CD137 intracellular domain of the disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of amino acid position 1 to amino acid position 12 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO:1.

[0090] In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:3.

[0091] The truncated CD137 intracellular domains described herein are referred to interchangeably throughout as "truncated CD137", "CD137t", "truncated 4-1BB", "4-1BBt", "truncated BB" or "BBt" unless otherwise indicated, e.g., when describing constructs or costimulatory molecules of the present application. In some embodiments, the mutant CD137 intracellular domain comprises a deletion of one, two, three or four lysine residues from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain of the disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutations from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain of the disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutations at amino acid positions selected from amino acid positions 1, 5, 6 and 12 at the N-terminus of the CD137 intracellular domain of the disclosure. In some embodiments, the one or more lysine mutations are lysine to alanine mutations. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO:1.

[0092] In some embodiments, a mutant CD137 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, a mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations from amino acid position 1 to amino acid position 12 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, a mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5, and 6 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, a mutant CD137 intracellular domain comprising one or more proximal basic amino acid mutations of the disclosure further comprises a lysine mutation at amino acid position 12 of the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, the lysine mutation is a lysine to alanine mutation. In some embodiments, a CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO:1.

[0093] As described herein, "CD134" is a member of the tumor necrosis factor (TNF) receptor family, and is also referred to as OX-40, ACT35, IMD16, TXGP1L, and tumor necrosis factor receptor superfamily member 4 (TNFRSF4). As described herein, the terms "CD134", "OX-40", "OX40", "OX-40 wild type", "OX-40 wt", "OX40 wild type", "OX40 wt", "40", "40 wild type" and "40wt" are used interchangeably throughout, unless otherwise indicated, e.g., when describing constructs or costimulatory molecules of the present application.

[0094] In some embodiments, the CD134 intracellular domain can be derived from a mammalian CD 134. In some embodiments, the mammalian CD134 can be human CD134, mouse CD134, rat CD134, or monkey CD134. In some embodiments, the CD134 intracellular domain may be derived from human CD134, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the human CD134 amino acid sequences according to GenBank Accession Nos. NP_003318, AAI05071, AAI05073, XP_016857721.1, XP_016857720.1, XP_011540377.1, XP_011540379.1, XP_011540378.1, XP_011540376.1, P43489.1, NP_001284491.1, NP_003317.1, EAW56278.1, and CAB96543.1. In some embodiments, the CD134 intracellular domain may be derived from mouse CD134, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the mouse CD134 amino acid sequences according to GenBank Accession Nos. NP_035789.1, AAI39267.1, AAI39240.1, NP_033478.1, XP_006538787.3, P47741.1, EDL15067.1, CAA79772.1, CAA59476.1, XP_021017102.2, and XP_021056714.1. In some embodiments, the CD134 intracellular domain may be derived from rat CD134, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the rat CD134 amino acid sequences according to GenBank Accession Nos. NP_035789.1, NP_037181.1, P15725.1, EDL81353.1, CAB96543.1, and CAA34897.1.In some embodiments, the CD134 intracellular domain is selected from the group consisting of GenBank accession numbers: XP_010375483.1, XP_001090870.1, XP_021523144.1, XP_017750744.1, XP_003939714.1, XP_026313229.1, XP_026313228.1, XP_003890998.2, XP_025242 473.1, XP_011768627.1, XP_005545179.1, XP_011886513.1, XP_011886512.1, XP_011857387.1 and XP_011811769.1, or an isoform or variant thereof.

[0095] In some embodiments, the CD134 intracellular domain described herein comprises the amino acid sequence starting at amino acid position 241 to the last amino acid at the C-terminus of the amino acid sequence of any one of the human CD134 proteins described herein. In some embodiments, the CD134 intracellular domain described herein comprises the amino acid sequence starting at amino acid position 236 to the last amino acid at the C-terminus of the amino acid sequence of the mouse CD134 protein described herein.

[0096] In some embodiments, the mutant CD134 intracellular domain described herein is derived from any one of the CD134 proteins described herein comprising one or more mutations, which may be additions / insertions, deletions / truncations or substitutions / replacements of one or more amino acids within the amino acid sequence of the CD134 protein. In some embodiments, the mutant CD134 intracellular domain described herein is any one of the CD134 intracellular domain sequences described herein comprising one or more mutations, which may be additions / insertions, deletions / truncations or substitutions / replacements of one or more amino acids within the amino acid sequence of the CD134 intracellular domain. In some embodiments, the mutant CD134 intracellular domain described herein is a CD134 intracellular domain described herein comprising a deletion or substitution of one or more amino acids within the amino acid sequence of the CD134 intracellular domain that may be targeted for ubiquitination. In some embodiments, the mutant CD134 intracellular domain described herein is a CD134 protein described herein comprising a deletion or substitution of one or more lysine residues within the amino acid sequence of the CD134 intracellular domain that may be targeted for ubiquitination. In some embodiments, a mutant CD134 intracellular domain described herein is a CD134 protein described herein that comprises a deletion or substitution of one or two lysine residues within the amino acid sequence of the CD134 intracellular domain that can be targeted for ubiquitination. In some embodiments, the lysine residues within the amino acid sequence of the CD134 intracellular domain described herein that can be deleted or replaced are at amino acid positions 252 and / or 276 of the CD134 intracellular domain.

[0097] In some embodiments, the mutant CD134 intracellular domain can be a truncated CD134 intracellular domain. The truncated CD134 intracellular domain described herein can be any one of the CD134 proteins described herein, in which a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50, 100, 200 or more amino acids has been deleted from the N-terminus of the CD137 protein described herein. The truncated CD134 intracellular domain described herein can be any one of the CD134 intracellular domain sequences described herein, in which a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids has been deleted from the N-terminus of the CD134 intracellular domain described herein. In some embodiments, the amino acids deleted from the N-terminus of the CD134 intracellular domain include one or more proximal polybasic amino acids of the CD134 intracellular domain.

[0098] In some embodiments, the truncated CD134 intracellular domain comprises an amino acid sequence according to amino acid position 15 to amino acid position 37 of the CD134 intracellular domain of the disclosure. In some embodiments, the truncated CD134 intracellular domain comprises a deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments, the truncated CD134 intracellular domain comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO:4.

[0099] In some embodiments, the mutant CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the mutant CD134 intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:6.

[0100] The truncated CD134 intracellular domains described herein are referred to as "truncated CD134," "CD134t," "truncated OX-40," "truncated OX40," "OX-40t," "OX40t," and "40t," which are used interchangeably throughout, unless otherwise indicated, e.g., when describing constructs or costimulatory molecules of the present application.

[0101] In some embodiments, the mutant CD134 intracellular domain comprises a deletion of lysine residues from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments, the mutant CD134 intracellular domain comprises a lysine mutation at amino acid position 12 at the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments, the lysine mutation is a lysine to alanine mutation. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO:4.

[0102] In some embodiments, the mutant CD134 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 at the N-terminus of a CD134 intracellular domain of the disclosure. In some embodiments, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutations from amino acid position 1 to amino acid position 14 at the N-terminus of a CD134 intracellular domain of the disclosure. In some embodiments, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, and 5 at the N-terminus of a CD134 intracellular domain of the disclosure. In some embodiments, the mutant CD137 intracellular domain further comprises a lysine mutation at amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO:4.

[0103] [Table 1] The membrane proximal polybasic region is shown in italics. Potential PI3K binding sites are bold and underlined. The TRAF1 / 2 binding motif, the major motif Px(Q / E)E and the minor motif Px(Q / E)x are underlined. Potential ubiquitination sites are shown in bold.

[0104] In some embodiments, the chimeric intracellular domain comprises a first signaling domain derived from a protein of the CD28 family, hi some embodiments, the first signaling domain is derived from any one of CD28, CD28H, ICOS, or a combination thereof.

[0105] In some embodiments, the chimeric intracellular domain comprises a first signaling domain derived from an ICOS protein.

[0106] The "ICOS protein" described herein is an inducible T cell costimulatory protein, also referred to as AILIM, CD278, CCLP, CRP-1, H4, Ly115 and CVID1. In some embodiments, the ICOS intracellular domain may be derived from a mammalian ICOS. In some embodiments, the mammalian ICOS may be human ICOS, mouse ICOS, rat ICOS or monkey ICOS. In some embodiments, the ICOS intracellular domain may be derived from human ICOS, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the human ICOS amino acid sequences according to GenBank Accession Nos. AAH28006.1, NP_036224.1, AIC51287.1, AIC60036.1, NP_036224.1, Q9Y6W8.1, EAW70357.1, EAW70356.1, EAW70355.1, AAL40934.1, AAL40933.1, CAC06612.1, AAX93073.1, AAM00909.1, AAH28210.1 and CAD59742.1. In some embodiments, the ICOS intracellular domain may be derived from mouse ICOS, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the mouse ICOS amino acid sequences according to GenBank Accession Nos. NP_059508.2, Q9WVS0.2, EDL00161.1, CAM13242.1, CAM13241.1, CAB71153.1, AAG48732.1, AAH34852.1, XP_006496203.1, XP_006496202.1, XP_006496201.1, ACX50464.1, ACX50463.1, AAH28006.1, XP_021052880.1, XP_029334968.1, and XP_021030282.1.In some embodiments, the ICOS intracellular domain is selected from the group consisting of those listed under GenBank Accession Nos.: NP_072132.1, Q9R1T7.1, XP_008765358.1, XP_006245100.1, XP_006245099.1, EDL98922.1, EDL98921.1, XP_038940099.1, XP_032755449.1XP_017457364.1, XP_006256324.1, XP_00624509 ... The amino acid sequence may be derived from rat ICOS, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the rat ICOS amino acid sequences according to 006256323.1, XP_006256322.1XP_029425757.1, XP_029425757.1, XP_021119236.1, XP_012929934.1, XP_012867370.1 and XP_012867363.1. In some embodiments, the ICOS intracellular domain is selected from the group consisting of the following: GenBank Accession Nos. XP_007964137.1, NP_001253918.1, XP_010350939.1, XP_012301785.1, XP_012301784.1, XP_017739861.1, XP_01033471 4.1, XP_003925677.1, AFH29328.1, XP_008997520.1, XP_023075107.1, XP_023075 099.1, XP_021779593.1, XP_003907887.1, XP_025260988.1, XP_025260987.1, XP_ 025260986.1, XP_011716287.1, XP_011716285.1, XP_005574075.1, XP_011903009.1, XP_011805288.1, XP_011805287.1, XP_011847867.1, XP_011847866.1, XP_017392362.1, XP_033086489.1, XP_032134414.1, XP_032134413.1 and XP_017802331.1, or an isoform or variant thereof.

[0107] In some embodiments, the human ICOS intracellular domain described herein comprises the amino acid sequence from amino acid position 133 to the last amino acid at the C-terminus of the amino acid sequence of a human ICOS protein described herein. In some embodiments, the human ICOS intracellular domain described herein comprises the amino acid sequence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to amino acid position 133 to the last amino acid at the C-terminus of the amino acid sequence of a human ICOS protein described herein. In some embodiments, the human ICOS intracellular domain described herein comprises the amino acid sequence from amino acid position 133 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to the last amino acid at the C-terminus of the amino acid sequence of a human ICOS protein described herein. In some embodiments, the human ICOS intracellular domain described herein comprises the amino acid sequence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to amino acid position 133, and from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to the last amino acid at the C-terminus of the amino acid sequence of a human ICOS protein described herein.

[0108] In some embodiments, the human ICOS(28) intracellular domain described herein comprises a portion of the ICOS domain amino acid sequence from amino acid position 133 to amino acid position 183 of the amino acid sequence of the human ICOS protein described herein, and a portion of the ICOS domain amino acid sequence from amino acid position 184 to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein described herein. In some embodiments, the human ICOS(28) intracellular domain described herein comprises a portion of the ICOS domain amino acid sequence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to amino acid position 133 to amino acid position 183 of the human ICOS protein described herein. In some embodiments, the human ICOS(28) intracellular domain described herein comprises a portion of the ICOS domain amino acid sequence from amino acid position 133 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids C-terminal to amino acid position 183 of the human ICOS protein described herein. In some embodiments, the human ICOS(28) intracellular domain described herein comprises a portion of the ICOS domain amino acid sequence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids N-terminal to amino acid position 133 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids C-terminal to amino acid position 183 of a human ICOS protein described herein.

[0109] The "CD28 protein," also referred to as Tp44, is a constitutively expressed receptor for the CD80 (B7.1) and CD86 (B7.2) proteins on naive T cells and is important for T cell activation. In some embodiments, the CD28 intracellular domain can be derived from a mammalian CD28. In some embodiments, the mammalian CD28 can be human CD28, mouse CD28, rat CD28, or monkey CD28. In some embodiments, the CD28 intracellular domain is selected from the group consisting of the following: GenBank Accession Nos. P10747.1, NP_001230007.1, NP_001230006.1, NP_006130.1, EAW70350.1, EAW70349.1, EAW70348.1, EAW70347.1, AIC48451.1, CAC29237.1, AAA51945.1, AAA51944.1, AAL40931.1, AAF33794 ... It may be derived from human CD28, or an isoform or variant thereof, comprising an amino acid sequence identical to any one of the human CD28 amino acid sequences according to AF33793.1, AAF33792.1, XP_011510499.1, XP_011510497.1, XP_011510496.1, AAI12086.1, AAH93698.1, ABK41938.1, AAY24123.1, CAD57003.1 and AAA60581. In some embodiments, the CD28 intracellular domain may be derived from mouse CD28, or an isoform or variant thereof, that comprises an amino acid sequence identical to any one of the mouse CD28 amino acid sequences according to GenBank Accession Nos.: AAA37396.1, NP_031668.3, P31041.2, AAH64058.1, EDL00156.1, CAM13249.1, XP_036012281.1, XP_021054806.1, XP_021027481.1, XP_036015651.1, and XP_030104805.In some embodiments, the CD28 intracellular domain may be derived from rat CD28, or an isoform or variant thereof, that comprises an amino acid sequence identical to any one of the rat CD28 amino acid sequences according to GenBank Accession Nos. CAA39003.1, NP_037253.2, P31042.1, XP_008765300.1, EDL98926.1, XP_032755445.1, XP_034354910.1, XP_019061859.2, XP_008844474.1, XP_004851403.1, and XP_012865504.1. In some embodiments, the CD28 intracellular domain may be derived from monkey CD28, or an isoform or variant thereof, that comprises an amino acid sequence identical to any one of the monkey CD28 amino acid sequences according to GenBank Accession Nos. ABH06891.1, ABH08508.1, ABH06892.1ABH08509.1, ABQ09493.1, NP_001274262.1NP_001036106.2, ABG77998.1, ABG77997.1, and XP_014966207.1.

[0110] "CD28H protein," also referred to as CD28 homolog, and transmembrane and immunoglobulin domain-containing protein 2, has costimulatory activity in T cells by binding to B7H7. CD28H was first described as a molecule involved in cell-cell interactions, cell migration, and angiogenesis of epithelial and endothelial cells (7, 8). CD28H has a single extracellular immunoglobulin domain followed by a transmembrane domain and a 110 amino acid long cytoplasmic region. In some embodiments, the CD28 intracellular domain can be from a mammalian CD28H. In some embodiments, the mammalian CD28 can be human CD28H, mouse CD28H, rat CD28H, or monkey CD28H. In some embodiments, the CD28H intracellular domain may be derived from human CD28H, or an isoform or variant thereof, that comprises an amino acid sequence identical to any one of the human CD28H amino acid sequences according to GenBank Accession Nos. NP_001295161.1, NP_001162597.1, Q96BF3.2, XP_024307127.1, and XP_016881773.1.

[0111] In some embodiments, a human CD28 intracellular domain described herein comprises the amino acid sequence from amino acid position 145 to the last amino acid at the C-terminus of the amino acid sequence of a human CD28 protein described herein. In some embodiments, a portion of a human CD28 intracellular domain described herein may comprise the amino acid sequence from about amino acid position 195 to about amino acid position 212 of the amino acid sequence of a human CD28 protein described herein. In some embodiments, a portion of a human CD28 intracellular domain described herein may comprise the amino acid sequence from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid positions N-terminal to amino acid position 195 to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid positions C-terminal to amino acid position 220 of the amino acid sequence of a human CD28 protein described herein.

[0112] In some embodiments, the first signaling domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the first signaling domain derived from ICOS comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 9.

[0113] In some embodiments, the chimeric intracellular domain comprises a first signaling domain comprising a portion of a CD28 intracellular domain combined with an ICOS protein according to SEQ ID NO:9 (ICOS(28) domain). In some embodiments, the ICOS(28) domain comprises a portion of a CD28 intracellular domain inserted N-terminal to the PI-3K binding site of an ICOS protein according to SEQ ID NO:9. In some embodiments, the ICOS(28) domain comprises a portion of CD28 inserted 1, 2, 3, 4 or 5 amino acid positions N-terminal to the PI-3K binding site of an ICOS protein according to SEQ ID NO:9. In some embodiments, the ICOS(28) domain comprises a portion of CD28 inserted C-terminal to the PI-3K binding site of an ICOS protein according to SEQ ID NO:9. In some embodiments, the ICOS(28) domain comprises a portion of CD28 inserted 1, 2, 3, 4 or 5 amino acid positions C-terminal to the PI-3K binding site of an ICOS protein according to SEQ ID NO:9.

[0114] In some embodiments, the portion of CD28 is inserted at any amino acid position before amino acid position 48 in an ICOS protein of an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at any amino acid position from amino acid position 1 to amino acid position 48 in an ICOS protein of an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 47 to amino acid position 48 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 46 to amino acid position 47 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 45 to amino acid position 46 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 44 to amino acid position 45 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 43 to amino acid position 44 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9.

[0115] In some embodiments, the portion of CD28 is inserted at any position after amino acid position 51 in an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at any amino acid position from amino acid position 51 to amino acid position 67 in an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 51 to amino acid position 52 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 53 to amino acid position 54 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 54 to amino acid position 55 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 56 to amino acid position 57 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9. In some embodiments, the portion of CD28 is inserted at amino acid position 57 to amino acid position 58 of an ICOS protein having an amino acid sequence according to SEQ ID NO:9.

[0116] In some embodiments, the portion of CD28 in the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 51 to amino acid position 68 of the CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 in the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 51 to amino acid position 76 of the full length CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 in the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 45 to amino acid position 68 of the CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises a PRRP motif. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:11.

[0117] In some embodiments, the ICOS(28) domain comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, ICOS(28) comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:12.

[0118] In some embodiments, the chimeric intracellular domain comprises a first signaling domain derived from CD28. In some embodiments, the first signaling domain derived from CD28 comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the first signaling domain derived from CD28 comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 10.

[0119] The signaling domain derived from CD28 described herein is referred to interchangeably throughout as "CD28" or "28."

[0120] [Table 2] In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 13. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to any one of SEQ ID NOs: 14-17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to any one of SEQ ID NOs: 14-17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 14. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 15. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 15. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 16. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 16. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:17.

[0121] In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to any one of SEQ ID NOs: 120-129. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to any one of SEQ ID NOs: 120-129. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 120. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 120. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 121. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 121. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 122. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 122.

[0122] In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 123. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 123. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 124. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 124. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 125. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 125. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 126. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 126. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 127. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 127. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 128. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 128. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 129.In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:129.

[0123] [Table 3-1] [Table 3-2] In some embodiments, the chimeric intracellular domain further comprises a third signaling domain. In some embodiments, the third signaling domain is derived from any one of a CD3 signaling domain, a CD2 signaling domain, or an interleukin 2 receptor binding (IL-2RB) protein signaling domain. In some embodiments, the CD3 signaling domain is derived from a CD3ζ or CD3ε domain, or a combination thereof.

[0124] In some embodiments, the chimeric intracellular domain further comprises a third signaling domain derived from a CD3ζ protein. In some embodiments, the third signaling domain of the chimeric intracellular domain is a CD3ζ protein of an amino acid sequence according to SEQ ID NO: 18. In some embodiments, the third signaling domain of the chimeric intracellular domain is a CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity according to SEQ ID NO: 18.

[0125] Human CD3ζ full length (CD3Z full length) (SEQ ID NO: 18) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR In some embodiments, the third signaling domain of the chimeric intracellular domain is a CD3 signaling domain comprising an amino acid sequence according to any one of SEQ ID NOs: 45, 46, 47, and 48. In some embodiments, the third signaling domain of the chimeric intracellular domain is a CD3 ζ comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the third signaling domain of the chimeric intracellular domain is a CD3 ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 45. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD3 ζ comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 46. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the third signaling domain of the chimeric intracellular domain is a combination of CD3ε and truncated CD3ζ domain (CD3ζε domain). In some embodiments, the third signaling domain of the chimeric intracellular domain is CD3ζε comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the third signaling domain of the chimeric intracellular domain is CD3ζε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

[0126] In some embodiments, the third signaling domain of the chimeric intracellular domain is a mutant CD2 signaling domain. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the third signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 49.

[0127] In some embodiments, the third signaling domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the third signaling domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:50.

[0128] In some embodiments, the chimeric intracellular domain further comprises a fourth signaling domain, hi some embodiments, the fourth signaling domain is derived from any one of a CD3 signaling domain, a CD2 signaling domain, or an interleukin-2 receptor binding (IL-2RB) protein signaling domain, or a combination thereof, and wherein the third and fourth signaling domains are not identical.

[0129] In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a CD3 signaling domain comprising an amino acid sequence according to any one of SEQ ID NOs: 45, 46, 47, and 48. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a CD3 ζ comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a CD3 ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 45. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD3 ζ comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 46. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a combination of CD3ε and truncated CD3ζ domain (CD3ζε domain). In some embodiments, the fourth signaling domain of the chimeric intracellular domain is CD3ζε comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is CD3ζε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

[0130] In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a mutant CD2 signaling domain. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 49.

[0131] In some embodiments, the fourth signaling domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the fourth signaling domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:50.

[0132] The terms T cell, T-cell, t cell, t-cell and T lymphocyte may be used interchangeably in this disclosure.

[0133] In some embodiments, the extracellular domain comprises a protein or a portion thereof that binds to a target and induces activation and / or proliferation of an immune cell. In some embodiments, the extracellular domain comprises any one of a) a component of a T cell receptor (TCR) complex, b) a component of a chimeric antigen receptor (CAR), c) a component of a T cell co-receptor, the component of the T cell co-receptor being a T cell co-stimulatory protein or a T cell inhibitory protein, d) a ligand that binds to a cell surface receptor or a component thereof, e) a component of a cytokine receptor, e) a component of a chemokine receptor, g) a component of an integrin receptor, h) a component of an endothelial cell surface protein receptor or a fragment thereof, i) a component of a neural guidance protein receptor, and f) a component of a complement receptor. In some embodiments, the component of the T cell co-receptor or CAR is a component of PD1, CD28, CD2, OX-40, ICOS, CTLA-4, CD28, CD3, CD4, CD8, CD40L, Lag-3, Tim-3, or TIGIT, or a combination thereof. In some embodiments, the T cell coreceptor or CAR ligand or component binds to CD19, B cell maturation Ag (BCMA), PD-L1, PD-L2, IL-10, proliferation-inducing ligand (APRIL), BAFF, OX-40L, ICOS-L, B7-1, B7-2, CD40, CD58, CD59, nectin, CD155, or CD112, or a combination thereof. In some embodiments, the cytokine receptor binds to IL-10, IL-27, TGF-β, IL-12, IL-1, IL-2, IL-4, IL-5, IFN-γ, or IFN-α / β, or a combination thereof. In some embodiments, the complement receptor component is a single C3aR, C5aR, CD46 / MCP, CD55, CD97, or DAF, or a combination thereof.

[0134] In some embodiments, the extracellular domain comprises the amino acid sequence of a component of any one of: a) a chemokine receptor, b) a cytokine receptor, c) a ligand for a cell surface receptor, d) an integrin receptor, e) a cell adhesion molecule or receptor thereof, f) an endothelial cell surface protein receptor or a fragment thereof, g) a complement receptor, and h) a neural guidance protein receptor. In some embodiments, the extracellular domain comprises the amino acid sequence of a component of any one of: epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), chemokine receptor (CCR) 4, CCR5, CCR7, CCR10, lymphocyte function-associated antigen-1 (LFA-1), leukocyte-specific β2 integrins (αLβ2, αMβ2, αXβ2, αDβ2), β7 integrins (α4β7 and αEβ7), extracellular matrix (ECM)-associated β1 integrin (α1-α6β1), L-selectin, or sialyl Lewis. x The amino acid sequence of any one of the components.

[0135] In some embodiments, the extracellular domain is a protein, peptide, glycoprotein, antibody or fragment thereof, hi some embodiments, the antibody or fragment thereof is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, an sdAb, an Fv, a VHH fragment, or a single chain Fv fragment.

[0136] In some embodiments, the extracellular domain comprises two or more binding sites for targeting two or more non-identical target antigens. In some embodiments, the extracellular domain comprises two or more binding sites for targeting two or more non-identical sites on a target antigen. In some embodiments, the extracellular domain comprises two antigen-binding domains or fragments of a bispecific antibody. In some embodiments, the extracellular domain comprises a F(ab)2 fragment of a bispecific antibody. In some embodiments, the extracellular domain comprises two or more antigen-binding domains or fragments of a multispecific antibody.

[0137] In some embodiments, the extracellular domain binds to a target that is a tumor antigen, a pathogen-associated protein, or an antigen associated with a disease or disorder that is cancer, an autoimmune disease or disorder, an infectious disease, an inflammatory disease, a renal disease or disorder, a pulmonary disease or disorder, a hepatic disease or disorder, a cardiovascular disease or disorder, a neurodegenerative disorder or disorder, or a metabolic disorder or disorder.

[0138] In some embodiments, the tumor antigen is any one of a tumor-associated antigen (TAA), a tumor-secreted antigen (TSA), or a non-conventional antigen (UCA). In some embodiments, the TAA is any one of a cancer germline antigen (CGA), a human endogenous retrovirus (HERV), a tissue differentiation antigen (TDA), and an overexpressed tumor antigen. In some embodiments, the TSA is derived from any one of a mosaic single nucleotide variation (mSNV), an insertion-deletion mutation (INDEL), a gene fusion, and a viral oncoprotein. In some embodiments, the UCA is derived from a non-coding region of the genome or a coding region of the genome. In some embodiments, the UCA is derived from aberrant transcription, translation, or post-translational modification.

[0139] In some embodiments, the TAA is associated with a solid tumor or cancer or a hematological cancer, hi some embodiments, the TAA is associated with a solid tumor or the cancer is selected from a sarcoma, carcinoma, or lymphoma that manifests as, leads to, or is associated with a solid tumor.

[0140] In some embodiments, the TAA is associated with a sarcoma that is a soft tissue sarcoma or a sarcoma of bone (osteosarcoma). In some embodiments, the TAA is associated with a sarcoma that is a soft tissue sarcoma or a sarcoma of bone (osteosarcoma). In some embodiments, the TAA is associated with a sarcoma that is a soft tissue sarcoma or a sarcoma of bone (osteosarcoma). In some embodiments, the TAA is associated with a sarcoma that is a soft tissue sarcoma or a follicular soft tissue sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear tissue sarcoma, dedifferentiated liposarcoma, hyperplastic small round cell tumor of connective tissue, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, inflammatory neuroblastoma (esthesia neuroblastoma), Ewing's sarcoma, extraskeletal rhabdomyosarcoma, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, childhood fibrosarcoma, inflammatory myofibroblastoma, Kaposi's sarcoma, leiomyosarcoma of bone, liposarcoma, inflammatory myofibroblastoma, fibrosarcoma ... The present invention relates to a sarcoma selected from liposarcoma, osteosarcoma, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH), malignant mesenchymal tumor, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxoid liposarcoma, myxoid inflammatory fibroblastic sarcoma, multiple tumors with perivascular epithelial differentiation, osteosarcoma, extraperiosteal osteosarcoma, tumors with perivascular epithelial differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing's tumor, rhabdomyosarcoma, small cell osteosarcoma, monofibroid tumor, synovial sarcoma, or telangiectatic osteosarcoma.

[0141] In some embodiments, the TAA is associated with a carcinoma selected from basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma, hi some embodiments, the TAA is associated with a carcinoma selected from adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, or small cell carcinoma.

[0142] In some embodiments, the TAA is associated with a solid tumor or cancer selected from anal cancer, appendix cancer, cholangiocarcinoma (i.e., biliary tract cancer), breast cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, colon polyp, primary carcinoma of unknown etiology (cup), esophageal cancer, eye cancer, fallopian tube cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0143] In some embodiments, the breast cancer is invasive ductal carcinoma, ductal carcinoma in situ, invasive lobular carcinoma, or lobular carcinoma in situ. In some embodiments, the pancreatic cancer is adenocarcinoma or islet cell carcinoma. In some embodiments, the colorectal cancer is adenocarcinoma. In some embodiments, the colonic polyps are associated with familial adenomatous polyposis. In some embodiments, the bladder cancer is transitional cell bladder cancer, squamous cell bladder cancer, or adenocarcinoma. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma, squamous cell lung carcinoma, or large cell lung carcinoma. In some embodiments, the non-small cell lung cancer is large cell lung carcinoma. In some embodiments, the lung cancer is small cell lung carcinoma. In some embodiments, the prostate cancer is adenocarcinoma or small cell carcinoma. In some embodiments, the ovarian cancer is ovarian epithelial carcinoma. In some embodiments, the cholangiocarcinoma is proximal cholangiocarcinoma or distal cholangiocarcinoma.

[0144] In some embodiments, the TAA is associated with any one of a hematological cancers selected from leukemia, myeloma, or lymphoma. In some embodiments, the TAA is associated with a leukemia selected from acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute promyelocytic leukemia (APL), mixed lineage leukemia (MLL), or myelodysplastic syndrome (MDS).

[0145] In some embodiments, the TAA is associated with a myeloma that is multiple myeloma. In some embodiments, the TAA is associated with a multiple myeloma selected from hyperdiploid (HMM) or non-hyperdiploid or hypodiploid subtypes of multiple myeloma. In some embodiments, the TAA is associated with a multiple myeloma selected from light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, or immunoglobulin E (IgE) myeloma.

[0146] In some embodiments, the TAA is associated with a lymphoma that is a Hodgkin's lymphoma or a non-Hodgkin's lymphoma. In some embodiments, the TAA is associated with a non-Hodgkin's lymphoma. In some embodiments, the TAA is associated with a non-Hodgkin's lymphoma selected from small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphoma, diffuse large cell lymphoma, follicular center cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, mantle cell lymphoma, or marginal zone B-cell lymphoma. In some embodiments, the TAA is associated with a lymphoma that is a Hodgkin's lymphoma. In some embodiments, the TAA is associated with a Hodgkin's lymphoma selected from nodular sclerosing classical Hodgkin's lymphoma, lymphocyte-rich classical Hodgkin's lymphoma, or lymphocyte-depleted classical Hodgkin's lymphoma.

[0147] In some embodiments, the TAA is an acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell, or FAB The cancer is associated with any one of the following: ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, hypercalcemia associated with paraneoplastic syndromes / malignancies, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis carcinoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, and hemangioma.

[0148] In some embodiments, the extracellular domain is an antigen that is associated with kallikrein 4, papillomavirus binding factor (PBF), preferentially expressed melanoma antigen (PRAME), Wilms' tumor-I (WTI), hydroxysteroid dehydrogenase-like I (HSDLI), mesothelin, cancer testis antigen (NY-ES0-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2 / neuroreceptor tyrosine kinase (Her2 / Neu), cancer-associated epithelial cell adhesion molecule (EpCAM), ovarian cancer, and and uterine cancer antigen (CAI25), folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), LI cell adhesion molecule (LICAM), mannan-MUC-1, human endogenous retrovirus K (HERV-K-MEL), Kitakyushu lung cancer antigen-I (KK-LC-1), human cancer / testis antigen (KM-HN-1), cancer testis antigen (LAGE-I), melanoma antigen-Al (MAGE-Al), sperm surface zona pellucida-binding protein (Spl 7), synovial sarcoma, X-breakpoint 4 (SSX-4), transient axonal glycoprotein-1 (TAG-I), transient axonal glycoprotein-2 (TAG-2), enabled homolog (ENAH), mammoglobin A, NY-BR-I, breast cancer antigen, (BAGE-1), B melanoma antigen, melanoma antigen-Al (MAGE-Al), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X-breakpoint 2 (SSX-2), taxol resistance associated gene-3 (TRAG-3), avian myelocytomatosis viral oncogene (c-myc), cyclin B1, mucin I (MUC I), p62, survivin, lymphocyte common antigen (CD45), Dickkopf WNT signaling pathway inhibitor I (DKKI), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxylesterase, α-fetoprotein, macrophage colony-stimulating factor (M-CSF), prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), cytochrome C oxidase assembly factor I homolog (COA-1), 0-linked β-N-acetylglucosamine transferase (OGT), amplified osteosarcoma 9, endoplasmic reticulum lectin (OS-9), transforming growth factor β receptor 2 (TGF-βRII), murine leukemia glycoprotein 70 (gp70),Calcitonin-related polypeptide alpha (CALCA), programmed cell death 1 ligand 1 (CD274), mouse double minute 2 homolog (mdm-2), alpha-actinin-4, elongation factor 2, malic enzyme 1 (MEI), nuclear transcription factor Y subunit C (NFYC), G antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), cancer-testis antigen XAGE-1b, six-transmembrane prostate epithelial antigen 1 (STEAP1), PAP, prostate-specific antigen (PSA), fibroblast growth factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTC 1), B-Raf proto-oncogene (B-RAF), serine / threonine kinase, beta-catenin, cell division cycle 27 homolog (Cdc27), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 12 (CDK12), cyclin-dependent kinase inhibitor 2A (CDKN2A), casein kinase 1 alpha 1 (CSNKlAl), fibronectin 1 (FNl), GruwihAnest specific 7 (GAS7), glycoprotein nonmetastatic melanoma protein B (GPNMB), HAUS augmin-like complex subunit 3 (HAUS3), LDLR-fucosyltransferase, melanoma antigen recognized by T cells 2 (MART2), myostatin (MSTN), melanoma-associated antigen (mutated) 1 (MUM-1-2-3), poly(A) polymerase gamma (neo- PAP), myosin type I, protein phosphatase 1 regulatory subunit 3B (PPP1R3B), peroxiredoxin-5 (PRDX5), receptor tyrosine protein phosphatase kappa (PTPRK), transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPDl, triosephosphate isomerase, ocular albinism type 1 protein (OAl), member of the RAS oncogene family (RAB38), tyrosinase-related protein 1-2 (TRP-1-2), melanoma antigen Gp75 (gp75), tyrosinase, Melan-A (MART-1), glycoprotein 100 melanoma antigen (gplOO), N-acetylglucosaminyltransferase V gene (GnTVf),Lymphocyte antigen 6 complex locus K (LY6K), melanoma antigen-AlO (MAGE-AlO), melanoma antigen-Al2 (MAGE-Al2), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, taxol resistance associated protein 3 (TRAG-3), BDZ-binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), breakpoint cluster region Abelson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, elongation factor TuGTP-binding domain containing 2 (EFTUD2), ETS variant gene 6 / acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), Cyclin A1, fibronectin type III domain containing 3B (FDNC3B), promyelocytic leukemia / retinoic acid receptor alpha fusion protein (pml-RARα), melanoma antigen-Cl (MAGE-Cl), membrane protein alternative splicing isoform (D393-CD20), melanoma antigen-A4 (MAGE-A4), or melanoma antigen-A3 (MAGE-A3).

[0149] In some embodiments, the autoimmune condition or disorder is any one of type 1 diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), celiac disease, Sjogren's syndrome, polymyalgia rheumatica, ankylosing spondylitis, alopecia areata, vasculitis, and temporal arteritis. In some embodiments, the tumor associated antigen (TAA) associated with the autoimmune condition or disorder is derived from any one of carboxypeptidase H, chromogranin A, glutamic acid decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, α-enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, S100-β, citrullinated protein, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, and Sm antigen of the U-1 small ribonucleoprotein complex.

[0150] In some embodiments, the pathogen associated antigen is an antigen derived from a bacterial, fungal or parasitic protein or fragment thereof, hi some embodiments, the pathogen associated antigen is associated with HIV infection, human cytomegalovirus infection, hepatitis B infection, hepatitis C infection, Ebola virus infection, dengue fever, yellow fever, listeriosis, tuberculosis, cholera, malaria, leishmaniasis, or trypanosoma infection, or a combination thereof.

[0151] In some embodiments, the neurodegenerative disorder or condition is any one of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), spinocerebellar degeneration (SCA) or spinal muscular atrophy (SMA). In some embodiments, the antigen associated with the neurodegenerative disorder or condition is any one of amyloid beta (Ab), tau, alpha-synuclein (α-syn), mHTT or prion PrPsc, or a combination thereof.

[0152] In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 fM to 100 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 pM to 100 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 pM to 10 pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10 pM to 50 pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10 pM to 100 pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 100 pM to 500 pM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 500 pM to 1 nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 nM to 10 nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10 nM to 100 nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 10 nM to 100 nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 100 nM to 500 nM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 500 nM to 1 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 μM to 10 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 1 μM to 5 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 5 μM to 7.5 μM. In some embodiments, the extracellular domain binds to the target with a binding affinity of 7.5 μM to 10 μM.

[0153] In some embodiments, the extracellular domain comprises a signal peptide at the N-terminus. In some embodiments, the signal peptide may be derived from a surface expressed protein or a secreted protein. In some embodiments, the signal peptide may be derived from preprolactin, HIV pre-Env, HCV polyprotein, CB virus polyprotein, pestivirus polyprotein, precalreticulin, pre-VSV-G, HLA type I histocompatibility antigen, or PD-1 signal peptide (PD-1 SP), interleukin 12 (IL12), GM-CSF, or CD8 alpha chain (CD8a). In some embodiments, the signal peptide is PD-1 signal peptide (PD-1 SP). In some embodiments, the signal peptide is an HLA type I histocompatibility antigen or a portion thereof. In some embodiments, the extracellular domain is derived from PD1. In some embodiments, the extracellular domain comprises an amino acid sequence from positions 1-163 of an amino acid sequence according to any one of SEQ ID NOs: 19-21. In some embodiments, the extracellular domain comprises an amino acid sequence from positions 1-163 of an amino acid sequence according to SEQ ID NO: 19. In some embodiments, the extracellular domain comprises the amino acid sequence from positions 1 to 163 of the amino acid sequence according to SEQ ID NO: 20. In some embodiments, the extracellular domain comprises the amino acid sequence from positions 1 to 163 of the amino acid sequence according to SEQ ID NO: 21.

[0154] In some embodiments, the extracellular domain comprises an amino acid sequence according to any one of SEQ ID NOs: 22-23. In some embodiments, the extracellular domain comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the extracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 22. In some embodiments, the extracellular domain comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the extracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 23.

[0155] [Table 4] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to any one of SEQ ID NOs:24-44 and 130-132.

[0156] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:24.

[0157] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 25. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:25.

[0158] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:26.

[0159] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 27. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 27.

[0160] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 28. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:28.

[0161] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 29. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 29.

[0162] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 30. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:30.

[0163] In some embodiments, the extracellular domain of the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 31. In some embodiments, the extracellular domain of the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 31.

[0164] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:32.

[0165] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 33. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:33.

[0166] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 34. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:34.

[0167] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 35. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:35.

[0168] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 36. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:36.

[0169] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 37. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:37.

[0170] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 38. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:38.

[0171] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 39. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:39.

[0172] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 40. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 40.

[0173] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 41. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 41.

[0174] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 42. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 42.

[0175] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 43. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 43.

[0176] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 44. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 44.

[0177] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 130. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 130.

[0178] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 131. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 131.

[0179] In some embodiments, the RTCR disclosed herein comprises an amino acid sequence according to SEQ ID NO: 132. In some embodiments, the RTCR disclosed herein comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 132.

[0180] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 6] In some embodiments, the extracellular domain is derived from a CD19 binding protein. In some embodiments, the CD19 binding protein is a CD19-binding chimeric antigen receptor (CAR). In some embodiments, the extracellular domain comprises an amino acid sequence according to SEQ ID NO: 51. In some embodiments, the extracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NO: 51.

[0181] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to any one of SEQ ID NOs: 52-69. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 52. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 52.

[0182] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 53. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:53.

[0183] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 54. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:54.

[0184] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 55. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:55.

[0185] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 56. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:56.

[0186] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:57.

[0187] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 58. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:58.

[0188] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 59. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:59.

[0189] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 60. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:60.

[0190] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 61. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:61.

[0191] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to any one of SEQ ID NOs: 62. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:62.

[0192] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:63.

[0193] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 64. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:64.

[0194] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 65. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:65.

[0195] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 66. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:66.

[0196] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 67. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 67.

[0197] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 68. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO:68.

[0198] In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 69. In some embodiments, the CD19-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 69.

[0199] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] In some embodiments, the extracellular domain comprises a hinge region. In some embodiments, the hinge region is derived from CD8, PD-1, CD28, ICOS, or IgG. In some embodiments, the transmembrane domain of the RTCR disclosed herein is derived from CD8, PD1, CD28, ICOS, or IgG.

[0200] The present disclosure also provides a nucleic acid encoding a RTCR disclosed herein. In some embodiments, the nucleic acid encoding a RTCR disclosed herein is according to SEQ ID NOs: 75-86 and 92-110. In some embodiments, the nucleic acid disclosed herein comprises a nucleic acid sequence encoding a chimeric intracellular domain. In some embodiments, the RTCR disclosed herein is for expression in a T cell, wherein the T cell co-expresses at least one or a combination of endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L.

[0201] The present disclosure also provides a vector, comprising the nucleic acid disclosed herein.In some embodiments, the vector disclosed herein is any one of a viral vector, a plasmid, a cosmid, a yeast artificial chromosome, a bacterial artificial chromosome, or a transposon / transposase system.In some embodiments, the viral vector is an adenoviral vector or a lentiviral vector.In some embodiments, the expression vector is a lentiviral vector.

[0202] The present disclosure also provides a cell comprising a nucleic acid or vector disclosed herein. In some embodiments, the cell disclosed herein is an engineered T cell. In some embodiments, the engineered T cell is an allogeneic T cell. In some embodiments, the engineered T cell is an autologous T cell. In some embodiments, the engineered T cell is a naive T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (T SCM ), central memory T cells (T CM ) and regulatory T cells (T reg )

[0203] In some embodiments, the extracellular domain is a B-cell maturation Ag (BCMA) binding protein. In some embodiments, the BCMA binding protein is a BCMA-specific T-cell receptor (TCR). In some embodiments, the BCMA binding protein is a BCMA-specific chimeric antigen receptor (CAR). In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to any one of SEQ ID NOs: 137-146.

[0204] In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 137. In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 137.

[0205] In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 138. In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 138.

[0206] In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 139. In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 139.

[0207] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 140. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 140.

[0208] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 141. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 141.

[0209] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 142. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 142.

[0210] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 143. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 143.

[0211] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 144. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 144.

[0212] In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 145. In some embodiments the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 145.

[0213] In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to SEQ ID NO: 146. In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 146.

[0214] In some embodiments, the extracellular domain is a B-cell maturation Ag (BCMA) binding protein. In some embodiments, the BCMA binding protein is a BCMA-specific T-cell receptor (TCR). In some embodiments, the BCMA binding protein is a BCMA-specific chimeric antigen receptor (CAR). In some embodiments, the BCMA-binding chimeric antigen receptor comprises an amino acid sequence according to any one of SEQ ID NOs: 141, 142, 145, and 146.

[0215] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

Table 9-22

[0216] In some embodiments, the artificial antigen receptor binds to a TAA associated with a solid tumor or a hematological cancer. In some embodiments, the artificial antigen receptor binds to a TAA associated with acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell, or FAB. The compound binds to a TAA associated with a cancer selected from any one of ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, hypercalcemia associated with paraneoplastic syndrome / malignancy, solid tumor, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis carcinoma, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, and hemangioma.

[0217] In some embodiments, the artificial antigen receptor is kallikrein 4, papillomavirus binding factor (PBF), preferentially expressed melanoma antigen (PRAME), Wilms' tumor-I (WTI), hydroxysteroid dehydrogenase-like I (HSDLI), mesothelin, cancer testis antigen (NY-ES0-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2 / neuroreceptor tyrosine kinase (Her2 / Neu), cancer-associated epithelial cell adhesion molecule (EpCAM), ovarian cancer, and and uterine cancer antigen (CAI25), folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), L1 cell adhesion molecule (LICAM), mannan-MUC-1, human endogenous retrovirus K (HERV-K-MEL), Kitakyushu lung cancer antigen-I (KK-LC-1), human cancer / testis antigen (KM-HN-1), cancer testis antigen (LAGE-I), melanoma antigen-Al (MAGE-Al), sperm surface zona pellucida-associated protein (Spl 7), synovial sarcoma, X-breakpoint 4 (SSX-4), transient axonal glycoprotein-1 (TAG-I), transient axonal glycoprotein-2 (TAG-2), enabled homolog (ENAH), mammoglobin A, NY-BR-I, breast cancer antigen, (BAGE-1), B melanoma antigen, melanoma antigen-Al (MAGE-Al), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X-breakpoint 2 (SSX-2), taxol resistance associated gene-3 (TRAG-3), avian myelocytomatosis viral oncogene (c-myc), cyclin B1, mucin I (MUC I), p62, survivin, lymphocyte common antigen (CD45), Dickkopf WNT signaling pathway inhibitor I (DKKI), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxylesterase, α-fetoprotein, macrophage colony-stimulating factor (M-CSF), prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), cytochrome C oxidase assembly factor I homolog (COA-1), 0-linked β-N-acetylglucosamine transferase (OGT), amplified osteosarcoma 9, endoplasmic reticulum lectin (OS-9), transforming growth factor β receptor 2 (TGF-βRII), murine leukemia glycoprotein 70 (gp70),Calcitonin-related polypeptide alpha (CALCA), programmed cell death 1 ligand 1 (CD274), mouse double minute 2 homolog (mdm-2), alpha-actinin-4, elongation factor 2, malic enzyme 1 (MEI), nuclear transcription factor Y subunit C (NFYC), G antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), cancer-testis antigen XAGE-1b, six-transmembrane prostate epithelial antigen 1 (STEAP1), PAP, prostate-specific antigen (PSA), fibroblast growth factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTC 1), B-Raf proto-oncogene (B-RAF), serine / threonine kinase, beta-catenin, cell division cycle 27 homolog (Cdc27), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 12 (CDK12), cyclin-dependent kinase inhibitor 2A (CDKN2A), casein kinase 1 alpha 1 (CSNKlAl), fibronectin 1 (FNl), GruwihAnest specific 7 (GAS7), glycoprotein nonmetastatic melanoma protein B (GPNMB), HAUS augmin-like complex subunit 3 (HAUS3), LDLR-fucosyltransferase, melanoma antigen recognized by T cells 2 (MART2), myostatin (MSTN), melanoma-associated antigen (mutated) 1 (MUM-1-2-3), poly(A) polymerase gamma (neo- PAP), myosin type I, protein phosphatase 1 regulatory subunit 3B (PPP1R3B), peroxiredoxin-5 (PRDX5), receptor tyrosine protein phosphatase kappa (PTPRK), transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPDl, triosephosphate isomerase, ocular albinism type 1 protein (OAl), member of the RAS oncogene family (RAB38), tyrosinase-related protein 1-2 (TRP-1-2), melanoma antigen Gp75 (gp75), tyrosinase, Melan-A (MART-1), glycoprotein 100 melanoma antigen (gplOO), N-acetylglucosaminyltransferase V gene (GnTVf),Lymphocyte antigen 6 complex locus K (LY6K), melanoma antigen-AlO (MAGE-AlO), melanoma antigen-Al2 (MAGE-Al2), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, taxol resistance associated protein 3 (TRAG-3), BDZ-binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), breakpoint cluster region Abelson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, elongation factor TuGTP-binding domain containing 2 (EFTUD2), ETS variant gene 6 / acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), Cyclin A1, fibronectin type III domain containing 3B (FDNC3B), promyelocytic leukemia / retinoic acid receptor alpha fusion protein (pml-RARα), melanoma antigen-Cl (MAGE-Cl), membrane protein alternative splicing isoform (D393-CD20), melanoma antigen-A4 (MAGE-A4), and melanoma antigen-A3 (MAGE-A3).

[0218] In some embodiments, the artificial antigen receptor binds to an antigen associated with an autoimmune condition or disorder selected from any one of type 1 diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or multiple sclerosis (MS). In some embodiments, the artificial antigen receptor binds to an antigen associated with an autoimmune condition or disorder selected from any one of carboxypeptidase H, chromogranin A, glutamic acid decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), proinsulin, α-enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, S100-β, citrullinated protein, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histone and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, and Sm antigen of the U-1 small ribonucleoprotein complex.

[0219] In some embodiments, the artificial antigen receptor binds to a pathogen-associated antigen derived from a bacterial, fungal, or parasitic protein or fragment thereof, hi some embodiments, the artificial antibody receptor binds to an antigen associated with HIV infection, human cytomegalovirus infection, hepatitis B infection, hepatitis C infection, Ebola virus infection, dengue fever, yellow fever, listeriosis, tuberculosis, cholera, malaria, leishmaniasis, or trypanosoma infection, or a combination thereof.

[0220] In some embodiments, the artificial antigen receptor binds to an antigen associated with a neurodegenerative disorder or condition selected from Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), spinocerebellar degeneration (SCA), or spinal muscular atrophy (SMA). In some embodiments, the antigen associated with a neurodegenerative disorder or condition is any one of amyloid beta (Aβ), tau, alpha-synuclein (α-syn), mHTT, or prion PrPSc, or a combination thereof.

[0221] In some embodiments, the therapeutic polypeptide is a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, an immunogenic polypeptide, or a cell surface protein that binds to a target on the surface of another cell, hi some embodiments, the immune cell modulating protein is a cytokine, a chemokine, a transcription factor, a protein kinase, a protease, a component of a cell signaling pathway, or an adaptor protein.

[0222] In some embodiments, the cells disclosed herein express a RTCR disclosed herein. In some embodiments, the cells disclosed herein stably or transiently express a RTCR disclosed herein. In some embodiments, the cells disclosed herein stably express a RTCR disclosed herein. In some embodiments, the cells disclosed herein transiently express a RTCR disclosed herein.

[0223] In some embodiments, the cells disclosed herein co-express at least one or a combination of endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L.

[0224] The present disclosure also provides a modified T lymphocyte (T cell), comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), the modification reducing or eliminating the level of expression or activity of the TCR; and (b) a recombinant T cell costimulatory receptor (RTCR) as disclosed herein. In some embodiments, the modification of the endogenous sequence encoding the T cell receptor (TCR) is performed using a nucleic acid modification system. In some embodiments, the nucleic acid modification system is one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease. In some embodiments, the modification of the endogenous sequence encoding the T cell receptor (TCR) is performed by non-homologous end joining repair. In some embodiments, the non-homologous end joining repair is generated by a zinc finger nuclease and introduced into the cell by physical means, a viral vector, or a non-viral vector. In some embodiments, the non-homologous end joining repair is generated by TALE nucleases and introduced into cells by physical means, viral vectors, or non-viral vectors. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of the TCR alpha chain. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of the TCR beta chain. In some embodiments, modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of both the alpha chain and the beta chain TCR alpha chain.

[0225] In some embodiments, the engineered T cells disclosed herein co-express at least one or a combination of endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L.

[0226] In some embodiments, the methods disclosed herein further comprise modifying an endogenous sequence encoding a component of the major histocompatibility complex (MHC) class I (MHC-I), where the modification reduces or eliminates the level of expression or activity of MHC-I. In some embodiments, the modification reduces or eliminates the expression or activity of β2-macroglobulin.

[0227] The present disclosure also provides a composition comprising the RTCR disclosed herein. The present disclosure also provides a composition comprising a nucleic acid encoding the RTCR disclosed herein. The present disclosure also provides a composition comprising a vector comprising a nucleic acid disclosed herein. The present disclosure also provides a composition comprising a cell disclosed herein. The present disclosure also provides a composition comprising a modified T cell disclosed herein.

[0228] The disclosure also provides a composition comprising a cell population, the population comprising a plurality of cells comprising a nucleic acid encoding an RTCR as disclosed herein or a vector comprising a nucleic acid encoding an RTCR as disclosed herein.The disclosure also provides a composition comprising a cell population, the population comprising a plurality of modified T cells as disclosed herein.

[0229] The present disclosure also provides a method of producing a plurality of modified T cells, the method comprising: a) providing a plurality of primary T cells as disclosed herein; b) providing a composition comprising an RTCR as disclosed herein, a nucleic acid encoding an RTCR as disclosed herein, or a vector comprising a nucleic acid encoding an RTCR as disclosed herein; and c) introducing the composition of (b) into the plurality of primary T cells of (a) to produce a plurality of modified T cells under conditions in which the RTCR is stably expressed in the plurality of modified T cells. In some embodiments, the method of producing a plurality of modified T cells as disclosed herein further comprises modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the endogenous TCR. In some embodiments, the method of producing a plurality of modified T cells as disclosed herein further comprises modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the endogenous TCR.

[0230] In some embodiments, the modification of the endogenous sequence encoding the T cell receptor (TCR) is performed using a nucleic acid modification system. In some embodiments, the modification of the endogenous sequence to reduce or eliminate the level of expression or activity is performed using a nucleic acid modification system. In some embodiments, the nucleic acid modification system is one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease. In some embodiments, the modification of the endogenous sequence is performed by non-homologous end joining repair. In some embodiments, the non-homologous end joining repair is generated by a zinc finger nuclease and introduced into the cell by physical means, a viral vector, or a non-viral vector. In some embodiments, the non-homologous end joining repair is generated by a TALE nuclease and introduced into the cell by physical means, a viral vector, or a non-viral vector. In some embodiments, the modification of the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression level of the alpha chain of the TCR. In some embodiments, modifying the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression levels of the beta chain of the TCR, in some embodiments, modifying the endogenous sequence encoding the T cell receptor (TCR) reduces or eliminates the expression levels of both the alpha chain and the beta chain TCR alpha chain.

[0231] In some embodiments, modifying an endogenous sequence reduces or eliminates the level of expression or activity of Major Histocompatibility Complex (MHC) Class I (MHC-I), where modifying the endogenous sequence reduces or eliminates the level of expression or activity of MHC-I. In some embodiments, modifying the endogenous sequence reduces or eliminates the expression or activity of β2-macroglobulin.

[0232] In some embodiments, the method of producing a plurality of modified T cells disclosed herein further comprises d) maintaining or expanding the plurality of modified T cells in a suitable cell culture medium; and e) either i) cryopreserving the plurality of modified T cells in a suitable cell freezing medium, or ii) preparing the plurality of modified T cells for administration to a subject suffering from a disease or disorder.

[0233] Cells or modified T cells of the present disclosure, and compositions comprising a plurality of modified T cells produced by the methods of the present disclosure, intended for administration to a subject may need to meet one or more "release criteria" that indicate that the composition is safe and effective for formulation as a pharmaceutical product and / or administration to a subject. Release criteria may include a requirement that a composition of the present disclosure (e.g., cells or modified T cells of the present disclosure) contain a certain percentage of cells or modified T cells that express the RTCR of the present disclosure on their cell surface. The expansion process should be continued until a certain criterion is met (e.g., achieving a certain total number of cells or modified T cells of the present disclosure, or a certain percentage of the total number of cells or modified T cells that express the RTCR of the present disclosure).

[0234] Certain criteria indicate the point at which the expansion process should end. For example, cells should be formulated, reactivated, or cryopreserved when they reach a cell size of 300 fL (otherwise, cells that reach a size above this threshold may begin to die). Cryopreservation immediately after a cell population reaches an average cell size of less than 300 fL may result in better cell recovery upon thawing and culture, since the cells have not yet reached full quiescence prior to cryopreservation (full quiescent size is approximately 180 fL). Prior to expansion, T cells of the present disclosure may have a cell size of approximately 180 fL, but after 3 days of expansion may more than four times their cell size to approximately 900 fL. Over the next 6-12 days, the population of T cells slowly decreases in cell size until they are fully quiescent at 180 fL.

[0235] The process for preparing a cell population for formulation may include, but is not limited to, enriching the cells of the cell population, washing the cells, and / or further selecting the cells via drug resistance or magnetic bead sorting for specific surface-expressed markers. The process for preparing a cell population for formulation may further include sorting steps to ensure safety and purity of the final product. For example, if patient-derived tumor cells are used to stimulate the modified T cells of the present disclosure or have been modified to stimulate the modified T cells of the present disclosure being prepared for formulation, it is important that the patient-derived tumor cells are not included in the final product.

[0236] In some embodiments, the cells disclosed herein or engineered T cells disclosed herein express an RTCR comprising a mutant CD137 or mutant CD134 intracellular signaling domain disclosed herein on the cell surface at a level that is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or 20-fold higher compared to the expression level of a costimulatory molecule comprising wild-type CD137 or wild-type CD134 intracellular domain, respectively.

[0237] Pharmaceutical Composition or Formulation In some embodiments, the compositions disclosed herein and the populations of engineered T cells produced using the methods disclosed herein are in the form of a pharmaceutical formulation (or composition). In some embodiments, the pharmaceutical formulations disclosed herein include a pharma- ceutically acceptable carrier. The pharmaceutical formulations of the present disclosure can be dispensed into bags for injection, cryopreservation, and / or storage.

[0238] The pharmaceutical formulations of the present disclosure can be cryopreserved using standard protocols and, optionally, injectable cryopreservation media. For example, DMSO-free cryopreservation agents (e.g., CryoSOfree™ DMSO-free cryopreservation media) can be used to reduce the toxicity associated with freezing. The cryopreserved pharmaceutical formulations of the present disclosure can be stored for injection into patients at a later date. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and therefore the pharmaceutical formulations can be cryopreserved but packaged in pre-portioned "doses" that can be separated for thawing of individual doses.

[0239] The pharmaceutical formulations of the present disclosure may be stored at room temperature. Effective treatment may require multiple administrations of the pharmaceutical formulations of the present disclosure, and therefore the pharmaceutical formulations may be packaged in pre-divided "doses" that may be stored together but separated for administration of individual doses.

[0240] The pharmaceutical formulations of the present disclosure may be stored for subsequent regrowth and / or selection for the production of additional doses to the same patient, for example in the case of allogeneic therapy that may require future administration following remission and recurrence of a condition.

[0241] As mentioned above, the present disclosure provides stable formulations, preferably comprising saline or phosphate buffer with selected salts, as well as preservative-containing preservative solutions and formulations, and versatile preservative formulations suitable for pharmaceutical or veterinary use, comprising at least one modified cell in a pharma- ceutically acceptable formulation. The preservative formulations contain at least one known preservative, or at least one of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, polymers, or mixtures thereof, in an aqueous diluent. Any suitable concentration or mixture can be used, for example, about 0.0015%, or any range, value, or fraction therein, as known in the art. Non-limiting examples include no preservatives, about 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., Examples of such compounds include 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkyl paraben (for example, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0242] As described above, the present disclosure provides an article of manufacture comprising packaging material and at least one vial containing a solution of at least one modified cell, optionally in an aqueous diluent, comprising a predetermined buffer and / or preservative of the at least one modified cell, the packaging material including a label indicating that such solution can be maintained for a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or more.

[0243] The products of the present disclosure are useful for administration over a period ranging from immediate to 24 hours or more. Thus, the presently claimed products provide significant benefits to patients. The formulations of the present disclosure can be safely stored at temperatures between about 2° C. and about 40° C. and can retain the biological activity of the protein for extended periods of time, thus allowing for packaging labels indicating that the solution can be stored and / or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more.

[0244] The products of the present disclosure include packaging materials that provide the conditions under which the product may be used, as well as information required by regulatory authorities.

[0245] The present disclosure also provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective number of cells comprising a nucleic acid encoding an RTCR disclosed herein or a vector comprising a nucleic acid encoding an RTCR disclosed herein, a therapeutically effective number of any one of the modified T cells disclosed herein, a therapeutically effective amount of any one of the compositions disclosed herein, or a therapeutically effective number of modified T cells produced by the methods disclosed herein.

[0246] In some embodiments, the subject is a mammal. In some embodiments, the mammal is any one of a human, a primate, a rodent, a dog, a cat, an ungulate, a horse, and a pig. In some embodiments, the mammal is a human. In some embodiments, the disease or disorder is any one of a cancer, an autoimmune disorder, an infectious disease, an inflammatory disease or condition, a renal disease or disorder, a pulmonary disease or disorder, a liver disease or disorder, a cardiovascular disease or disorder, a neurodegenerative disorder or condition, or a metabolic disorder or condition. In some embodiments, the cancer is a solid tumor or a blood cancer. In some embodiments, the infectious disease is caused by a bacteria, a virus, a fungus, a protozoan, or a parasite. In some embodiments, the neurodegenerative disorder or condition is any one of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and disorders associated with PD, a prion disease, a motor neuron disease (MND), Huntington's disease (HD), spinocerebellar degeneration (SCA), or spinal muscular atrophy (SMA).

[0247] The present disclosure provides chimeric costimulatory intracellular proteins (CIPs) comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least the second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

[0248] The present disclosure also provides a chimeric costimulatory intracellular protein (CIP) comprising a first and at least a second signaling domain, wherein the first and at least a second signaling domain are not identical, and wherein the at least the second signaling domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

[0249] In some embodiments of the CIPs disclosed herein, the mutant intracellular signaling domain of the TNFR family protein is any one of a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain. In some embodiments, the CIP further comprises a transmembrane domain. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain is a truncated CD137 intracellular domain. In some embodiments of the CIPs disclosed herein, the truncated CD137 intracellular domain comprises an amino acid sequence according to amino acid position 13 to amino acid position 42 of the CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the truncated CD137 intracellular domain comprises a deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the N-terminus of the CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the truncated CD137 intracellular domain comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the CD137 intracellular domain of the disclosure comprises an amino acid sequence according to SEQ ID NO:1.

[0250] In some embodiments of the CIPs disclosed herein, the truncated CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO:3.

[0251] In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises a deletion of one, two, three, or four lysine residues from amino acid position 1 to amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises one or more lysine mutations from amino acid position 1 to amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises one or more lysine mutations at amino acid positions selected from amino acid positions 1, 5, 6, and 12 at the N-terminus of a CD137 intracellular domain of the disclosure.

[0252] In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations from amino acid position 1 to amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5, and 6 at the N-terminus of a CD137 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD137 intracellular domain further comprises a lysine mutation at amino acid position 12 at the N-terminus of a CD137 intracellular domain of the disclosure.

[0253] In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain is a truncated CD134 intracellular domain. In some embodiments of the CIPs disclosed herein, the truncated CD134 intracellular domain comprises an amino acid sequence from amino acid position 15 to amino acid position 37 of the CD134 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the truncated CD134 intracellular domain comprises a deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the truncated CD134 intracellular domain comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain of the disclosure.

[0254] In some embodiments of the CIPs disclosed herein, the truncated CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO:6.

[0255] In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain comprises a deletion of lysine residues from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain comprises a lysine mutation at amino acid position 12 at the N-terminus of the CD134 intracellular domain of the disclosure.

[0256] In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 at the N-terminus of a CD134 intracellular domain of the disclosure. In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutations from amino acid position 1 to amino acid position 14 at the N-terminus of a CD134 intracellular domain. In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, and 5 at the N-terminus of a CD134 intracellular domain. In some embodiments of the CIPs disclosed herein, the mutant CD134 intracellular domain further comprises a lysine mutation at amino acid position 12 at the N-terminus of a CD134 intracellular domain. In some embodiments, the CIPs disclosed herein comprise a first signaling domain derived from a protein of the CD28 family, hi some embodiments, the CIPs disclosed herein comprise a first signaling domain derived from any one of CD28, CD28H, ICOS, or a combination thereof.

[0257] In some embodiments, the CIP disclosed herein comprises a first signaling domain derived from ICOS. In some embodiments, the first signaling domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO:9.

[0258] In some embodiments, the CIPs disclosed herein comprise a first signaling domain comprising a portion of the CD28 intracellular domain combined with an ICOS domain according to SEQ ID NO:9. In some embodiments of the CIPs disclosed herein, the first signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs:12 or 109. In some embodiments, the CIPs disclosed herein comprise a first signaling domain derived from CD28. In some embodiments of the CIPs disclosed herein, the first signaling domain derived from CD28 comprises an amino acid sequence according to SEQ ID NO:10. In some embodiments of the CIPs disclosed herein, the first signaling domain derived from CD28 comprises an amino acid sequence according to any one of SEQ ID NOs:121-122. In some embodiments, the CIPs comprise an amino acid sequence according to any one of SEQ ID NOs:14-17.

[0259] In some embodiments, the CIPs disclosed herein further comprise a third signaling domain. In some embodiments, the CIPs disclosed herein further comprise a third signaling domain derived from any one of a CD3 signaling domain, a CD2 signaling domain, or an interleukin 2 receptor binding (IL-2RB) protein signaling domain, or a combination thereof. In some embodiments, the CD3 signaling domain of the CIPs disclosed herein is derived from a CD3ζ or CD3ε domain, or a combination thereof. In some embodiments, the CD3 signaling domain of the CIPs disclosed herein is a CD3 domain comprising an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47, and 48.

[0260] In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence according to SEQ ID NO: 18. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity according to SEQ ID NO: 18. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity according to SEQ ID NO: 45. In some embodiments, the third signaling domain of a CIP disclosed herein is a truncated CD3 ζ domain comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the third signaling domain of a CIP disclosed herein is a truncated CD3 ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity according to SEQ ID NO: 46. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 epsilon domain comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 epsilon domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the third signaling domain of a CIP disclosed herein is a combination of CD3ε and a truncated CD3ζ domain (CD3ζε domain).In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 ζε domain comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD3 ζε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

[0261] In some embodiments, the third signaling domain of a CIP disclosed herein is a CD2 signaling domain. In some embodiments, the third signaling domain of a CIP disclosed herein is a mutant CD2 signaling domain. In some embodiments, the mutant CD2 signaling domain is a truncated CD2 signaling domain. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the third signaling domain of a CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NO: 49.

[0262] In some embodiments, the third signaling domain of a CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the third signaling domain of a CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NO: 50.

[0263] In some embodiments, the CIPs disclosed herein further comprise a fourth signaling domain. In some embodiments, the CIPs disclosed herein further comprise a fourth signaling domain derived from any one of a CD3 signaling domain, a CD2 signaling domain, or an interleukin 2 receptor binding (IL-2RB) protein signaling domain, or a combination thereof, wherein the third and fourth signaling domains are not identical. In some embodiments, the fourth signaling domain of the CIPs disclosed herein is derived from a CD3ζ or CD3ε domain, or a combination thereof. In some embodiments, the fourth signaling domain of the CIPs disclosed herein is a CD3 domain comprising an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47, and 48.

[0264] In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 18. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 zeta domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 45. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a truncated CD3 ζ domain comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the third signaling domain of a CIP disclosed herein, the fourth signaling domain of a CIP disclosed herein is a truncated CD3 ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity according to SEQ ID NO: 46. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 epsilon domain comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 epsilon domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a combination of CD3ε and a truncated CD3ζ domain (CD3ζε domain).In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 ζε domain comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD3 ζε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

[0265] In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD2 signaling domain. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a mutant CD2 signaling domain. In some embodiments, the mutant CD2 signaling domain is a truncated CD2 signaling domain. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the fourth signaling domain of a CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NO: 49.

[0266] In some embodiments, the fourth signaling domain of a CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the fourth signaling domain of a CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NO: 50.

[0267] In some embodiments, the CIPs disclosed herein are for expression in T cells, wherein the T cells co-express at least one or a combination of endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L.

[0268] In some embodiments, the CIPs disclosed herein are co-expressed with a T cell receptor (TCR) in a T cell. In some embodiments, the TCR is an endogenous TCR. In some embodiments, the TCR is an artificial TCR. In some embodiments, the artificial TCR is a high affinity TCR. In some embodiments, the CIPs, when co-expressed with a TCR in a T cell, provide a second activation signal to induce T cell activation and proliferation, and the first activation signal is provided by antigen binding by the TCR.

[0269] In some embodiments, a CIP disclosed herein is expressed in a T cell as a component of an artificial receptor for a target. In some embodiments, the artificial receptor is a chimeric antigen receptor (CAR), a receptor for a ligand or a component thereof, an antibody or a fragment thereof. In some embodiments, a CIP disclosed herein is expressed as a component of a CAR. In some embodiments, a CIP disclosed herein is expressed as a component of an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, an sdAb, an Fv, a VHH fragment, or a single chain Fv fragment. In some embodiments, a CIP expressed in a T cell as a component of an artificial receptor as disclosed herein induces T cell activation and proliferation upon target binding by the artificial receptor.

[0270] The term "about" or "approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of a stated value. In some embodiments, "about" or "approximately" can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of a stated value. In some embodiments, "about" or "approximately" can be understood as within 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of a stated value. In some embodiments, "about" or "approximately" can be understood as within 1%, 0.5%, 0.1%, 0.05%, or 0.01% of a stated value.

[0271] The following examples are provided to better illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. To the extent that specific materials are mentioned, they are merely for illustrative purposes and are not intended to limit the present disclosure. Those skilled in the art may develop equivalent means or reactants without exercising inventive capacity and without departing from the scope of the present disclosure. EXAMPLES

[0272] Materials and Methods Media and cell lines Complete DMEM was made by supplementing DMEM with penicillin / streptomycin / glutamine, 20 mM HEPES, 10 μg / mL gentamicin, and 10% FBS. Complete RPMI was made by supplementing RPMI with penicillin / streptomycin / glutamine, 20 mM HEPES, 10 μg / mL gentamicin, 10% FBS, and 50 uM 2-ME. T cell growth medium was made by supplementing complete RPMI with 50 ng / ml IL2, 10 ng / mL IL7, and 10 ng / mL IL15 (Peprotech). Cytokine medium was made by supplementing X-Vivo15 with 1% human serum, 20 mM HEPES, penicillin / streptomycin / glutamine, and 10 μg / mL gentamicin. Human PBMCs were purchased from iSpecimen and cultured in complete RPMI. 293FT was purchased from Invitrogen. K562 and A375 cells were purchased from ATCC and cultured in complete DMEM.

[0273] Plasmids and cloning A lentiviral plasmid containing the PGK promoter driving a truncated human EGFR receptor (huEGFRt), followed by the MSCV promoter driving GFP, followed by the WPRE sequence, was ordered from Vector Builder. The costimulatory molecule, followed by the P2A sequence, was ordered as a single gene block (Invitrogen) and placed in frame with the huEGFRt sequence using NEB Builder homology-based recombination. The CAR and TCR sequences were constructed from three gene block fragments (Invitrogen) and cloned using NEB Builder downstream of the MSCV promoter after GFP excision. PD-L1_P2A and HLA-A2 were cloned in frame with huEGFRt, respectively, in place of GFP.

[0274] P2A amino acid sequence (SEQ ID NO:111) GSGATNFSLLKQAGDVEENPGP Human EGFRt amino acid sequence (alias: huEGFRt (AA112)) (SEQ ID NO: 112) MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTK IISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM HLA-A2 signal peptide (SEQ ID NO: 113) MAVMAPRTLVLLLSGALALTQTWA huGMCSF signal peptide, amino acid sequence (SEQ ID NO:119) MLLLVTSLLLCELPHPAFLLIP P2A nucleic acid sequence (SEQ ID NO:114) GGATCCGGCGCCACCAATTTCAGCCTGCTGAAACAGGCTGGCGACGTGGAAGAGAACCCTGGACCT Human EGFRt nucleic acid sequence (SEQ ID NO: 115) HLA-A2 signal peptide nucleic acid sequence (SEQ ID NO:116) ATGGCTGTGATGGCCCCTAGAACACTGGTGCTGCTGCTGTCTGGTGCCCTGGCTCTGACTCAGACATGGGCC CD8a signal peptide nucleic acid sequence (SEQ ID NO: 148) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCTAGACCT CD8a hinge (SEQ ID NO: 149) ACCACCACCCCGCCCCAGACCCCCCACCCCGCCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCCGCCGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGC huGMCSF signal peptide, nucleic acid sequence (SEQ ID NO: 150) ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGCTGATCCCC Lentivirus production and preparation of RetroNectin plates VSV pseudotyped lentivirus was produced in 6-well plates. Briefly, 293FT was added to 0.9 × 10 6 or 1.4 x 10 6Cells were seeded at 1000 x 1000 cells / well. Once cells had attached and reached at least 80% confluency, a mixture of lentiviral plasmid, packaging vector (psPAX2) and VSV-G envelope expression plasmid (PMD2.G) was transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. After 18 hours, the medium was replaced with 3 mL of fresh DMEM. 48 hours after medium replacement, viral supernatant was collected and spun down at 1500 RPM to remove 293FT cells / debris. Retronectin was coated on 24-well non-tissue culture treated plates at 20 μg / well in PBS- / - for 2 hours at 37°C or overnight at 4°C. Retronectin was removed and washed once with PBS before adding lentiviral SN (2 mL). Plates were then spun at 1500G at 32°C for 90 minutes to concentrate the viral particles on Retronectin. Lentiviral SN was removed immediately prior to transduction of primary T cells or tumor cells. Alternatively, T cells were transduced with 8ug / mL polybrene by spinfection at 800G for 2 hours at 32°C.

[0275] T cell culture, transduction, and isolation Human PBMCs were activated in T cell expansion medium containing CD3 / 28 microbeads (Invitrogen) in complete RPMI (100 ul beads / 50x10 6PBMCs). After 48 hours of activation, activated PBMCs were transferred to lentivirus-coated retronectin plates for 48 hours and then transferred to 6-well plates containing fresh T cell growth medium. After an additional 24 hours in culture, cell transduction was determined by flow cytometry and transduced cells were enriched based on huEGFRt expression. To isolate cells based on EGFR expression, T cell cultures were harvested and activation beads were removed. Cells were then stained in 1:100 anti-EGFR-APC antibody in MACS buffer for 30 minutes at 4°C. Cells were then washed and incubated with anti-APC microbeads (Miltenyi) for 15–30 minutes at 4°C. Unbound microbeads were then removed by centrifugation and huEGFRt cells were isolated by positive selection on a mini-macs column. Cells were eluted from the mini-MACS column, returned to culture in T cell growth medium and used for experiments within 2 weeks. To generate stable cell lines, cells were harvested and transduced similarly to primary T cells. EGFR selection was performed twice at 2-week intervals.

[0276] T cell stimulation When T cells were stimulated with plate-bound antibodies, maxisorp flat-bottom plates (Invitrogen) were coated with the indicated amount of anti-human CD3 antibody (HIT3a-Biolegend) in PBS- / - for 2 h at 37 °C. Plates were washed twice with basal RPMI before use. For K562 stimulation, K562 cells were harvested, resuspended in cytokine medium, and aliquoted into U-bottom plates. Similarly, A375 cells were plated 1 day before adding T cells in DMEM in 96-well flat-bottom plates. Medium was changed before addition of cognate T cells. After EGFR+ selection, T cells were harvested, counted, resuspended at the appropriate concentration in cytokine medium, and distributed into wells with antibody or APC. For K562 experiments, anti-CD3 (HIT3a / Biolegend) was added at the indicated doses 1-2 h after K562 / T cell interaction at 37 °C. For tracking T cell proliferation, T cells were labeled with Violet Tracking Dye (CTV) according to Biolegend's protocol prior to addition to stimulation plates. Supernatants were collected 18-36 hours after stimulation to assess cytokine secretion and proliferation / T cell death after 96 hours of stimulation.

[0277] Cytokine multiplex assay Following collection of T cell supernatants, cytokines were measured using the Legendplex Multi-Analyte Flow Assay Kit for Human Th or Th1 Cytokines (Biolegend). The manufacturer's protocol was followed with the following exceptions: 75 μL of T cell SN was used to measure cytokines, and 2 μL of each reagent / well was used. Secreted cytokines were measured by flow cytometry, and multiple experiments were combined and analyzed, with values ​​normalized to the maximum response of the control group to normalize for variability between experiments and donors.

[0278] Conjugation assay To assess conjugation of T cells to target cells, T cells were labeled with CFSE (Biolegend) and K562_HLA-A2 or K562_HLA-A2_PD-L1 cells were labeled with CTV according to the manufacturer's protocol. T cells and APCs were mixed at a 1:2 ratio and briefly centrifuged in a 1.5 mL Eppendorf tube to facilitate conjugation. The cell pellet was incubated at 37°C for 30 min, then the cell pellet was gently resuspended by repeated pipetting (20x) with a p200 and cut-off pipette tip and immediately assessed by flow cytometry.

[0279] Flow cytometry Cells were collected and washed in MACS buffer (PBS- / -, 1% FBS, 1 mM EDTA) and then stained in MACS buffer containing the relevant antibodies. Anti-EGFR-APC, anti-mouse TCRβ-FITC, anti-human PD1-PE, anti-CD3 APC-Cy7, and anti-CD8 PE-Cy7 were all purchased from biolegend. After addition of antibodies, cells were stained for 30–60 min at 4°C. For detection of CD-19 CAR expression, cells were incubated with CD-19Fc recombinant protein in MACS buffer at 1 μg / mL for 30 min at room temperature. Cells were then washed and incubated with anti-human FC antibody at a 1:100 dilution. Cells were then washed three times in MACS buffer and analyzed on an Acea NovoCyte flow cytometer. Cells were collected in a constant volume, allowing accurate cell counts to be obtained.

[0280] Example 1: Design of costimulatory molecules containing chimeric intracellular signaling domains The disclosure herein provides for the design of costimulatory molecules that contain intracellular signaling domains that include or are derived from CD137 / 4-1BB or CD134 / OX-40 receptors as shown in Figure 1. Examination of the CD137 family of sequences in the cytoplasmic tail (Figure 1) revealed a common membrane-proximal polybasic domain, as well as several lysine residues that may function as ubiquitination sites, and a TRAF-binding domain that serves to activate the NF-κB signaling pathway after receptor ligation. Without being bound by theory, it is believed that the conserved lysine residues may function as ubiquitination sites that can control the ubiquitination and degradation of CD134 / CD137, and that the disrupted location of the CD137 or CD134 cytoplasmic tail in potential CD28 / ICOS-CD134 CAR or CD28 / ICOS-CD137 CAR receptors, respectively, may affect the localization or half-life of the resulting molecules, either through the polybasic domain or the conserved lysine residues. New fusion domains between ICOS / CD28 intracellular domain and the cytoplasmic domain of CD137 or OX-40 lacking the polybasic sequence and conserved lysine residues, as well as their wild-type (WT) counterparts, were generated (Figures 2A and 2B). A portion of the cytoplasmic domain of CD28 involved in binding Lck and Vav3, which may enhance stimulation, was also included. The extracellular domain of PD-1 was used to create either a dominant negative (DN) version by omitting the intracellular tail, or an inhibitory switch receptor that changes the negative regulatory signal to a positive regulatory signal, thus providing cell-intrinsic PD-1 blockade. The cytoplasmic tails described herein, i.e., the intracellular costimulatory domains, can be expanded through the use of the cytoplasmic tails of other signaling proteins of interest to create new CAR receptors or different inhibitory switch receptors, or to express other immunoregulatory extracellular domains, as detailed in Figure 3.

[0281] Example 2: Generation and testing of in vitro functionality of checkpoint costimulatory molecules The disclosure herein provides the design of costimulatory molecules and validation of their effects on the function of high affinity TCR. The costimulatory molecules described herein were designed as shown in Figures 2A and 2B. In all recombinant receptors, the PD1 signal peptide (SP) was replaced with a signal peptide from HLA-A2, which increases the surface expression of the receptor. As controls, PD1-WT and a truncated PD-1 lacking the ITIM-containing intracellular tails (TLs) were included. Two second generation receptors containing the transmembrane and intracellular domains of CD28 or ICOS were included as second generation control receptors. In addition, 15 third generation receptors were generated that contain the transmembrane and intracellular domains derived from ICOS and / or CD28 linked to the intracellular signaling domain of a TNF receptor superfamily member. The first generation contained the wild-type CD137 / 4-1BB intracellular domain (HLA A2-SP_PD1_ICOS_BB: SEQ ID NO: 26), whereas the second and third generations contained either the CD137 / 4-1BB domain or the CD134 / OX-40 intracellular domain with key mutations engineered to increase surface expression (HLA A2-SP_PD1_ICOS_BBt: SEQ ID NO: 27 and HLA A2-SP_PD1_ICOS_OX40t: SEQ ID NO: 28, respectively). Additionally, the third generation receptor described herein contains a chimeric intracellular domain comprising a portion of a CD28 intracellular domain inserted into an ICOS intracellular domain further linked to either a mutated CD137 domain (ICOS4BBt) or a mutated CD134 / OX40 domain (ICOS-OX40t) (HLA A2-SP_PD1_ICOS(28)_BBt: SEQ ID NO: 29 and HLA A2-SP_PD1_ICOS(28)_OX40t: SEQ ID NO: 30). Two further third generation receptors were generated as described herein containing a CD28 intracellular domain linked to either a mutated CD137(28_BBt) or a mutated CD134 / OX40(28_OX40t) domain (HLA A2-SP_PD1_28_BBt: SEQ ID NO: 131 and HLA A2-SP_PD_28_OX40t: SEQ ID NO: 132) (Figure 2A).These vectors were cloned into lentiviral vectors and fused to truncated huEGFR receptors (huEGFRt) by a self-truncating peptide to track and magnetically select transduced cells. When these receptors were expressed by lentiviral transduction in primary T cells, each receptor expressed significantly above endogenous PD-1 levels (Figure 4A). Although second generation costimulatory molecules were expressed well, inclusion of the CD137 (4-1BB) intracellular domain considerably reduced surface expression of the recombinant receptors. The disclosure herein shows that inclusion of a mutant intracellular domain that maintains the TRAF binding domain rescues surface expression of these optimized third generation receptors. Surface expression of huEGFRt and costimulatory molecules demonstrates increased expression of the truncated CD137 (4-1BB) design (ICOS_BBt) compared to the non-truncated version (ICOS_BBwt). Similar mutations in the cytoplasmic domain of CD134 (OX-40) also resulted in high surface expression of costimulatory molecules (Figure 4B).

[0282] After transduction, T cells were isolated to >90% purity based on expression of huEGFRt and used for restimulation experiments. The results disclosed herein demonstrate that engagement of costimulatory molecules in vitro enhanced T cell cytokine production and proliferation, especially at low doses of anti-CD3 antibody (Figures 5A-5D). To generate a more physiological system, either HLA-A2 alone or HLA-A2 and PD-L1 were overexpressed on K562 cells and incubated with costimulatory receptor-transduced T cells and the indicated doses of anti-CD3. Incubation with K562:PD-L1 cells reduced the amount of secreted cytokines, especially in T cells overexpressing PD1_WT (Figure 6A). Expression of either PD1_TL or PD1_ICOS_BBwt had little effect on the secretion of IL-2, TNF, or IFNγ, whereas expression of PD1_28 or PD1_ICOS increased effector cytokine secretion 3-4 fold over GFP controls in the presence of PD-L1-expressing K562 cells. Notably, both PD1_ICOS_BBt and PD1_ICOS_OX40t costimulatory molecules further improved this effect, increasing effector cytokine secretion 2-3 fold over PD1_ICOS-expressing cells in the presence of PD-L1 (Figure 6C). Expression of PD1_28, PD1_28_BBt, and PD1_28_OX40t resulted in comparable effector cytokine secretion (Figure 6B). None of the constructs were constitutively active and had minimal effect on cytokine secretion in the absence of PD-L1 or anti-CD3, indicating that both PD-1 and antigen are required to initiate a T cell response. Fitting the cytokine data, T cells expressing a) PD1_28, PD1_28_BBt, and PD1_28_OX40t (Figure 7A, lower panel, and Figure 7C), and b) PD1_ICOS_BBt and PD1:ICOS_OX40t (Figure 7B, lower panel, and Figure 7C) proliferated best in response to and were best able to kill PD-L1-expressing K562 cells (Figures 7A-7B, upper panels).Applying the cytokine data, T cells expressing PD1_ICOS_BBt and PD1_ICOS_OX40t proliferated best in response to PD-L1 expressing K562 cells and were best able to kill PD-L1 expressing K562 cells (Figure 7C-7E). Again, this response required both anti-CD3 and PD-L1 expression. The costimulatory molecules demonstrated costimulatory capacity as their expression increased T cell proliferation when cells were stimulated on 96-well plates coated with anti-CD3 and anti-PD1 (Figure 8).

[0283] The effect of the receptor with the polybasic and lysine residue mutations was smaller than that of the PD1_ICOS_BBt costimulatory molecule on both the surface expression of costimulatory molecules (Figures 9A-9B), effector cytokine production in response to stimulation with anti-CD3 antibodies (Figure 9C), and T cell proliferation in response to stimulation with target cells expressing PD-L1 (Figure 9D). The PD1_ICOS_OX40t receptor (with a truncated OX40 intracellular domain) had an effect comparable to that of the wild-type PD1_ICOS_OX40wt receptor (containing the wild-type OX40 intracellular domain) on both the surface expression of costimulatory molecules (Figures 10A-10B), effector cytokine production in response to stimulation with anti-CD3 antibodies (Figure 10C), and T cell proliferation in response to stimulation with target cells expressing PD-L1 (Figure 10D).

[0284] Furthermore, ICOS-based costimulatory molecules promoted T cell:PD-L1-expressing (PD-L1+) target cell interactions in flow-based conjugation assays, suggesting that these receptors promote long-term T cell-APC interactions while scanning for cognate antigens, a useful property when scanning for low-abundance antigens in the TME (Figures 11A-11B).

[0285] The present disclosure shows that costimulatory molecules based on the modified third generation intracellular signaling domains disclosed herein are superior to the existing PD1_28 costimulatory molecule in enhancing T cell effector function in response to PD-L1+ target cells, including increased T cell proliferation, cytokine secretion, and target cell killing. The third generation intracellular signaling domains disclosed herein can be successfully combined with TCR-T therapy targeting TAAs.

[0286] Example 3: In vitro preclinical studies Described herein are T cells expressing a specific HLA-A2 / NY-ESO-specific TCR and a costimulatory molecule comprising ICOS and a mutant CD137 signaling domain that increase expression of the costimulatory molecule on the T cell surface (FIG. 12A), effector cytokine production (FIG. 12B), and killing of NY-ESO-expressing target cells (FIG. 12C) compared to T cells expressing the specific HLA-A2 / NY-ESO-specific TCR alone.

[0287] Described herein are CD-19 CAR constructs comprising modified third generation intracellular signaling domains as disclosed herein. CD-19 (FMC63scFV) CARs with the third generation intracellular signaling constructs described herein include constructs comprising intracellular chimeric domains: CD28-CD137-CD3ζ (28_ΒΒwt_z), CD28-CD137 mutant-CD3ζ (28_ΒΒt_z), CD28-CD134 mutant-CD3ζ (28_OX40t_Z), ICOS-CD137-CD3ζ (ICOS_ΒΒ_z), ICOS-CD137 mutant-CD3ζ (ICOS_ΒΒt_z), and ICOS-CD134 mutant-CD3ζ (ICOS_OX40t-z). Also provided are CD-19CARs with third generation intracellular signaling constructs with a portion of CD28 inserted within the ICOS domain: ICOS(28)-CD137-CD3ζ (ICOS(28)_ΒΒwt_z), ICOS(28)-CD137 mutant-CD3ζ (ICOS(28)_ΒΒt_z), and ICOS(28)-CD134 mutant-CD3ζ (ICOS(28)_OX40t-z). Second generation constructs including CD137-CD3ζ (ΒΒwt_z), CD28-CD3ζ(28_z), and ICOS-CD3ζ (ΙCOS_z) are used as controls. Similar to the studies described herein using PD-1 3 generation intracellular signaling constructs, CD19 CAR constructs with CD137 and CD134 mutant domains showed higher expression compared to the corresponding constructs with wild-type CD137 and CD134 domains (28_BBwt_z and ICOS_BBwt_z, respectively) (Figures 13A-13B). The in vitro studies described herein show increased killing of CD19 expressing cells (CD19+) (Figures 14A-14B and Figures 15C-15D), increased effector cytokine production (Figure 14C, right panel), and increased T cell proliferation and persistence (Figures 15A-15B and 15E) by primary T cells transduced with 3rd generation CD28- and ICOS-based CD19 CARs.

[0288] The present disclosure also shows that expression of a CD28-based receptor containing a mutated CD134 / CD137 signaling domain and an ICOS-based receptor containing a mutated CD134 / CD137 signaling domain increased binding of BCMA-specific T cells (BCMA CAR T cells) to a target antigen (BCMA-Fc) (Figures 16A-16B). The present disclosure also shows that expression of a CD28-based receptor containing a mutated CD134 / CD137 signaling domain and an ICOS-based receptor containing a mutated CD134 / CD137 signaling domain increased proliferation and effector cytokine production (Figures 16C and 16E) and target cell killing (Figure 16D) by BCMA-specific T cells in response to a myeloma cell line expressing BCMA.

Claims

1. A recombinant T cell costimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signaling domain, wherein said first and said at least a second signaling domain are not identical, and wherein said at least a second signaling domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

2. the mutant CD137 intracellular domain (1) A truncated CD137 intracellular domain comprising: 1a) the amino acid sequence from amino acid position 13 to amino acid position 42 of the CD137 intracellular domain; 1b) deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the N-terminus of the CD137 intracellular domain; 1c) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain; and / or 1d) the amino acid sequence according to SEQ ID NO:3; (2) deletion of one, two, three, or four lysine residues from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain; (3) one or more lysine mutations from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain, optionally one or more lysine mutations at amino acid positions selected from amino acid positions 1, 5, 6 and 12 at the N-terminus of the CD137 intracellular domain; (4) a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain; and / or (5) one or more proximal basic amino acid mutations at amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, optionally one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5, and 6 of the N-terminus of the CD137 intracellular domain, and optionally a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain; The RTCR of claim 1 .

3. the mutant CD134 intracellular domain is (1) A truncated CD134 intracellular domain comprising: 1a) the amino acid sequence from amino acid position 15 to amino acid position 37 of the CD134 intracellular domain; 1b) deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from the N-terminus of the CD134 intracellular domain; 1c) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain; and / or 1d) the amino acid sequence according to SEQ ID NO:6; (2) deletion of lysine residues from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain; (3) a lysine mutation at amino acid position 12 of the N-terminus of the CD134 intracellular domain; (4) a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain; and / or (5) one or more proximal basic amino acid mutations at amino acid positions 1 to 14 of the N-terminus of the CD134 intracellular domain, optionally at one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, and 5 of the N-terminus of the CD134 intracellular domain; 2. The RTCR of claim 1, wherein the mutant CD134 intracellular domain further comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain.

4. the chimeric intracellular domain comprises a first signaling domain derived from a protein of the CD28 family, optionally wherein the first signaling domain is derived from CD28, CD28H, ICOS, or a combination thereof, and optionally: a) said first signaling domain is derived from ICOS and optionally comprises an amino acid sequence according to SEQ ID NO: 9, optionally said chimeric intracellular domain comprises a first signaling domain comprising a portion of a CD28 intracellular domain combined with an ICOS domain according to SEQ ID NO: 9, and optionally said first signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 12 and 109; or b) said first signaling domain is derived from CD28, optionally comprising an amino acid sequence according to SEQ ID NO: 10, optionally said first signaling domain comprising an amino acid sequence according to any one of SEQ ID NOs: 121-122, and optionally said chimeric intracellular domain comprising an amino acid sequence according to any one of SEQ ID NOs: 14-17; 4. An RTCR according to any one of claims 1 to 3.

5. The chimeric intracellular domain further comprises a third signaling domain, optionally wherein the third signaling domain is derived from a CD3 signaling domain, a CD2 signaling domain, an interleukin-2 receptor binding (IL-2RB) protein signaling domain, or a combination thereof, and optionally comprises: a) the CD3 signaling domain is derived from the CD3ζ or CD3ε domain, or a combination thereof, optionally wherein the CD3 signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47, and 48; b) said CD2 signaling domain is a mutant CD2 signaling domain, optionally said mutant CD2 signaling domain is a truncated CD2 signaling domain, and optionally said CD2 signaling domain comprises an amino acid sequence according to SEQ ID NO: 49; or c) the IL-2RB protein signaling domain comprises an amino acid sequence according to SEQ ID NO:50; The RTCR according to any one of claims 1 to 4.

6. the chimeric intracellular domain further comprises a fourth signaling domain, optionally wherein the fourth signaling domain is derived from a CD3 signaling domain, a CD2 signaling domain, an interleukin-2 receptor-binding (IL-2RB) protein signaling domain, or a combination thereof, wherein the third and fourth signaling domains are not identical, and optionally; a) the CD3 signaling domain is derived from the CD3ζ or CD3ε domain or a combination thereof, optionally wherein the CD3 signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47 and 48; b) said CD2 signaling domain is a mutant CD2 signaling domain, optionally said mutant CD2 signaling domain is a truncated CD2 signaling domain, and optionally said CD2 signaling domain comprises an amino acid sequence according to SEQ ID NO: 49; or c) the IL-2RB protein signaling domain comprises an amino acid sequence according to SEQ ID NO:50; The RTCR according to any one of claims 1 to 5.

7. The extracellular domain comprises a protein or a portion thereof that induces activation and / or proliferation of an immune cell, and optionally the extracellular domain comprises a) components of the T cell receptor (TCR) complex; b) components of a chimeric antigen receptor (CAR); c) a component of a T cell co-receptor, the component being a T cell co-stimulatory protein or a T cell inhibitory protein; d) a ligand that binds to a cell surface receptor or a component thereof; e) components of cytokine receptors; f) components of chemokine receptors; g) components of integrin receptors; h) components of endothelial cell surface protein receptors or fragments thereof; i) a component of a neural guidance protein receptor, and j) components of complement receptors; The RTCR according to any one of claims 1 to 6, comprising any one of the following:

8. The extracellular domain comprises: a) the component of the T cell co-receptor or the CAR is a component of PD1, CD28, CD2, OX-40, ICOS, CTLA-4, CD28, CD3, CD4, CD8, CD40L, Lag-3, Tim-3, TIGIT, or a combination thereof; b) the T cell co-receptor or the component of the CAR binds to CD19, B cell maturation Ag (BCMA), PD-L1, PD-L2, IL-10, proliferation-inducing ligand (APRIL), BAFF, OX-40L, ICOS-L, B7-1, B7-2, CD40, CD58, CD59, nectin, CD155, or CD112, or a combination thereof; c) the cytokine receptor binds IL-10, IL-27, TGF-β, IL-12, IL-1, IL-2, IL-4, IL-5, IFN-γ, IFN-α / β, or a combination thereof; d) the component of a complement receptor is a component of C3aR, C5aR, CD46 / MCP, CD55, CD97, or DAF, or a combination thereof; and / or e) the extracellular domain is selected from the group consisting of epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), chemokine receptor (CCR) 4, CCR5, CCR7, CCR10, netrin-1 receptor, semaphorin receptor, lymphocyte function-associated antigen-1 (LFA-1), leukocyte-specific β2 integrin (αLβ2, αMβ2, αXβ2, or αDβ2β), β7 integrin (α4β7 or αEβ7), extracellular matrix (ECM)-bound β1 integrin (α1-α6β1), L-selectin, or sialyl Lewis ｘ comprising the amino acid sequence of the component; Optionally, the extracellular domain is a polypeptide, a glycoprotein, or an antibody or fragment thereof, optionally the antibody or fragment thereof is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, an sdAb, an Fv, a VHH fragment, or a single chain Fv fragment, and further optionally, the extracellular domain binds to a target selected from a tumor antigen, a pathogen-associated protein, and an antigen associated with an autoimmune, inflammatory, metabolic, or neurodegenerative condition or disorder, optionally the tumor antigen is a tumor-associated antigen (TAA), a tumor-secreted antigen (TSA), or a non-conventional antigen (UCA), optionally the extracellular domain binds to the target with a binding affinity of 1 fM to 100 μM, optionally the extracellular domain binds to the target with a binding affinity of 1 μM to 10 μM, and Further optionally, the extracellular domain comprises: a) a signal peptide at the N-terminus, said signal peptide being derived from a surface protein or a secreted protein, optionally said signal peptide being: i) PD-1 signal peptide (PD-1 SP); or ii) an HLA type I histocompatibility antigen or a portion thereof; Contains; b) a hinge region, optionally said hinge region being derived from CD8, PD-1, CD28, ICOS, or IgG; 8. The RTCR of claim 7.

9. The RTCR of any one of claims 1 to 8, wherein the transmembrane domain is derived from CD8, PD1, CD28, COS, or IgG.

10. 10. The RTCR of any one of claims 1 to 9, wherein the RTCR is for expression in a T cell, and the T cell co-expresses at least one of the endogenous costimulatory molecules selected from CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, CD40L, and combinations thereof.

11. A nucleic acid encoding the RTCR according to any one of claims 1 to 10, or a vector comprising said nucleic acid.

12. A cell comprising the nucleic acid or vector of claim 11, optionally the cell is a modified T cell or a modified natural killer T cell (NK-T cell), optionally the modified T cell is an allogeneic T cell or an autologous T cell, optionally the modified T cell is a naive T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (T ＳＣＭ ), central memory T cells (T ＣＭ ), or regulatory T cells (T ｒｅｇ ), optionally the cell further comprises a sequence encoding an artificial antigen receptor, a therapeutic polypeptide, an immune cell modulating protein, or a combination thereof, optionally the artificial antigen receptor comprises a chimeric antigen receptor (CAR), optionally the cell transiently or stably expresses an RTCR, and optionally the cell co-expresses the RTCR and at least one or more of the endogenous costimulatory molecules selected from CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L, and combinations thereof.

13. 1. A modified T lymphocyte (T cell), comprising: (a) modification of an endogenous sequence encoding a T cell receptor (TCR), which reduces or eliminates the level of expression or activity of the TCR; and (b) a recombinant T cell costimulatory receptor (RTCR) according to any one of claims 1 to 10. and optionally further comprising a modification of an endogenous sequence encoding a component of the major histocompatibility complex (MHC) class I (MHC-I), wherein said modification reduces or eliminates the level of expression or activity of said MHC-I, and optionally said modified T cell co-expresses said RTCR and at least one of the endogenous costimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L, or a combination thereof.

14. The following: a) an RTCR according to any one of claims 1 to 10; b) a nucleic acid according to claim 11; c) the vector according to claim 11; d) a cell according to claim 12; e) a modified T cell according to claim 13; and / or f) a cell population, said population comprising a plurality of the cells of claim 12 or said population comprising a plurality of the modified T cells of claim 13; A composition comprising:

15. 1. A method of producing a plurality of modified T cells, the method comprising: a) providing a plurality of primary T cells; b) providing a composition comprising an RTCR according to any one of claims 1 to 10, a nucleic acid or a vector according to claim 11; and c) introducing the composition of (b) into the plurality of primary T cells of (a) to produce a plurality of modified T cells under conditions for stably expressing the RTCR in the plurality of modified T cells, optionally wherein the method further comprises modifying an endogenous sequence encoding an endogenous T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the endogenous TCR, optionally wherein the method further comprises modifying an endogenous sequence of the plurality of primary T cells, wherein the modification reduces or eliminates the level of expression or activity of major histocompatibility complex (MHC) class I (MHC-I), and optionally wherein the method further comprises d) maintaining or expanding the plurality of modified T cells in a suitable cell culture medium; e) Below i) cryopreserving the plurality of modified T cells in a suitable cell freezing medium; or ii) preparing the plurality of modified T cells for administration to a subject in need thereof. Either The method further comprises:

16. 15. A method of treating a disease or disorder comprising administering a therapeutically effective number of the cells of claim 12, a therapeutically effective number of the engineered T cells of claim 13, a therapeutically effective amount of the composition of claim 14, or a therapeutically effective number of engineered T cells produced by the method of claim 15 to a subject in need thereof, optionally wherein the subject is a mammal, and optionally wherein the mammal is a human, and optionally wherein the disease or disorder is cancer, an autoimmune disease or disorder, an infectious disease, an inflammatory disease, a renal disease or disorder, a pulmonary disease or disorder, a liver disease or disorder, a cardiovascular disease or disorder, a neurodegenerative disease or disorder, or a metabolic disease or disorder, and optionally wherein the cancer is a solid tumor or a hematological cancer, and optionally wherein the solid tumor is a sarcoma, carcinoma, or melanoma, or wherein the hematological cancer is a leukemia, lymphoma, or myeloma, and optionally wherein a) the cancer is acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB selected from ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndromes (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, hypercalcemia associated with paraneoplastic syndromes / malignancies, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis carcinoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, and hemangioma; b) the infection is caused by a bacterium, a virus, a fungus, a protozoan, or a parasite; c) the neurodegenerative disorder is Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), spinocerebellar atrophy (SCA), or spinal muscular atrophy (SMA); or d) the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, or multiple sclerosis; method.

17. 1. A chimeric costimulatory intracellular protein (CIP) comprising a first and at least a second signaling domain, the first and the at least second signaling domains are not identical; The CIP, wherein the at least a second signaling domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein, wherein the mutant intracellular signaling domain of the TNFR family protein is a mutant CD134 (4-1BB) intracellular domain or a mutant CD137 (OX-40) intracellular domain, and optionally the CIP further comprises a transmembrane domain.

18. 18. The CIP of claim 17, comprising: a) the mutant CD137 intracellular domain is: (1) A truncated CD137 intracellular domain, optionally comprising: 1i) the amino acid sequence from amino acid position 13 to amino acid position 42 of the CD137 intracellular domain; 1ii) deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the N-terminus of the CD137 intracellular domain; 1iii) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain; and / or (1) the amino acid sequence according to SEQ ID NO:3; (2) deletion of one, two, three, or four lysine residues from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain; (3) one or more lysine mutations from amino acid position 1 to amino acid position 12 at the N-terminus of the CD137 intracellular domain, optionally one or more lysine mutations at amino acid positions selected from amino acid positions 1, 5, 6 and 12 at the N-terminus of the CD137 intracellular domain; (4) a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain; (5) one or more proximal basic amino acid mutations at amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, optionally one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5, and 6 of the N-terminus of the CD137 intracellular domain, and optionally a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain; Contains; or b) the mutant CD134 intracellular domain is (1) A truncated CD134 intracellular domain, optionally comprising: 1i) the amino acid sequence from amino acid position 15 to amino acid position 37 of the CD134 intracellular domain; 1ii) a deletion of a contiguous stretch of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from the N-terminus of the CD134 intracellular domain; 1iii) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acids from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain; and / or 1iv) comprises an amino acid sequence according to SEQ ID NO:6; (2) deletion of lysine residues from amino acid position 1 to amino acid position 14 at the N-terminus of the CD134 intracellular domain; (3) a lysine mutation at amino acid position 12 of the N-terminus of the CD134 intracellular domain; (4) a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain; and / or (5) one or more proximal basic amino acid mutations at amino acid positions 1 to 14 of the N-terminus of the CD134 intracellular domain, optionally at one or more proximal basic amino acid mutations at amino acid positions selected from amino acid positions 1, 2, and 5 of the N-terminus of the CD134 intracellular domain; and optionally the mutant CD134 intracellular domain further comprises a lysine mutation at amino acid position 12 at the N-terminus of the CD134 intracellular domain.

19. the chimeric intracellular domain comprises a first signaling domain derived from a protein of the CD28 family, optionally the first signaling domain is derived from CD28, CD28H, ICOS, or a combination thereof, and optionally a) the first signaling domain is derived from ICOS, and optionally i) the first signaling domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO:9; ii) the first signaling domain comprises a portion of the CD28 intracellular domain combined with an ICOS domain according to SEQ ID NO:9; or iii) the first signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 12 or 109; or b) the first signaling domain is derived from CD28, wherein optionally i) the first signaling domain derived from CD28 comprises an amino acid sequence according to SEQ ID NO: 10; or ii) the first signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 121-122; 19. The CIP of any one of claims 17 or 18, further optionally comprising an amino acid sequence according to any one of SEQ ID NOs: 14 to 17.

20. The CIP further comprises a third signaling domain, optionally wherein the third signaling domain is derived from a CD3 signaling domain, a CD2 signaling domain, an interleukin-2 receptor-binding (IL-2RB) protein signaling domain, or a combination thereof, and optionally a) the CD3 signaling domain is derived from the CD3ζ or CD3ε domain or a combination thereof, optionally the CD3 signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47 and 48; b) said CD2 signaling domain comprises a mutant CD2 signaling domain, optionally said mutant CD2 signaling domain is a truncated CD2 signaling domain and optionally said CD2 signaling domain comprises an amino acid sequence according to SEQ ID NO: 49; or c) The CIP according to any one of claims 17 to 19, wherein the IL-2RB protein signaling domain comprises an amino acid sequence according to SEQ ID NO:

50.

21. the chimeric intracellular domain further comprises a fourth signaling domain, optionally the fourth signaling domain is derived from a CD3 signaling domain, a CD2 signaling domain, or an interleukin-2 receptor-binding (IL-2RB) protein signaling domain, or a combination thereof, and the third and fourth signaling domains are not identical; and optionally a) the CD3 signaling domain is derived from the CD3ζ or CD3ε domain or a combination thereof, and optionally the CD3 signaling domain comprises an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47, and 48; b) said CD2 signaling domain is a mutant CD2 signaling domain, optionally said mutant CD2 signaling domain is a truncated CD2 signaling domain, optionally said CD2 signaling domain comprises an amino acid sequence according to SEQ ID NO: 49; or c) the IL-2RB protein signaling domain comprises an amino acid sequence according to SEQ ID NO:50; A CIP according to any one of claims 17 to 20.

22. 22. The CIP of any one of claims 17 to 21, wherein the CIP is for expression in a T cell, and the T cell co-expresses at least one of the endogenous costimulatory molecules selected from CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8, and CD40L, and combinations thereof.

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