Allogeneic cell compositions and methods of use

Abstract

Disclosed are chimeric stimulatory receptors (CSRs), cell compositions comprising CSRs, methods of making and methods of using same for the treatment of a disease or disorder in a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims the priority to, and benefit of, U.S. Provisional Application No. 62 / 727,498, filed on September 5, 2018, U.S. Provisional Application No. 62 / 744,073, filed on October 10, 2018, U.S. Provisional Application No. 62 / 815,334, filed on March 7, 2019, and U.S. Provisional Application No. 62 / 815,880, filed on March 8, 2019. The contents of each of these applications are hereby incorporated by reference in their entireties. FIELD OF THE DISCLOSURE

[02] The disclosure is directed to molecular biology, and more, specifically, to chimeric receptors, allogeneic cell compositions, methods of making and methods of using the same. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[03] The contents of the file named "‘POTH-046 00 IWO SecjuenceListing.txt”, which was created on September 5, 2019, and is 55.7 MB in size are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION

[04] There has been a long-felt but unmet need in the art for an allogeneic cell composition that overcomes the challenges presented by eliminating genes involved in a graft versus host response and host versus graft response. The disclosure provides allogeneic cell compositions, methods of making and methods of using these compositions which comprise non-naturally occurring structural improvements to restore responsiveness of allogeneic cells to environmental stimuli as well as reduce or prevent rejection by natural killer cell-mediated cytotoxicity. SUMMARY OF THE INVENTION

[05] The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[06] The activation component can comprise a portion of one or more of a component of a T~ cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitor}' protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activation component binds. The activation component can comprise a CD2 extracellular domain or a portion thereof to which an agoni st binds.

[07] The signal transduction domain can comprise one or more of a component of a human signal transduction domain, T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain can comprise a CD3 protein or a portion thereof. The CD3 protein can comprise a CD3^ protein or a portion thereof.

[08] The endodomain can further comprise a cytoplasmic domain. The cytoplasmic domain can be isolated or derived from a third protein. The first protein and the third protein can be identical. The ectodomain can further comprise a signal peptide. The signal peptide can be derived from a fourth protein. The first protein and the fourth protein can be identical. The transmembrane domain can be isolated or derived from a fifth protein. The first protein and the fifth protein can be identical.

[09] In some aspects, the activation component does not bind a naturally-occurring molecule. In some aspects, the activation component binds a naturally-occurring molecule but the CSR does not transduce a signal upon binding of the activation component to a naturally-occurring molecule. In some aspects, the activation component binds to a non-naturally occurring molecule. In some aspects, the activation component does not bind a naturally-occurring molecule but binds a non-naturally occurring molecule. The CSR can selectively transduces a signal upon binding of the activation component to a non-naturally occurring molecule. In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory' receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof, and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3£ protein or a portion thereof. In some aspects, the non-naturally CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 17062. In a preferred aspect, the non-naturally occurring CSR comprises an amino acid sequence of SEQ ID NO: 17062.

[010] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) wherein the ectodomain comprises a modification. The modification can comprise a mutation or a truncation of the amino acid sequence of the activation component or the first protein when compared to a wild type sequence of the activation component or the first protein. The mutation or a truncation of the amino acid sequence of the activation component can comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. The mutation or truncation of the CD2 extracellular domain can reduce or eliminate binding with naturally occurring CD58. In some aspects, the CD2 extracellular domain comprising the mutation or truncation comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 17119. In a preferred aspect, the CD2 extracellular domain comprising the mutation or truncation comprises an amino acid sequence of SEQ ID NO: 17119.

[011] In a preferred aspect, the present disclosure provides non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds and wherein the CD2 extracellular domain or a portion thereof to which an agonist binds comprises a mutation or truncation; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3£ protein or a portion thereof. In some aspects, the non-naturally CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95% or at least 99%o identical to SEQ ID NO: 17118. In a preferred aspect, the non-naturally occurring CSR comprises an amino acid sequence of SEQ ID NO: 17118.

[012] The present disclosure provides a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

[013] The present disclosure provides a cell comprising any CSR disclosed herein. The present disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

[014] A modified cell disclosed herein can be an allogeneic cell or an autologous cell. In some preferred aspects, the modified cell is an allogeneic cell. In some preferred aspects, the modified cell is an allogeneic T-cell or a modified allogeneic CAR T-cell.

[015] The present disclosure provides a composition comprising any CSR disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein or a composition comprising a plurality of modified cells disclosed herein.

[016] The present disclosure provides a modified T lymphocyte (T-cell), comprising: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity' of the TCR; and (b) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[017] The modified T-cell can further comprise an inducible proapoptotic polypeptide. The modified T-cell can further comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

[018] The modified T-cell can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E can further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding the HLA-E.

[019] The modified T-cell can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor can comprise a chimeric antigen receptor (CAR).

[020] The CSR can be transiently expressed in the modified T-cell. The CSR can be stably expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be transiently expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be stably expressed in the modified T-cell. The inducible proapoptotic polypeptide can be transiently expressed in the modified T-cell. The inducible proapoptotic polypeptide can be stably expressed in the modified T-cell. The non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be transiently expressed in the modified T-cell. The non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be stably expressed in the modified T-cell.

[021] The modified T-cell can be an autologous cell. The modified T-cell can be an allogeneic cell. The modified T-cell can be an early memory T cell, a stem cell-like T cell, a stem memory T cell (Tscm), a central memory T cell (Tcm) or a stem cell-like T cell.

[022] The present disclosure provides a composition comprising any modified T-cell disclosed herein. The present disclosure also provides a composition comprising a population of modified T lymphocytes (T-cells), wherein a plurality of the modified T-cells of the population comprise the CSR disclosed herein. The present disclosure also provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise the modified T-cell disclosed herein.

[023] The present disclosure provides methods of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of any composition disclosed herein; or a composition for use in the treatment of a disease or disorder. In one aspect, the composition is a modified T-cell or population of modified T-cells as disclosed herein. The present disclosure also a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds the CSR.

[024] The present disclosure provides a method of producing a population of modified T-cells compri sing, consisting essential of, or consisting of introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The present disclosure provides a composition comprising a population of modified T-cells produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%s, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm) or a TscM-like cell; and wherein the one or more cellsurface marker(s) compri se CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (Tcm) or a TcM-like cell; and wherein the one or more cell-surface inarker(s) comprise CD45RO and CD62L. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition produced by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition produced by the method. The method of treating can further comprising administering an activator composition to the subject to activate the population of modified T-cells in vivo, to induce cell division of the population of modified T-cells in vivo, or a combination thereof.

[025] The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modifi ed T-cells. The present disclosure provides a composition comprising a population of modified T-cells produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%s, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cellsurface marker(s) of a stem memory’ T cell (Tscm) or a TscM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (Tcm) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition produced by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition produced by the method. In some aspects, the modified T-cells within the population of modified T-cells administered to the subject no longer express the CSR.

[026] The present disclosure provides a method of expanding a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that stably express the CSR within the plurality of modified T-celis and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not stably expressing the CSR under the same conditions. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm) or a Tscm-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cellsurface marker(s) of a central memory T cell (Tcm) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The present disclosure provides a composition comprising a population of modified T-cells expanded by the method. The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition expanded by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition expanded by the method. The method of treating can further comprising administering an activator composition to the subject to activate the population of modified T-cells in vivo, to induce cel I division of the population of modified T-cells in vivo, or a combination thereof.

[027] The present disclosure provides a method of expanding a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing the CSR under the same conditions. The present disclosure provides a composition comprising a population of modified T-cells expanded by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm) or a TscM-like cell; and wherein the one or more cellsurface marker(s) comprise CD45RA and CD62L. some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory T cell (Tcm) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L.The composition can be for use in the treatment of a disease or disorder. The present disclosure also provides for use of a composition expanded by the method for the treatment of a disease or disorder. The present disclosure further provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeutically-effective amount of the composition expanded by the method. In some aspects, the modified T-cells within the population of modified T-cells administered to the subject no longer express the CSR.

[028] Any of the above aspects can be combined with any other aspect.

[029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be 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. About 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 the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[030] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[032] FIG. 1 is a schematic diagram depicting a T-cell receptor (TCR) and co-receptors CD28 and CD2.

[033] FIG. lisa schematic diagram depicting primary and secondary co-stimulation is delivered to T-cell via binding of agonist mAbs (anti-CD3, anti-CD28, and anti-CD2). Full T-cell activation critically depends on TCR engagement in conjunction with a second signal by costimulatory receptors that boost the immune response. Primary and secondary co-stimulation can be delivered to T-cell via treatment with and engagement of surface receptors with agonist mAbs (E.g. anti-CD3, anti-CD28, and anti-CD2).

[034] FIG. 3 is a schematic diagram showing that, in absence of TCR, only secondary costimulation is delivered to T-cell via binding of agonist mAbs. Since full T-cell activation is critically dependent on primary stimulation via CD3y in conjunction with a second signal by costimulatory receptors, T cell activation and expansion is suboptimal and thus reduced.

[035] FIG. 4 is a schematic diagram showing that, in absence of TCR, stimulation is enhanced with expression of Chimeric Stimulatory Receptors (CSRs). In the absence of TCR, but in the presence of surface-expressed CSR / s, primary and secondary co-stimulatory signals are delivered when T cell is treated with standard agonist mAbs. Since a fuller T-cell activation is achieved via CSR-mediated stimulatory signals, T cell activation and expansion is enhanced.

[036] FIG. 5 is a schematic diagram depicting an exemplary CSR CD28z of the disclosure.

[037] FIG. 6 is a schematic diagram depicting an exemplary CSR CD2z of the disclosure.

[038] FIG. 7 is a schematic of a strategy for mutation of CSR CD2z to eliminate natural ligand (CD58) binding. A panel of CSR. CD2z mutants was designed within the extracellular domain of CD2. The goal of this panel was to identify mutants that no longer bind CD58 but retain their receptivity to being bound by the anti-CD2 activator reagent. This may be desirable for two main reasons: 1) CD58 expression by activated T cells may interact with the wild type (WT) CD2z CSR and possibly interfere with the optimal performance of the CSR, and 2) since the WT CD2z CSR might function as a natural ligand CAR, it is possible that T cells expressing the CSR may mediate cytotoxic activity against CD58-expressing cells, including activated T cells. Thus, a mutant CD2z CSR that cannot interact with CD58 but retains its ability to bind activating anti-CD2 reagent for optimal cell expansion is desired.

[039] FIG. 8 is a schematic diagram depicting an exemplary CSR CD2z-Dl 1IH of the disclosure. A DI 1 IH mutation is within the CD2 extracellular domain of the CSR CD2z-Dl 1 IH construct.

[040] FIGS. 9A-9B are a series of plots showing that piggyBac18’ delivery of CSR enhances the expansion of TCRb / b2M double-knockout CAR-T cells. Pan T cells isoiated from normal donor blood were genetically modified using the piggyBac1® DNA modification system in combination with the Cas-CLOVER™ gene-editing system. Cells were electroporated in a single reaction with a transposon encoding a CAR, selection gene and a CSR (either CD28z or CD2z), an mRNA. encoding the super piggyBac™ transposase enzyme, an mRNA encoding Cas-CLOVER™, and multiple guide RNA (gRNA) targeting TCRb and b2M in order to knockout the TCR and MHCI (double-knockout; DKO). The cells were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 16 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). In the samples expressing either CD2z or CD28z CSR, a greater degree of expansion of the DKO cells was observed as a greater frequency of the CAR alone DKO cells (FIG. 9A and 9B). In DKO CAR-T cell samples expressing either CD2z or CD28z CSR, at least a two fold expansion of the cells was observed in comparison to DKO CAR-T cells alone.

[041] FIGS. 10A-10B are a series of plots showing that CSR CD2z or CD28z in purified DKO CAR-T cells results in enhanced expansion upon re-stimulation. After initial genetic modification and a first round of stimulation and expansion, cells from each group (Mock (WT CAR-T cells), DKO CAR-T cells, DKO CAR-T cells + CD2z CSR, and DKO CAR-T cells + CD28z CSR) were purified for TCR'MHCT cells using magnetic beads. The purified cells were then re-stimulated using anti-CD2, anti-CD3, and anti-CD28 agonist mAbs. At the end of the 14 day culture period, TCR and MHCI expression (A) as well as magnitude of cell population expansion (B) was determined. After this secondary expansion, all purified DKO cells, including those expressing either CD2z or CD28z CSR, were still extremely pure for DKO cells (>98.8% DKO). DKO CAR-T cells expressing either CD2z or CD28z CSR resulted in enhanced expansion when compared to those not expressing either CSR.

[042] FIG. 11 is a graph showing that cytokine supplementation can further expand purified DKO CAR-T cells expressing CSR upon re-stimulation. After initial genetic modification and a first round of stimulation and expansion, cells expressing CSRs were purified for DKO cells using magnetic beads. The purified cells were then re-stimulated using anti-CD2, anti~CD3, and anti-CD28 agonist mAbs in the presence exogenous purified recombinant IL7 and IL 15. At the end of the 14 day culture period, magnitude of cell population expansion was determined . After a secondary expansion, all purified DKO cells, including those expressing either CD2z or CD28z CSR, were still extremely pure for TCRMHCT cells (>98.8% double knockout (data not shown)). In addition, cells grew robustly in the presence of IL7 and IL 15, which was greater than that without supplementation. These data demonstrate that exogenous cytokines may be added to further expand WT CAR-T cells expressing CSR.

[043] FIG. 12 is a graph showing that surface expression of CAR is not significantly affected by co-expression of CSR in DKO cells. After secondary expansion, cells (Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z CSR, and DKO CAR-T cells + CD28z CSR) were stained for the surface-expression of CAR and compared to control WT CAR-T cells and Mock T cells. Expression of CD2z or CD28z CSR does not have a significant impact on expression of CAR molecule on the surface of T cells.

[044] FIG. 13 is a graph showing that expression of CSRs does not significantly affect DKO CAR-T cell cytotoxicity in vitro. After secondary' expansion, cells (Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z CSR, and DKO CAR.-T cells + CD28z CSR) were co-cultured with engineered K562-BCMA-Luciferase (eK562-Luc.BCMA) or negative control line K562-PSMA-Luciferase (eK562-Luc.PSMA) for 48 hours at 10:1, 3:1, or 1:1 E:T ratios. Luciferase signal was measured to determine cytotoxicity. Killing of eK562-Luc.PSMA is shown in dotted lines, while killing of eK562-Luc.BCMA is shown in solid lines. All CAR+ T cells expressed an anti-BCMA specific CAR. DKO CAR-T cells exhibit similar in vitro cytotoxicity as WT CAR-TCR cells. This activity is not significantly affected by CD2z or CD28z CSR co-expression.

[045] FIG. 14 is a graph showing that expression of CSRs does not significantly affect DKO CAR-T cell secretion of IFNg in vitro. Supernatants from the 48 hour killing assay were assayed for secreted IFNg as a measure of antigen-specific functionality of the BCMA CAR T cells. All CAR-T cells, either with or without CD2z or CD28z CSR expression secrete IFNg in response to co-culture with target cells expressing BCMA (eK562-Luc.BCMA), but not those expressing an irrelevant target (eK562-Luc.PSMA).

[046] FIG. 15 is a series of plots showing that expression of CSRs does not significantly affect DKO CAR-T cell proliferation in vitro. Mock (WT T-cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z CSR, and DKO CAR-T cells + CD28z CSR cells were labelled with Cell Trace Violet (CTV), which is diluted as cells proliferate. The cells were cocultured for 5 days with eK562-Luc.PSMA or eK562-Luc.BCMA cells at a 1:2 E:T ratio. All CAR-T cells, either with or without CD2z or CD28z proliferate in response to target cells expressing BCMA (eK562-Luc.BCMA) but not those expressing an irrelevant antigen (eK562-Luc.PSMA).

[047] FIG. 16 is a pair of graphs showing that the memory phenotype of DKO CAR-T is not significantly affected with CD2z CSR co-expression. WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z, and DKO CAR-T cells + CD28z were stained for expression of surface CD45RA, CD45RO, and CD62L to define Tscm, Tcm, Tern, and Teff cells; Tscm (CD45RA+CD45RO CD62L+), Tcm (CD45RA'CD45RO+CD62L+), Tem (CD45RA' CD45RO+CD62L‘), Teff (CD45RA+CD45RO’CD62L’). WT and DKO CAR-T cells with or without CD2z are comprised predominantly of exceptionally high levels of favorable Tscm and Tcm cells. However, when CD28z is expressed in DKO CAR-T cells, the phenotype is significantly more differentiated, favoring Tcm and Tem cells. This phenotype may have a negative impact on the in vivo functionality of these CAR T cells since they appear to be more differentiated.

[048] FIG. 17 is a series of graphs showing that the expression of activation / exhaustion markers in DKO CAR-T is not significantly affected with CD2z CSR co-expression. Mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z, and DKO CAR-T cells + CD28z were examined by flow cytometry for the expression of important exhaustion molecules Lag3, PD1, and Tim3. WT and DKO CAR-T cells with or without CD2z have little to no expression of exhaustion molecules when compared to mock T cells. However, expression of CD28z CSR in DKO CAR-T during the expansion process leads to significant upregulation of exhaustion markers Lag3, PD1, and Tim3. This phenotype may have a negative impact on the in vivo functionality of these CAR T cells since they appear to be more exhausted. By contrast, CD2z expression has little to no effect on the exhaustion phenotype of DKO CAR-T cells while significantly enhancing the expansion capability of the cells.

[049] FIG. 18 is a graph showing that delivery of CSR enhances the expansion of CAR-T cells. CSRs were delivered to CAR-T cells either transiently by mRNA or stably by piggyBac Pan T cells isolated from the blood of a normal donor were genetically modified using the piggyBac® DNA modification system and the standard Poseida process. Cells were coelectroporated in a single reaction with mRNA encoding the Super piggyBactransposase enzyme (SPB), a transposon encoding a BCM A CAR and selection gene, along with an additional mRNA encoding a CSR (either CD28z or CD2z; resulting in transient expression) or a CD 19 mRNA control, or, with a transposon encoding a BCMA CAR, selection gene and a CSR (either CD28z or CD2z; resulting in stable expression). The cells ’were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). Bars represent total live CAR-T cells in well and numbers indicate foldenhancement of expansion above CAR-T cells produced in the absence of a CSR or a CD 19 mRNA control. In the samples expressing either CD2z or CD28z CSR, either transiently or stably, a greater degree of expansion of the CAR-T cells.

[050] FIG. 19 is a series of bar graphs showing that expression of CSRs does not significantly affect CAR-T cell cytotoxicity. CSRs were delivered to CAR-T cells either transiently by mRNA or stably by piggyBac®. Pan T cells isolated from the blood of a normal donor were genetically modified using the piggyBac® DNA modification system and the standard Poseida process. Cells were co-electroporated in a single reaction with mRNA encoding the Super piggyBac™ transposase enzyme (SPB), a transposon encoding a BCMA CAR and selection gene, along with an additional mRNA encoding a CSR (either CD28z or CD2z; resulting in transient expression), or, with a transposon encoding a BCMA CAR, selection gene and a CSR (either CD28z or CD2z; resulting in stable expression). The cells were subsequently stimulated with agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19 day culture period. At the end of the initial culture period all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). To assess CAR-T cell ability to kill, cells were co-cultured with engineered K562-BCMA-Luciferase (eK562-Luc.BCMA) or negative control line K562-Luciferase (eK562-Luc) for 48 hours at 10:1, 3:1, or 1:1 E:T ratios. Luciferase signal was measured to determine cytotoxicity. Killing of eK562-Luc is shown in bar graph on left, while killing of eK562-Luc.BCMA is shown in bar graph on right. All CAR+ T cells expressed an anti-BCMA specific CAR and exhibited similar in vitro cytotoxicity against BCM A+ target cells. In summary, this activity was not significantly affected by transient or stable CSR co-expression.

[051] FIG. 20 is a schematic diagram showing that, in presence of TCR, stimulation is enhanced with expression of Chimeric Stimulatory Receptors (CSRs). In the presence of surface-expressed CSR / s, either transiently or stably expressed, enhanced primary and secondary costimulatory signals are delivered when T cell is treated with reagents displaying agonist mAbs. In one aspect, this schematic diagram represents an autologous cell. Since a fuller T-cell activation is achieved via CSR-mediated stimulatory signals, T cell activation and expansion is enhanced.

[052] FIG. 21 is a series of graphs showing that CSRs are expressed on the surface of T cells and do not lead to cellular activation in the absence of exogenous stimulation. Pan T cells from normal blood donors were stimulated with anti~CD3 / anti~CD28 beads in standard T cell culture media, then rested. These cells were then electroporated (BTX ECM 830 electroporator @ 500V for 700 ps) with 10 pg of mRNA encoding either CD28 CSR, CD2 CSR, or wild-type CD 19 control. Two days later the electroporated cells ’were examined by flow cytometry for surfaceexpression of each molecule and data are shown as stacked histograms. In addition, cell size (FSC-A) and CD69 expression was evaluated as a possible indication of cellular activation above the Mock electroporated control cells. Increased surface expression of CD28, CD2, and CD19 were detected in T cells electroporated either with CD28z CSR, CD2z CSR or CD19, respectively. Expression of these molecules on the surface of T cells did not intrinsically activate the cells in the absence of exogenous stimulation.

[053] FIG. 22 is a series of line graphs showing that CSR molecules can be delivered transiently during manufacturing for the enhanced expansion of CAR-T cells. Pan T cells isolated from healthy donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) for the production of allogeneic (Allo) CAR-T cells, or without CC gene-editing for the production of autologous (Auto) CAR-T cells; auto CAR-T cells were produced by nucleofection of an mRNA encoding the super piggyBac® transposase enzyme (SPB) and a transposon encoding a CAR, selection gene and a safety switch. For production of Allo CAR-T, cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNAs (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and the CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR. For CAR-T cells that did not receive a CSR encoded in the transposon for stable integration, the CD2z CSR was provided to the cells transiently as an mRNA only once in the initial EP reaction, at varying amounts of 5 pg, 10 pg, and 20 pg of mRNA in a 100 pl EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of a 19-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). Data for each is shown in line graph at various days of production. In the samples where the CD2z CSR was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a greater degree of expansion of the CAR-T cells was observed as compared to the CAR-T cells produced without a CSR. These data show that the CSR can be delivered transiently as mRNA during manufacturing for enhanced expansion of both autologous and allogeneic CAR-T products.

[054] FIG. 23A is a bar graph showing CSR CD2z mutant staining data. A panel of CSR CD2z mutants was designed, constructed, and tested for surface expression and binding to several anti-CD2 antibody reagents. To do so, each mutant was synthesized, subcloned into an in-house mRNA production vector, and then high-quality mRNA was produced for each. K562 cells were electroporated with 9 pg of mRNA, and surface-expression of each molecule was analyzed by flow cytometry the next day and data are shown as bar graphs. Each molecule was stained with anti-CD2 activator reagent, anti-CD2 monoclonal antibody (clone TS1 / 8), or anti- CD2 polyclonal antibody reagent (goat anti-human CD2). Variable binding was observed for each construct and data are summarized in FIG. 23C.

[055] FIG. 23B is a series of bar graphs showing CSR CD2z mutant degranulation data. The panel of CSR CD2z mutants was tested for the capability of mediating degranulation against CD58-positive cell targets. T cell degranulation is a surrogate of T cell killing that can be measured by FACS staining for intracellular CD 107a expression following coculture with target cell lines expressing target antigen. Specifically, pan T cells from normal blood donors were stimulated with anti-CD3 / anti-CD28 beads in standard T cell culture media, then rested. These cells were then electroporated (BTX ECM 830 electroporator @ 500V for 700 ps) with 9 pg of mRNA expressing CSR CD2z mutants and cultured overnight. The next day, the cells were cocultured for 4-6 hours in the presence of various target cell lines. Positive target cell lines included K562 cells or Rat2 cells that were electroporated or lipofected, respectively, with mRNA encoding human CD58, while negative controls were either Rat2 cells that were not electroporated or CSR CD2z mutant expressing T cells alone. Only T cells expressing CSR CD2z mutants that recognized surface-expressed human CD58 v / ere capable of degranulating at levels above background. Little reactivity was observed for the DI 11H, K67R / Y110D, K67R / Q70K / Y110D / D111H, Delta K106-120, CD3z deletion and mock control, and data are summarized in FIG. 23C.

[056] FIG. 23C is a summary of staining and degranulation data. Data from surfaceexpression and binding studies, as well as those from degranulation experiments for each CSR CD2z mutant is summarized in the table. Two candidates that are expressed on the surface and / or retain binding to the anti-CD2 activator reagent that do not mediate anti-CD58 degranulation activity are the Dll 1H and K67R / Y1101) CSR CD2z mutants. Only the DI 11H mutant is strongly bound by all staining reagents on the cell surface while completely abrogating anti-CD58 degranulation activity.

[057] FIG. 23D is a series of flow7 cytometry plots showing the expression of CD48, CD58 or CD59 on K562 and Rat2 cells. To confirm possible ligands for the CSR WT CD2z molecule, a panel of known and suspected ligands including human CD48, CD58, and CD59 were tested. Degranulation of engineered T cells was evaluated against the cell lines K562 and Rat2 that were made to overexpress the target ligands and confirmed for expression by FACS staining. Red histograms are unstained cells and blue histograms are cells that were electroporated / lipofected with mRNA and then stained for expression of the respective marker by FACS.

[058] FIG. 23E is a bar graph showing that CSR CD2z recognizes human CD58, but not CD48 or CD59. To confinn possible ligands for the CSR WT CD2z molecule, a panel of known and suspected ligands including human CD48, CD58, and CD59 were tested. Degranulation of engineered T cells was evaluated against the cell lines K562 and Rat2 that were made to overexpress the target ligands and confirmed for expression by FACS staining. Cells were electroporated / lipofected with mRNA and then stained for expression of the respective marker by FACS. As a control, a BCMA CAR. was included as well as a K562 cell line overexpressing BCMA. In addition, T cells transfected with GFP were also included as a control. T cell degranulation is a surrogate of T cell killing that can be measured by FACS staining for intracellular CD 107a expression following coculture with target cell lines expressing target antigen. Pan T cells from normal blood donors were stimulated with anti-CD3 / anti-CD28 beads in standard T cell culture media, then rested. These T cells were then electroporated with mRNA expressing CSR WT CD2z, BCMA CAR, or GFP and cultured overnight. The next day, the cells were cocultured for 4-6 hours in the presence of the various target cell lines that were electroporate / lipofected with mRNA encoding human CD48, CD58 or CD59, while negative controls were either K562 or Rat2 cells that were not electroporated / lipofected, or each of the electroporated T cells alone. T cells expressing either the CSR WT CD2z or BCMA CAR were capable of degranulating at levels above background when cocultured with cell lines overexpressing human CD58 or BCMA, respectively, and not against human CD48 or CD59. Little reactivity was observed for the T cells expressing GFP.

[059] FIG. 24A is a bar graph showing that the delivery of CSR CD2z-Dl 11H mutant enhances the expansion of Allo CAR-T cells. Pan T cells isolated from healthy donor blood were genetically modified using the piggyBac DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) for the production of allogeneic (Allo) CAR-T cells, or without CC gene-editing, as a control, for the production of autologous (Auto) CAR-T without a CSR (No CSR); auto CAR-T cells were produced by nucleofection of an mRNA encoding the super piggyBac™ transposase enzyme (SPB) and a transposon encoding a CAR, selection gene and a safety switch. For production of Allo CAR-T, cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNAs (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and either the WT or mutant (DI 11H) CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR, For the latter, Allo CAR-T cells that did not receive a CSR encoded in the transposon for stable integration, the WT or mutant (DI 11H) CSR CD2z was provided to the cells transiently as an mRNA only once in the initial EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of up to a 15-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown), and then all nonedited TCR-positive cells were depleted via negative selection to yield a population of Allo CAR-T cells that were >99% TCR-negative (data not shown). All samples were performed in duplicate, except the Auto (No CSR.) control, and data for peak expansion for each (day of peak expansion is displayed) is shown in bar graph where error bars represent standard deviation. In the samples where either the WT or mutant (DI 11H) CD2z was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a greater degree of expansion of the Allo CAR-T cells was observed as compared to the Allo CAR-T cells produced without a CSR.

[060] FIG. 24B is a series of bar graphs showing that the delivery of CSR CD2z-Dl 11H mutant does not inhibit gene editing. Pan T cells isolated from healthy donor blood ’were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene-editing system (CC) to produce allogeneic (Allo) CAR-T cells. Cells were electroporated (EP) in a single reaction with an mRNA encoding the SPB enzyme, an mRNA encoding CC, multiple guide RNA (gRNA) targeting TCRb and b2M for the knockout of TCR and MHCI, and a transposon encoding either a CAR, selection gene and either the WT or mutant (DI 11H) CSR CD2z, or a transposon encoding a CAR, selection gene and a safety switch that did not encode a CSR. For the latter, cells that did not receive a CSR encoded in the transposon for stable integration, the WT or mutant (DI 11H) CSR CD2z was provided transiently as an mRNA only once in the initial EP reaction. Following EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3 and anti-CD28, and were later selected for genetic modification over the course of up to a 14-day culture period using the selection gene. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection for genetically-modified cells (data not shown). All samples were performed in duplicate, and data is shown in bar graph where error bars represent standard deviation. In the samples where either the WT or mutant (Dll 1H) CD2z was provided stably (as encoded in the transposon (Stable)) or transiently (as encoded in mRNA (mRNA)), a similar or greater degree of gene editing of the Allo CAR-T cells was observed as compared to the Allo CAR-T cells produced without a CSR.

[061] FIG. 24C is a bar graph showing that the memory' phenotype of Allo CAR-T is not significantly affected by delivery of CD2z CSRs. Allo CAR-T cells with no CSR and Allo CARTs with CSR that was delivered either stably or transiently were stained for expression of surface CD45RA, CD45RO, and CD62L to define Tscm, Tcm, Tern, and Teff cells; Tscm (CD45RA+CD45RO'CD62I / ), Tcm (CD45RA'CD45RO+CD62L+), Tern (CD45RA’ CD45RO+CD62L"), Teff (CD45RA~CD45RO'CD62L'). All samples were performed in duplicate, and data is shown in bar graph where error bars represent standard deviation. Delivery of CSRs did not dramatically affect the levels of favorable Tscm and Tcm cells in the products.

[062] FIG. 25 is a schematic diagram depicting an exemplary HLA-bGBE composition of the disclosure.

[063] FIG. 26 is a schematic diagram depicting an exemplary7 HLA-gBE composition of the disclosure.

[064] FIG. 27 is a pair of graphs showing that expression of single-chain HLA-E diminishes NK cell-mediated cytotoxicity against HLA-deficient T cells. B2M and TCRaP was knocked-out of T cells (Jurkat) using CRISPR. B2M / TCRaP double-knockout (DKO) T cells were electroporated with mRNA encoding an HLA-E molecule (HLA-bGBE), expressed on a single chain with B2M and the peptide VMAPRETLIL (SEQ ID NO: 17127) (B2M / peptide / HLA-E). DKO T cells electroporated with varying amounts of mRNA encoding single chain HLA-E were used as targets for artificial antigen presenting cell (aAPC)-expanded NK cells in a 3 hour coculture. % cytotoxicity was calculated based on the number of target cells remaining after 3 hours compared to target cells alone. These data demonstrate that surface expression of HLA-E in DKO T cells reduces the total level of cell killing by NK cells in a dose-dependent manner.

[065] FIG. 28 is a listing of gRNA sequences (from top to bottom) and primer sequences (from top to bottom)

[066] FIG. 29 is a series of flow cytometry plots showing that targeted knockout of endogenous HLA-ABC, but not HLA-E. Since we showed that surface expression of HLA-E in MHCI KO T cells can increase their resistance to NK cell-mediated cytotoxicity, we explored additional strategies beyond introduction of a single-chain HLA-E gene. To do so, multiple guide RNA (gRNA) were designed to disrupt the expression of the main targets of host versus graft (HvG), HLA-A, HLA-B and HLA-C, while minimizing disruption of endogenous HLA-E. Specifically, guides were designed to target a conserved region occurring in all the three MHCI protein targets, but not in HLA-E. Pan human T cells were electroporated with mRNA encoding CRISPR Cas9 in combination with various gRNAs and efficiency of MHCI knockout was measured by surface HLA-A and HLA-E expression. FACS analysis of HLA-A and HLA-E expression was performed after a single round of T cell expansion and data are displayed below7. These data demonstrate that gene-editing technology can be used to target disruption of MHCI while retaining levels of endogenous HLA-E on the surface of gene-edited T cells.

[067] FIG. 30 is a schematic diagram of the missing-self hypothesis of natural killer mediated toxicity towards MHCI-KO cells.

[068] FIG. 31 is a schematic depiction of the Csy4-T2A-Clo051-G4Slinker-dCas9 construct map (Embodiment 2).

[069] FIG. 32 is a schematic depiction of the pRTl-Clo051-dCas9 Double NLS construct map (Embodiment 1).

[070] FIG. 33 is a schematic diagram showing an exemplary method for the production of allogeneic CAR-Ts of the disclosure.

[071] FIG. 34A is a graph showing high efficiency gene editing of endogenous TCRa in proliferating Jurkat cells and in resting primary human pan T cells as an exemplary method for the production of allogeneic and universal CAR-Ts using Cas-CLOVER™ (an RNA-guided fusion protein comprising a dCas9-Clo051). Cas-CLOVER system disrupted TCRa expression in rapidly proliferating Jurkat T cells and non-dividing resting T cells at comparably high levels.

[072] FIG. 34B is a series of flow cytometry graphs showing efficient gene editing of endogenous TCRa, TCRb, and B2M in resting primary human pan T cells using Cas-CLOVER™. Critical targets TCRa, TCRB, and B2M that mediate al loreactivity were efficiently edited by Cas-CLOVER in resting human T cells.

[073] FIG. 35 is a series of flow cytometry plots showing that Cas-CLOVER can be multiplexed by co-delivering reagents for TCRP and P2M into primary' human T cells. TCRp / p2M double knock-out (DKO) cells were further enriched using antibody-beads based purification, and purified cells were analyzed by FACS for downregulation of surface expressed CDS and p2M.

[074] FIG. 36 is a series of graphs demonstrating reduced alloreactivity after KO of TCR and MHCI. Alloreactivities of WT or DKO (TCR and MHCI) CAR-T cells was analyzed by mixed lymphocyte reaction (MLR) and IFNy by ELISpot assay. On the left, WT or gene-edited DKO CAR-T cells were labeled with celltrace violet (CTV) and mixed at 1:1 ratio with irradiated peripheral blood mononuclear cells (PBMC)s and incubated for 12 days or 20 hr before analysis of proliferation or activation-induced secretion of IFNy by ELISpot assay, respectively. WT or DKO CAR-T cells were incubated with PBMCs from either allogenic (Donor #1 PBMC and Donor #2 PBMC) or autologous (Autologous PBMC) donors at 1:1 ratio. After 12 days, CTV dye dilution was assessed by FACS and results showed significant proliferation of WT CAR-T cells when incubated with allogeneic PBMCs; proliferative rates of 40% and 39% by WT CART cells was observed when cultured with allogeneic PBMCs from two different donors in comparison to only 2% when WT CAR-T cells were incubated with autologous PBMCs. On the other hand, DKO CAR-T cells did not proliferate when incubated with allogeneic PBMCs, demonstrating that KO of TCR and MHCI resulted in the elimination of graft-versus-host alloreactivity. This was also true in the short-term IFNy by ELISpot assay (lower left) which showed that only WT CAR-T cells became activated and secreted IFNy when incubated with allogeneic PBMCs, but not the DKO CAR-T cells. On the right, irradiated WT or DKO CAR-T cells were mixed at 1:1 ratio with PBMCs labeled with CFSE and incubated for 12 days or 20hr before analysis of proliferation or activation-induced secretion of IFNy by ELISpot assay, respectively. After 12 days, CFSE dye dilution was assessed by FACS and showed significant proliferation of PBMCs (most likely T cells) when incubated with allogeneic CAR-T cells; 37% and 9% of PBMCs proliferated in comparison to only 2% when incubated with autologous CART cells. On the other hand, PBMCs did not proliferate above background when incubated with allogeneic CAR-T cells, demonstrating that KO of TCR and MHCI resulted in the elimination of host-versus-graft al loreactivity. This was also true in the short-term IFNy by ELISpot assay (lower left) which showed that only WT CAR-T cells caused activation and secretion of IFNy by PBMCs when incubated with allogeneic CAR-Ts, not the DKO CAR-T cells.

[075] FIG. 37 is a series of graphs showing that DKO and WT CAR-Ts have similar CARexpression and stem-like phenotypes. Gene editing does not affect CAR-T cell phenotype. BCMA CAR-expressing TCRp / p2M DKO and WT T cells were analyzed for phenotype. CAR expression was comparable in WT and DKO. WT and DKO CAR-T cells were analyzed by FACS for expression of CD45RA and CD62L, markers for T stem cell memory (TSCM). These data demonstrate that gene editing of allo CAR-Ts does not significantly reduce the composition of memory CAR-T cells, retaining the exceptionally high and predominantly Tscm phenotype.

[076] FIG. 38 is a series of graphs showing that DKO CAR-Ts are highly functional. Gene editing does not affect CAR-T cell functionality. BCMA CAR-expressing TCRp / p2M DKO and WT T cells were analyzed for function. Proliferation against H929 (BCMA+) tumor lines was assessed by mixing CAR-T cells with H929 cells, incubated for 7 days, and analyzed for tumorspecific proliferation by FACS. Cytotoxicity and IFNg secretion against H929 (BCMA+) tumor lines was assessed by mixing CAR-T cells with H929 cells at various ratios, incubated for 24hrs and analyzed for tumor-specific killing by FACS. Cytotoxicity data are normalized to the tumor cell only sample. These data show7 that gene editing to produce DKO CAR-T cells does not significantly affect their functional capacity.

[077] FIG. 39A is a schematic diagram showing preclinical evaluation of the P-PSMA-101 transposon when delivered by a full-length plasmid (FLP) versus a nanotransposon (NT) at ‘stress’ doses using the Murine Xenograft Model. The murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSGmice was utilized to assess in vivo anti-tumor efficacy of the P-PSMA-101 transposon as delivered by a full-length plasmid (FLP) or a nanotransposon (NT) at two different ‘stress’ doses (2.5xlOA6 or 4xlOA6) of total CAR-T cells from two different normal donors. All CAR-T cells were produced using piggyBac^ (PB) deliver}'- of P-PSMA-101 transposon using either FLP or NT deliver}'. Mice were injected in the axilla with LNCaP and treated when tumors were established (100-200 mm3 by caliper measurement). Mice were treated with two different ‘stress’ doses (2.5xlOA6 or 4x10A6) of P-PSMA-101 CAR-Ts by IV injection for greater resolution in detecting possible functional differences in efficacy between transposon delivery? by the FLP and the NT.

[078] FIG. 39B are a series of graphs showing the tumor volume assessment of mice treated as described in FIG. 34A. Tumor volume assessment by caliper measurement for control mice (black), Donor #1 FLP mice (red), Donor #1 NT mice (blue), Donor #2 FLP mice (orange), and Donor #2 NT mice (green) as displayed as group averages with error bars (top) and individual mice (bottom). The y-axis shows the tumor volume (mm3) assessed by caliper measurement. The x-axis shows the number of days post T cell treatment. Delivered by NT, P-PSMA-101 transposon at a ‘stress’ dose demonstrated enhanced anti-tumor efficacy as measured by caliper in comparison to the FLP and control mice against established SC LNCaP.luc solid tumors. DETAILED DESCRIPTION OF THE INVENTION

[079] The present disclosure provides a non-naturally occurring chimeric stimulatory? receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[080] The activation component can comprise, consist essential of, or consist of one or more of a component of a human transmembrane receptor, a human cell-surface receptor, a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The activation component can comprise, consist essential of, or consist of: a portion of one or more of a component of a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor to which an agoni st of the activation component binds.

[081] The ectodomain can comprise, consist essential of, or consist of: a CD2 extracellular domain or a portion thereof to which an agonist binds or the ectodomain can comprise, consist 25 essential of, or consist of: a CD28 extracellular domain or a portion thereof to which an agonist binds. The activation component can comprise, consist essential of, or consist of: a CD2 extracellular domain or a portion thereof to which an agonist binds or the activation component can comprise, consist essential of, or consist of: a CD28 extracellular domain or a portion thereof to which an agonist binds. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17111. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17111. The CD2 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17111. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which an agonist binds comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17099.

[082] The signal transduction domain can comprise, consist essential of, or consist of: one or more of a component of a human signal transduction domain, T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR costimulatory protein, a component of a TCR. inhibitory protein, a cytokine receptor, or a chemokine receptor. The second protein can comprise, consist essential of, or consist of: a CD3 protein or a portion thereof. The signal transduction domain can comprise, consist essential of, or consist of a CDS protein or a portion thereof. The CDS protein can comprise, consist essential of, or consist of a CD3£ protein or a portion thereof. The CD3g protein comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3C protein comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3£ protein comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17102.

[083] The endodomain of a CSR of the present disclosure can further comprise, consist essential of, or consist of a cytoplasmic domain. The cytoplasmic domain can be isolated or derived from a third protein. In some aspects, the first protein and the third protein of a CSR of the present disclosure are identical. The cytoplasmic domain can comprise, consist essential of, or consist of: a CD2 cytoplasmic domain or a portion thereof or the cytoplasmic domain can comprise, consist essential of, or consist of: a CD28 cytoplasmic domain or a portion thereof.

[084] The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17113. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17101.

[085] The endodomain of a CSR of the present disclosure can further comprise, consist essential of, or consist of a signal peptide. The signal peptide can be isolated or derived from a fourth protein. In some aspects, the first protein and the fourth protein of a CSR of the present disclosure are identical. The signal peptide can comprise, consist essential of, or consist of: a CD2 signal peptide or a portion thereof; the signal peptide can comprise, consist essential of, or consist of: a CD28 signal peptide or a portion thereof or the signal peptide can comprise, consist essential of, or consist of: a CD8a signal peptide or a portion thereof. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17110. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17098. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17037.

[086] The transmembrane domain of a CSR of the present disclosure can be isolated or derived from a fifth protein. In some aspects, the first protein and the fifth protein of a CSR of the present disclosure are identical. The transmembrane domain can comprise, consist essential of, or consist of: a CD2 transmembrane domain or a portion thereof or the transmembrane domain can comprise, consist essential of, or consist of: a CD28 transmembrane domain or a portion thereof. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17112. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17100.

[087] In some aspects, the activation component of the CSR of the present disclosure does not bind or is incapable of binding a naturally-occurring molecule. In some aspects, the activation component of the CSR of the present disclosure binds or is capable of binding a naturally-occurring molecule and the CSR transduces a signal upon binding of the activation component to the naturally-occur! ng molecule. In other aspects, the activation component of the CSR of the present disclosure can bind a naturally-occurring molecule but the CSR does not transduce a signal upon binding of the activation component to a naturally-occurring molecule. In preferred aspects, the activation component of the CSR of the present disclosure binds or is capable of binding to a non-naturally occurring molecule. The activation component of the CSR of the present disclosure selectively transduces a signal upon binding of a non-naturally occurring molecule to the activation component. In one aspect, the naturally occurring molecule is an naturally occurring agonist / activating agent for the activation component of the CSR. The naturally occurring agonist / activating agent that can bind a CSR activation component can be any naturally occurring antibody or antibody fragment. The naturally occurring antibody or antibody fragment can be a naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the naturally occurring agonist / activating agent that can bind a CSR activation component can be one or more of an anti-human CDS monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In one aspect, the non-naturally occurring molecule is an non-naturally occurring agonist / activating agent for the activation component of the CSR. The non-naturally occurring agonist / activating agent that can bind a CSR activation component can be any non-naturally occurring antibody or antibody fragment. The non-naturally occurring antibody or antibody fragment can be a non-naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the non-naturally occurring agonist / activating agent that can bind a CSR activation component can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist / activating agent that can bind a CSR activation component can be selected from the group consisting of anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab)(Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009).

[088] In some aspects, the ectodomain of the CSR of the present disclosure can comprise a modification. The modification can comprise a mutation or a truncation in the amino acid sequence of the activation component or the first protein when compared to a wild type amino acid sequence of the activation component or the first protein. The mutation or a truncation in the amino acid sequence of the activation component or the first protein can comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. The mutation or truncation of the CD2 extracellular domain reduces or eliminates binding with naturally occurring CD58.

[089] A reduction in binding is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the binding ability of the mutated or truncated CD2 extracellular domain is reduced when compared to the naturally occurring wild-type counterpart. An elimination in binding is when 100% of the binding ability of the mutated or truncated CD2 extracellular domain is reduced when compared to the naturally occurring wild-type CD2 extracellular domain.

[090] The mutated or truncated CD2 extracellular domain binds anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind naturally occurring CD58. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17119 . The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain compri ses, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17118.

[091] The present disclosure also provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein and wherein the activation component binds to a non-naturally occurring molecule but does not bind a naturally-occurring molecule; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[092] The present disclosure also provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is i solated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein the CSR does not transduce a signal upon binding of a naturally-occurring molecule to the activation component.

[093] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein the CSR transduces a signal upon binding of a non-naturally-occurring molecule to the activation component.

[094] The present disclosure also provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3£ protein or a portion thereof.

[095] The present disclosure also provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activation component comprising the amino acid sequence of SEQ ID NO: 17111, (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an endodomain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signal transduction domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 80% identical to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulator}' receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 90% identical to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 95% identical to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulator}' receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 99% identical to SEQ ID NO: 17062. The non- naturally occurring chimeric stimulator}' receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence of SEQ ID NO: 17062.

[096] The present disclosure further provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a mutation or truncation of a wild-type CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3g protein or a portion thereof. In one aspect, the mutation or truncation of the CD2 extracellular domain reduces or eliminates binding with naturally occurring CD58. In another aspect, the mutated or truncated CD2 extracellular domain binds anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind naturally occurring CD58.

[097] The present disclosure further provides a non-naturally occurring chimeric stimulator}' receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and a activation component comprising the amino acid sequence of SEQ ID NO: 17119; (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an endodomain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signal transduction domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulator}' receptor (CSR) can comprise, consist essential of) or consist of an amino acid sequence at least 80% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulator}' receptor (CSR) can comprise, consist essential of, or consist of an amino acid sequence at least 90% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an acid sequence at least 95% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) can comprise, consist essential of, or consist of an acid sequence at least 99% identical to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulator} / receptor (CSR) can comprise, consist essential of, or consi st of an acid sequence of SEQ ID NO: 17118.

[098] The present disclosure also provides a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides transposon, a vector, a donor sequence or a donor plasmid comprising, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector.

[099] The present disclosure also provides a cell comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a cell comprising, consisting essential of or consisting of a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a cell comprising, consisting essential of or consisting of a transposon, a vector, a donor sequence or a donor plasmid compri sing, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector. A cell of the present disclosure comprising, consisting essential of or consisting of any chimeric stimulatory' receptor (CSR) disclosed herein can be an allogeneic cell or an autologous cell. In some preferred embodiments, the cell is an allogeneic cell.

[0100] The present disclosure also provides a composition comprising, consisting essential of or consisting of any chimeric stimulatory' receptor (CSR) disclosed herein. The present disclosure also provides a composition comprising, consisting essential of or consisting of a nucleic acid sequence encoding an amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides a composition comprising, consisting essential of or consisting of a transposon, a vector, a donor sequence or a donor plasmid comprising, consisting essential of or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulatory receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector. The present disclosure also provides a composition comprising, consisting essential of or consisting of a cell or a plurality of cells comprising, consisting essential of or consisting of any chimeric stimulatory receptor (CSR) disclosed herein.

[0101] The present disclosure provides a modified cell comprising, consisting essential of, or consisting of a chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0102] The present disclosure also provides a modified cell comprising, consisting essential of, or consisting of (a) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (b) an inducible proapoptotic polypeptide.

[0103] The present disclosure also provides a modified cell comprising, consisting essential of, or consisting of: (a) a chimeric stimulatory' receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; (b) a sequence encoding an inducible proapoptotic polypeptide, and wherein the cell is a T-cell, (c) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR.

[0104] The present disclosure provides a modified cell comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); and (b) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

[0105] The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and (b) chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0106] The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) chimeric stimulatory receptor (CSR.) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (c) a non-naturally occurring chimeric antigen receptor.

[0107] The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); and (c) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain, and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0108] The present disclosure provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein, (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (d) a non-naturally occurring chimeric antigen receptor.

[0109] The present disclosure also provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0110] The present disclosure also provides a modified T lymphocyte (T-cell) comprising, consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (e) a non-naturally occurring chimeric antigen receptor.

[0111] The present disclosure also provides a modified T lymphocyte (T-cell), consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; (b) a modification that reduces or eliminates a level of expression or activity of a HLA class I histocompatibility antigen, alpha chain A (HLA-A), HLA class I histocompatibility antigen, alpha chain B (HLA-B), HLA class I histocompatibility antigen, alpha chain C (HLA-C), or a combination thereof; and (c) a chimeric stimulatory' receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0112] The present disclosure also provides a modified T lymphocyte (T-cell), consisting essential of, or consisting of: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR, (b) a modification that reduces or eliminates a level of expression or activity of a HLA class I histocompatibility antigen, alpha chain A (HLA-A), HLA class I histocompatibility antigen, alpha chain B (HLA-B), HLA class I histocompatibility antigen, alpha chain C (HLA-C), or a combination thereof; (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein, (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0113] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of an inducible proapoptotic polypeptide. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible proapoptotic polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 14641.

[0114] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). A reduction of a level of expression or activity is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the MHC-I in a cell or the functional activity of the MHC-I in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell. A reduction of a level of expression or activity is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the MHC-I in a T-cell or the functional activity of the MHC-I in a T-cell is reduced when compared to a naturally occurring wild-type T-cell. An elimination a level of expression or activity is when 100% of the expression of the MHC-I in a cell or the functional activity of the MHC-I in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell. An elimination a level of expression or activity is when 100% of the expression of the MHC-I in a T-cell or the functional activity of the MHC-I in a T-cell is reduced when compared to the naturally occurring wild-type T-cell.

[0115] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E). The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 1713 1.

[0116] The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a B2M signal peptide. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17126. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The B2M signal peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17131.

[0117] The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a B2M polypeptide. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17129.

[0118] The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a linker molecule (referred to herein as a linker). The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17130.

[0119] The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a peptide and a B2M polypeptide. The peptide comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17127.

[0120] The non-naturally occurring polypeptide comprising a HLA-E can further comprise, consist essential of, or consist of a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the HLA-E polypeptide. The first linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17128. The second linker comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17130.

[0121] In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consi sts essential of, or consists of a B2M signal peptide, a peptide, a first linker, a B2M polypeptide, a second linker and an HLA-E polypeptide. The peptide can be positioned between the B2M signal peptide and the first linker, the B2M polypeptide can be positioned between the first linker and the second linker and the second linker can be positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17065.

[0122] In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consi sts of a B2M signal peptide, a B2M polypeptide, a linker and an HLA-E polypeptide. The B2M polypeptide can be positioned between the B2M signal peptide and the linker, the linker can be positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17067.

[0123] In one aspect, the non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of a B2M signal peptide and an HLA-E polypeptide. The B2M signal peptide can be positioned before (e.g. 5’ in the context of a nucleic acid sequence or amino terminus in the context of an amino acid sequence) HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA-E comprises, consists essential of, or consists of the amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA- E comprises, consists essential of, or consists of the amino acid sequence of SEQ ID NO: 17068. The non-naturally occurring polypeptide comprising an HLA-E can be encoded by the nucleic acid have the sequence of SEQ ID NO: 17069.

[0124] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of, or consist of a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In a preferred aspect, the non-naturally occurring antigen receptor comprises, consists essential of or consists of a chimeric antigen receptor (CAR). The CAR comprise, consist essential of, or consist of (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. The ectodomain of the CAR can further comprise, consist essential of, or consist of a signal peptide. The ectodomain of the CAR can further comprise, consist essential of, or consist of a hinge between the antigen recognition region and the transmembrane domain. The endodomain of the CAR can further comprise, consist essential of, or consist of a human CD3g endodomain. The at least one costimulatory domain of the CAR can further comprise, consist essential of, or consist of a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In a preferred aspect, at least one costimulatory domain comprises a human CD28 and / or a 4-IBB costimulatory domain.

[0125] A modified cell of the present disclosure can be an immune cell or an immune cell precursor. The immune cell can be a lymphoid progenitor cell, a natural killer (NK) cell, a cytokine induced killer (CIK) cell, a T lymphocyte (T-cell), a B lymphocyte (B-cell) or an antigen presenting cell (APC). In preferred aspects, the immune cell is a T cell, an early memory’ T cell, a stem cel I-like T cell, a stem memory T cell (Tscm), a central memory T cell (Tcm) or a stem cell-like T cell. The immune cell precursor can a hematopoietic stem cell (HSC). The modified cell can be a stem cell, a differentiated cell, a somatic cell or an antigen presenting cell (APC). The modified cell can be an autologous cell or an allogeneic cell. In one aspect, the cell is a modified allogeneic T-cell. In another aspect, the cell is modified allogeneic T-cell expressing a chimeric antigen receptor (CAR), a CAR T-cell.

[0126] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a CSR of the present disclosure transiently or stably. In one aspect, a CSR of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In one aspect, a CSR of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0127] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure transiently or stably. In one aspect, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In one aspect, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed in a modified cell of the present di scl osure (preferably a modified T-cell of the present disclosure).

[0128] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express an induci ble proapoptotic poly peptide of the present disclosure transiently or stably. In one aspect, an inducible proapoptotic polypeptide of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In a preferred aspect, an inducible proapoptotic polypeptide of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0129] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can express a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure transiently or stably. In one aspect, a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure). In a preferred aspect, a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein of the present disclosure is stably expressed in a modifi ed cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0130] In one aspect, a CSR of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0131] In one aspect, a CSR of the present disclosure is stably expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0132] In one aspect, a CSR of the present disclosure is stably expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0133] In one aspect, a CSR of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and the non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0134] In one aspect, a CSR of the present disclosure is transiently expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0135] In one aspect, a CSR of the present disclosure is transiently expressed, a non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed, the inducible proapoptotic polypeptide of the present disclosure is stably expressed and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present di scl osure (preferably a modified T-cell of the present disclosure).

[0136] The present disclosure provides a modified cell (preferably a modified T-cell comprising, consisting essential of, or consisting of (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or acti vity of the TCR; and (b) a sequence encoding a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0137] The modified cell further can further comprise, consist essential of or consist of a sequence encoding an inducible proapoptotic polypeptide. The modified cell can further comprise, consist essential of or consist of a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor can comprise, consist essential of or consist of a chimeric antigen receptor (CAR).

[0138] A transposon, a vector, a donor sequence or a donor plasmid can comprise, consist essential of or consist of the sequence encoding the CSR, the sequence encoding the inducible proapoptotic polypeptide, or a combination thereof. The transposon, the vector, the donor sequence or the donor plasmid can further comprise, consist essential of or consist of a sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein. The transposon, the vector, the donor sequence, or the donor plasmid can further comprise, consist essential of or consist of a sequence encoding a selection marker. The transposon can be a piggyBac^ transposon, a piggy-Bac1® like transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon or a TcBuster transposon. The sequence encoding the CSR can be transiently expressed in the cell. The sequence encoding the CSR can be stably expressed in the cell. The sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. The sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In some aspects, the sequence encoding the CSR can be transiently expressed in the cell and the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. In some aspects, the sequence encoding the CSR can be stably expressed in the cell and the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell. In some aspects, the sequence encoding the CSR can be transiently expressed in the cell, the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell and sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In some aspects, the sequence encoding the CSR can be stably-expressed in the cell, the sequence encoding the inducible proapoptotic polypeptide can be stably expressed in the cell and sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the cell. In one aspect, the vector can be a viral vector. In one aspect, a viral vector can be an an adenoviral vector, adeno-associated viral (AAV) vector, retroviral vector, lentiviral vector or a chimeric viral vector.

[0139] A first transposon, a first vector, a first donor sequence, or a first donor plasmid can comprise, consist essential of or consist of the sequence encoding the CSR. The first transposon, the first vector, the first donor sequence, or the first donor plasmid can further comprise, consist essential of or consist of a sequence encoding a first selection marker.

[0140] A second transposon, a second vector, a second donor sequence, or a second donor plasmid can comprise, consist essential of or consist of one or more of the sequence encoding the inducible proapoptotic polypeptide, the sequence encoding a non-naturally occurring antigen receptor, and the sequence encoding a therapeutic protein. The second transposon, the second vector, the second donor sequence, or the second donor plasmid can further comprise, consistessential of or consist of a sequence encoding a second selection marker. The first selection marker and the second selection marker are identical. The first selection marker and the second selection marker are not identical. The selection marker can comprise, consist essential of or consist of a cell surface marker. The selection marker can comprise, consist essential of or consist of a protein that is active in dividing cells and not active in non-dividing cells. The selection marker can comprise, consist essential of or consist of a metabolic marker.

[0141] In one aspect, the selection marker can comprise, consist essential of or consist of a dihydrofolate reductase (DHFR) mutein enzyme. The DHFR mutein enzyme can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17012.

[0142] The DHFR mutein enzyme of SEQ ID NO: 17012 can further comprise, consist essential of or consist of a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme of SEQ ID NO: 17012 can further comprise, consist essential of or consist of one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80; a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

[0143] A modified cell of the present disclosure (preferably a modified T-cell of the present disclosure) can further comprise, consist essential of or consist of a gene editing composition. The gene editing composition can comprise, consist essential of or consist of a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The gene editing composition can be expressed transiently by the modified cell. The gene editing composition can be expressed stably by the modified cell.

[0144] The gene editing composition can comprise, consist essential of or consist of a sequence encoding a nuclease protein or a sequence encoding a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise, consist essential of or consist of a DNA sequence, an RNA sequence, or a combination thereof The nuclease or the nuclease domain thereof can comprise, consist essential of or consist of one or more of a CRISPR / Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. The CRISPR / Cas protein can comprise, consist essential of or consist of a nuclease-inactivated Cas (dCas) protein. The nuclease or the nuclease domain thereof can comprise, consist essential of or consist of a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise, consist essential of or consist of a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise, consist essential of or consist of a fusion protein. The fusion protein can comprise, consist essential of or consist of a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The fusion protein can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17013. The fusion protein is encoded by a nucleic acid comprising, consisting essential of or consisting of the sequence of SEQ ID NO: 17014. The fusion protein can comprise, consist essential of or consist of the amino acid sequence of SEQ ID NO: 17058. The fusion protein is encoded by a nucleic acid comprising, consisting essential of or consisting of the sequence of SEQ ID NO: 17059.

[0145] The gene editing composition can further comprise, consist essential of or consist of a guide sequence. The guide sequence can compri se, consist essential of or consist of an RNA sequence. In aspects when the modified cell is a T-cell, the guide RNA can comprise, consist essential of or consist of a sequence complementary to a target sequence encoding an endogenous TCR. The guide RNA can comprise, consist essential of or consist of a sequence complementary to a target sequence encoding a B2M polypeptide. The guide RNA can compri se, consist essential of or consist of a sequence complementary to a target sequence within a safe harbor site of a genomic DNA sequence.

[0146] The transposon, the vector, the donor sequence or the donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

[0147] The first transposon, the first vector, the first donor sequence or the first donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

[0148] The second transposon, the second vector, the second donor sequence or the second donor plasmid can further comprise, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

[0149] A third transposon, a third vector, a third donor sequence or a third donor plasmid can compri se, consist essential of or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

[0150] The Clo051 nuclease or a nuclease domain thereof can induce a single or double strand break in a target sequence. The donor sequence or a donor plasmid can integrate at a position of single or double strand break or at a position of cellular repair within a target sequence, or a combination thereof.

[0151] The present disclosure provides a composition comprising, consisting essential of, or consisting of a modified cell of the present disclosure (preferably a modified T-cell of the present disclosure).

[0152] The present disclosure provides a plurality of modified cells comprising any non-naturally occurring chimeric stimulatory' receptor (CSR) disclosed herein and provides a plurality' of modified cells comprising any modified cell disclosed herein. The plurality' of modified cells can comprise, consist essential of, or consist of immune cells or an immune cell precursors. The plurality of immune cells can comprise, consist essential of, or consist of lymphoid progenitor cells, natural killer (NK) cells, cytokine induced killer (CIK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells) or antigen presenting cells (APCs).

[0153] The present disclosure provides a composition comprising a population of modified cells, wherein a plurality of the modified cells of the population comprise any non-naturally occurring chimeric stimulatory' receptor (CSR) disclosed herein and provides a composition compri sing a population of modified cells, wherein a plurality of the modified cells of the population comprise any modified cell disclosed herein. The population of modified cells can comprise, consist essential of, or consist of immune cells or an immune cell precursors. The population of immune cells can comprise, consist essential of, or consist of lymphoid progenitor cells, natural killer (NK) cells, cytokine induced killer (CIK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells) or antigen presenting cells (APCs). The composition can comprise a pharmaceutically-acceptable carrier.

[0154] The present disclosure provides a composition comprising a population of modified T lymphocytes (T-cells), wherein a plurality of the modified T-cells of the population comprise any non-naturally occurring chimeric stimulatory' receptor (CSR) disclosed herein and provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise any modified T-cell disclosed herein. The composition can comprise a pharmaceutically-acceptable carrier.

[0155] Preferably, the present disclosure provides a composition comprising a population of T lymphocytes (T-cells), wherein a plurality of the T-cells of the population comprise a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essential of, or consisting of: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical. The composition can comprise a pharmaceutically-acceptable carrier. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the CSR.

[0156] The plurality of the T-cells of the population can further comprise an inducible proapoptotic polypeptide. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the inducible proapoptotic polypeptide.

[0157] The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the modification of the endogenous sequence encoding the TCR, wherein the modifi cation reduces or eliminates a level of expression or activity of the TCR.

[0158] The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the modification of the endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-I.

[0159] The plurality of the T-cells of the population can further comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR and a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

[0160] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise both modification of the endogenous sequence encoding the TCR, wherein the modification reduces or eliminates a level of expression or activity of the TCR and the modification of the endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-I.

[0161] The plurality of the T-cells of the population can further comprise a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the non-naturally occurring sequence comprising the HLA-E polypeptide.

[0162] The plurality of the T-cells of the population can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprise the non-naturally occurring antigen receptor, the sequence encoding a therapeutic polypeptide, or a combination thereof. In preferred aspects, the non-naturally occurring antigen receptor is a chimeric antigen receptor (CAR).

[0163] The plurality of the T-cells of the population can comprise an early memory T cell, a stem cell-like T cell, a stem memory T cell (Tscm), a central memory T cell (Tcm) or a stem celllike T cell. In some aspects, one or more of a stem cell-like T cell, a stem cell memory T cell (Tscm) and a central memory T cell (Tcm) comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%s, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population of modified T-cells.

[0164] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR expresses one or more cell-surface marker(s) of a stem memory T cell (Tscm) or a Tscm-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L.

[0165] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more cell-surface marker(s) of a central memory' T cell (Tcm) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L.

[0166] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population expresses one or more of CD127, CD45RO, CD95 and IL-2Rp cell-surface marker(s).

[0167] The present disclosure provides compositions for use in the treatment of a disease or disorder disclosed herein or the use of a composition for the treatment of any disease or disorder disclosed herein. The present disclosure also provides methods of treating a disease or disorder comprising, consisting essential of, or consisting of administering to a subject in need thereof a therapeutically-effective amount of a composition disclosed herein. The compositions can comprise, consist essential of or consist of any of the modified cells or populations of modified cells disclosed herein. Preferably, any of the modified T-cells or CAR T-cells disclosed herein .

[0168] The present disclosure provides a method of producing a modified T-cell comprising, consisting essential of, or consisting of, introducing into a primary human T-cell a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a modified T-cell under conditions that stably express the CSR within the modified T-cell and preserve desirable stem-like properties of the modified T-cell. The primary human T-cell can be a resting primary human T-cell. The present disclosure provides a modified T-cell produced by the disclosed method. The present disclosure provides a method of administering the modified T-cell comprising the stably expressed CSR produced by the disclosed method. The present disclosure provides the method of administering the modified T-cell comprising the stably expressed CSR produced by the disclosed method to treat a disease or disorder.

[0169] The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality' of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The primary’ human T-cells can comprise resting primary human T-cells. The present disclosure provides a population of modified T-cells produced by the disclosed method. The present disclosure provides a method of administering the population of modified T-cells comprising the stably expressed CSR produced by the disclosed method. The present disclosure provides a method of administering the population of modified T-cells comprising the stably expressed CSR produced by the disclosed method to treat a disease or disorder.

[0170] The present disclosure provides a method of producing a modified T-cell comprising, consisting essential of, or consisting of, introducing into a primary human T-cell a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a modified T-cell under conditions that transiently express the CSR. within the modified T-cell and preserve desirable stem-like properties of the modified T-cell. The primary human T-cell can be a resting primary' human T-cell. The present disclosure provides a modified T-cell produced by the disclosed method. The present disclosure provides a method of administering the modified T-cell comprising the transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method of administering the modified T-cell produced by the disclosed method after the modified T-cell no longer expresses the CSR. The present disclosure provides a method of administering a modified T-cell comprising the transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one aspect, the present disclosure provides a method of administering the modified T-cell produced by the disclosed method after the modified T-cell no longer expresses the CSR to treat a disease or disorder.

[0171] The present disclosure provides a method of producing a population of modified T-cells comprising, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality of modified T-cells under conditions that transiently express the CSR within the plurality’ of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells. The primary human T-cells can comprise resting primary human T-cells. The present disclosure provides a population of modified T-cell produced by the disclosed method. The present disclosure provides a method of administering the population of modified T-cells comprising the transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method of administering the population of modified T-cells produced by the disclosed method after the plurality of T-cells no longer express the CSR. The present disclosure provides a method of administering the population of modified T-cells comprising the transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one aspect, the present disclosure provides a method of administering the population of modified T-cells produced by the disclosed method after the plurality of modified T-cells no longer express the CSR to treat a disease or disorder.

[0172] The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR. The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-1). In some aspects, the method of producing a modified T-cell or producing a population of modified T-cells can further comprising introducing both a modification of an endogenous sequence encoding TCR, wherein the modification reduces or eliminates a level of expression or activity of the TCR and introducing a modification of an endogenous sequence encoding B2M, wherein the modification reduces or eliminates a level of expression or activity of MHC-1.

[0173] The method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing into the primary7 human T-cell or plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same. In one aspect, the antigen receptor is a non-naturally occurring antigen receptor. In a preferred aspect, the method of producing a modified T-cell or producing a population of modified T-cells can further comprise introducing into the primary human T-cell or plurality of primary7 human T cells a composition comprising a Chimeric Antigen Receptor (CAR) or a sequence encoding the same. The method can further comprise introducing into the primary human T-cell or plurality of primary7 human T cells a composition comprising an inducible proapoptotic polypeptide or a sequence encoding the same. The method of producing a modifi ed T-cell or producing a population of modified T-cells can further comprise introducing into the primary7 human T-cell or plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding the same and a composition comprising an inducible proapoptotic polypeptide or a sequence encoding the same.

[0174] The method of producing a modified T-cell or producing a population of modified T-cells can further comprise contacting the modified T-cell or population of modifi ed T-cells with an activator composition. The activator composition can comprise, consist essential of, or consist of one or more agonists or activating agents that can bind a CSR activation component of the modified T-cell or plurality of modified T-cells. The agonist / activating agent can be naturally occurring or non-naturally occurring. In preferred aspects, the agonist / activating agent is an antibody or antibody fragment. The agonist / activating agent can be one or more of an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist / activating agent that can be one or more of an anti-human CD3 monospecific tetrameric antibody complex., an anti-human CD2 monospecific tetrameric antibody complex, an antihuman CD28 monospecific tetrameric antibody complex, or a combination thereof. The agonist / activating can contact the modified T-cell or population of modified T-cells in vitro, ex vivo or in vivo. In a preferred aspect, the agonist / activating activates the modified T-cell or population of modified T-cells, induces cell division in the modified T-cell or population of modified T-cells, increases cell division (e.g., cell doubling time) in the modified T-cell or population of modified T-cells, increases fold expansion in the modified T-cell or population of modified T-cells, or any combination thereof.

[0175] The present disclosure provides a method of expanding a population of modified T-cells comprising, consisting essenti al of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality'- of modified T-cells under conditions that stably express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not stably expressing a CSR of the present disclosure under the same conditions. The method wherein the expansion of the plurality of modified T-cells is at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold or at least 10 fold higher than the expansion of a plurality of wild-type T-cells not stably expressing a CSR of the present disclosure under the same conditions.

[0176] The present disclosure provides a method of expanding a population of modified T-cells compri sing, consisting essential of, or consisting of, introducing into a plurality of primary human T-cells a composition comprising a Chimeric Stimulator Receptor (CSR) of the present disclosure or a sequence encoding the same to produce a plurality' of modified T-cells under conditions that transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modifi ed T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing a CSR of the present disclosure under the same conditions. The method wherein the expansion of the plurality of modified T-cells is at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least eight fold, at least nine fold or at least 10 fold higher than the expansion of a plurality of wild-type T-cells not transiently expressing a CSR of the present disclosure under the same conditions.

[0177] The activator composition of the methods of expanding a population of can comprise, consist essential of, or consist of one or more agonists or activating agents that can bind a CSR activation component of the modified T-cell or plurality of modified T-cells. The agonist / activating agent can be naturally occurring or non-naturally occurring. In preferred aspects, the agonist / activating agent is an antibody or antibody fragment. The agonist / activating agent can be one or more of an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist / activating agent that can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof.

[0178] The conditions can comprise culturing the modifi ed T-cell or plurality of modifi ed T-cells in a media comprising a sterol; an alkane; phosphorus and one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid. The culturing can be in vivo or ex vivo. The modified T-cell can be an allogeneic T-cell or the plurality of modified T-cells can be allogeneic T-cells. The modified T-cell can be an autologous T-cell or the plurality of modified T-cells can be autologous T-cells.

[0179] In some aspects, the media can comprise one or more of octanoic acid at a concentration of between 0.9 mg / kg to 90 mg / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints, and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive of the endpoints.

[0180] In some aspects, the media can comprise one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg and a sterol at a concentration of about 1 mg / kg.

[0181] In some aspects, the media can comprise one or more of octanoic acid at a concentration of between 6.4 pmol / kg and 640 pmol / kg, inclusive of the endpoints, palmitic acid at a concentration of between 0.7 umol / kg and 70 pmol / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 umol / kg and 75 pmol / kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol / kg and 25 umol / kg, inclusive of the endpoints.

[0182] In some aspects, the media can comprise one or more of octanoic acid at a concentration of about 64 umol / kg, palmitic acid at a concentration of about 7 pmol / kg, linoleic acid at a concentration of about 7.5 umol / kg, oleic acid at a concentration of about 7.5 pmol / kg and a sterol at a concentration of about 2.5 pmol / kg.

[0183] The present disclosure provides compositions comprising any modified T-cell produced by a method dislosed herein. The present disclosure provides compositions comprising any population of modified T-cell produced by a method dislosed herein. The present disclosure provides compositions comprising any modified T-cell expanded by a method dislosed herein. The present disclosure provides compositions comprising any population of modified T-cell expanded by a method dislosed herein.

[0184] The present disclosure provides compositions for use in the treatment of a disease or disorder disclosed herein or the use of a composition for the treatment of any disease or disorder disclosed herein. The present disclosure also provides methods of treating a disease or disorder comprising, consisting essential of, or consisting of administering to a subject in need thereof a therapeutically-effective amount of a composition disclosed herein and at least one non-naturally occurring molecule which binds to the activation component of a CSR disclosed herein. The compositions can comprise, consist essential of or consist of any of the modified cells or populations of modified cells disclosed herein. Preferably, any of the modified T-cells or CAR T-cells disclosed herein. Any non-naturally occurring molecule capable of binding to the activation component of the CSR of the present disclosure and selectively transducing a signal upon binding can be administered. Preferably, the non-naturally occurring molecule is an non-naturally CSR agonist / activating agent for the activation component. The non-naturally occurring agonist / activating agent that can bind a CSR activation component can be any non-naturally occurring antibody or antibody fragment. The non-naturally occurring antibody or antibody fragment can be a non-naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the non-naturally occurring agonist / activating agent that can bind a CSR activation component can be one or more of an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist / activating agent that can bind an activation component can be selected from the group consisting of anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab)(Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009). In some aspects, administration of non-naturally occurring molecule capable of binding to the activation component of the CSR stimulates cell division of the modified cells in vivo. Thus, the present disclosure provides a method of stimulating cell division of a modified cell of the present disclosure in vivo by administering a non-naturally CSR agonist / activating agent for the activation component to a subject harboring the modified cell of the present disclosure.

[0185] In some aspects, the disease or disorder is a cell proliferation disease or disorder. In some aspects, the cell proliferation disease or disorder is cancer. The cancer can be a solid tumor cancer or a hematologic cancer. In some aspects, the solid tumor is prostate cancer or breast cancer. In preferred aspects, the prostate cancer is castrate-resistant prostate cancer. In some aspects, the hematologic cancer is multiple myeloma.

[0186] The modified cells or population of modified cells comprised within the disclosed compositions can be cultured in vitro or ex vivo prior to administration to a subject in need thereof. The modified cells can be allogenic modified cells or autologous modified cells. In some aspects, the cells are allogeneic modified T-cells or autologous modified T-cells. In some aspects, the cells are allogeneic modified CAR T-cells or autologous modified CAR T-cells. In some aspects, the cells are allogeneic modified CAR T-cells comprising a CSR of the present disclosure or autologous modified CAR T-cells comprising a CSR of the present disclosure.

[0187] The modified cell compositions or the compositions comprising populations of modified cells can be administered to the patient by any means known in the art. In some aspects, the composition is administered by systemic administration. In some aspects, the composition is administered by intravenous administration. The intravenous administration can be in an intravenous injection or an intravenous infusion. In some aspects, the composition is administered by local administration. In some aspects, the composition is administered by an intraspinal, intracerebroventricular, intraocular or intraosseous injection or infusion.

[0188] The therapeutically effective amount can be a single dose or multiple doses of modified cell compositions or the compositions comprising populations of modified cells. In some aspects, the therapeutically effective dose is a single dose and wherein the allogeneic cells of the composition engraft and / or persist for a sufficient time to treat the disease or disorder. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured simultaneously

[0189] In some aspects, the uses and methods for the treatment of a disease or disorder further provide that subjects do not develop graft v host (GvH) disease, host v graft (HvG) disease, or a combination thereof, following administration of modified cell compositions disclosed herein or the compositions comprising populations of modified cells disclosed herein.

[0190] Allogeneic cells of the disclosure are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell.

[0191] In some embodiments of the composition and methods of the disclosure, allogeneic cells are stem cells. In some embodiments, allogeneic cells are derived from stem cells. Exemplary stem cells include, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), multipotent stem cells, pluripotent stem cells, and hematopoetic stem cells (HSCs).

[0192] In some embodiments of the composition and methods of the disclosure, allogeneic cells are differentiated somatic cells.

[0193] In some embodiments of the composition and methods of the disclosure, allogeneic cells are immune cells. In some embodiments, allogeneic cells are T lymphocytes (T cells). In some embodiments, allogeneic cells are T cells that do not express one or more components of a naturally-occurring T-cell Receptor (TCR). In some embodiments, allogeneic cells are T cells that express a non-naturally occurring antigen receptor. Alternatively, or in addition, in some embodiments, allogeneic cells are T cells that express a non-naturally occurring Chimeric Stimulatory Receptor (CSR). In some embodiments, the non-naturally occurring CSR comprises or consists of a switch receptor. In some embodiments, the switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain of the switch receptor binds to a TCR co-stimulatory molecule and transduces a signal to the intracellular space of the allogeneic cell that recapitulates TCR signaling or TCR co-stimulatory signaling. Chimeric Stimulatory Receptors (CSRs)

[0194] Adoptive cell compositions that are “universally” safe for administration to any patient requires a significant reduction or elimination of alloreactivity.

[0195] Towards this end, allogeneic cells of the disclosure are modified to interrupt expression or function of a T-cell Receptor (TCR) and / or a class of Major Histocompatibility Complex (MHC). The TCR mediates graft vs host (GvH) reactions whereas the MHC mediates host vs graft (HvG) reactions. In preferred embodiments, any expression and / or function of the TCR is eliminated in allogeneic cells of the disclosure to prevent T-cell mediated GvH that could cause death to the subject. Thus, in particularly preferred embodiments, the disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g. each cell of the composition expresses at a level so low as to either be undetectable or non-existent).

[0196] In preferred embodiments, expression and / or function of MHC class I (MHC-I, specifically, HLA-A, HLA-B, and HLA-C) is reduced or eliminated in allogeneic cells of the disclosure to prevent HvG and, consequently, to improve engraftment of allogeneic cells of the disclosure in a subject. Improved engraftment of the allogeneic cells of the disclosure results in longer persistence of the cells, and, therefore, a larger therapeutic window for the subject. Specifically, in the allogeneic cells of the disclosure, expression and / or function of a structural element of MHC-I, Beta-2-Microglobulin (B2M), is reduced or eliminated in allogeneic cells of the disclosure.

[0197] The above strategies for generating an allogeneic cell of the disclosure induce further challenges. T Cell Receptor (TCR) knockout (KO) in T cells results in loss of expression of CD3-zeta (CD3z or CD3Q, which is part of the TCR complex. The loss of CD3c in TCR-KO T-cells dramatically reduces the ability of optimally activating and expanding these cells using standard stimulation / activation reagents, including, but not limited to, agonist anti-CD3 mAb. When the expression or function of any one component of the TCR complex is interrupted, all components of the complex are lost, including TCR-alpha (TCRa), TCR-beta (TCRP), CD3-gamma (CD3y), CD3-epsilon (CD3s), CD3-delta (CD35), and CD3-zeta (CD3Q. Both CD3e and CD3l are required for T cell activation and expansion. Agonist anti-CD3 mAbs typically recognize CD3s and possibly another protein within the complex which, in turn, signals to CD3g. CD3C provides the primary stimulus for T cell activation (along with a secondary' costimulatory7 signal) for optimal activation and expansion. Under normal conditions, full T-cell activation depends on the engagement of the TCR in conjunction with a second signal mediated by one or more co-stimulatory receptors (e.g. CD28, CD2, 4-1BBL, etc...) that boost the immune response. However, when the TCR is not present, T cell expansion is severely reduced when stimulated using standard activation / stimulation reagents, including agonist anti-CD3 mAb. In fact, T cell expansion is reduced to only 20-40% of the normal level of expansion when stimulated using standard activation / stimulation reagents, including agonist anti-CD3 mAb.

[0198] The disclosure provides a Chimeric Stimulatory Receptor (CSR) to deliver CD3z primary' stimulation to allogeneic T cells in the absence of an endogenous TCR (and, consequently, an endogenous CD3Q when stimulated using standard activation / stimulation reagents, including agonist anti-CD3 mAh.

[0199] In the absence of an endogenous TCR, Chimeric Stimulatory' Receptors (CSRs) of the disclosure provide a CD3£ stimulus to enhance activation and expansion of allogeneic T cells. In other words, in the absence of an endogenous TCR, Chimeric Stimulatory Receptors (CSRs) of the disclosure rescue the allogeneic cell from an activation-based disadvantage when compared to non-allogeneic T-cells that express an endogenous TCR. In some embodiments, CSRs of the disclosure comprise an agonist mAb epitope extracellularly and a CD3C stimulatory domain intracellularly and, functionally, convert, an anti-CD28 or anti-CD2 binding event on the surface into a CD3z signaling event in an allogeneic T cell modified to express the CSR. In some embodiments, a CSR comprises a wild type CD28 or CD2 protein and a CD3z intracellular stimulation domain, to produce CD28z CSR and CD2z CSR, respectively. In preferred embodiments, CD28z CSR and / or CD2z CSR further express a non-naturally occurring antigen receptor and / or a therapeutic protein. In preferred embodiments, the non-naturally occurring antigen receptor comprises a Chimeric Antigen Receptor.

[0200] The data provided herein demonstrate that modified allogeneic T cells of the disclosure comprising / expressing a CSR of the disclosure improve or rescue, the expansion of allogeneic T cells that no longer express endogenous TCR when compared to those cells that do not comprise / express a CSR of the disclosure.

[0201] A wildtype / natural human CD28 protein (NCBI: CD28 HUMAN; UniProt / Swiss-Prot: P10747.1) comprises or consists of the amino acid sequence of: MLRLLLALNLFPSIQVTGNKTLVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVE VCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKS NGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFUFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 17096)

[0202] A nucleotide sequence encoding wildtype / natural CD28 protein (NCBI: CCDS2361.1) comprises or consists of the nucleotide sequence of: AT GC T CAGGC T GC T C T T GGC T C TCAAC T TAT T CCC T TCAAT T CAAGTAACAGGAAACAAGAT T T TGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTC CTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAA GTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACT GTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAAC AGAT AT TTACTTCTG CAAAAT T GAAG T TAT G TAT C C T C C T C C T TAG C T AGAC AAT GAGAAGAG C AATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTT CTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG ACTTCGCAGCCTATCGCTCCTGA (SEQ ID NO: 17097)

[0203] An exemplaty CSR CD28z protein of the disclosure comprises or consists of the amino acid sequence of (CD28 Signal peptide, CD28 Extracellular Domain, CD28 Transmembrane domain, CD28 Cytoplasmic Domain, CD3z Intracellular Domain): MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVE VCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKS NGTlIHVKGKHLCPSPLFPGPSKPEWFLVEVGAFLACYSLAEPTVAFTI^^ NMTPRRPGPTRKHZQFYAPFKDFAaYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 17060) CD28 Signal peptide: MLRLLLALNLFPSIQVTG (SEQ ID NO: 17098) CD28 Extracellular Domain'. NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCWYGNYSQQLQVYSKT GFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF PGPSKP (SEQ ID NO: 17099) CD28 Transmembrane domain: FWVLVWGGVLACYSLLVTVAFIIFWV (SEO ID NO: 17100) CD28 Cytoplasmic Domain'. RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEO ID NO: 17101) CD3z Intracellular Domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17102)

[0204] An exemplary nucleotide sequence encoding a CSR CD28z protein of the disclosure comprises or consists of the nucleotide sequence of (CD28 Signal peptide, CD 2 8 Extracellular Domain, CD28 Transmembrane domain. CD28 Cytoplasmic Domain, CD3z Intracellular Domain): ATGC TGAGAC TGC TGC TGGCCC TGAATC TGT TCCCCAGCATCCAAGTGACCGGCAACAAGATCC TGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACAACGCCGTGAACCTGAGCTGCAAGTACAG CTACAACCTGTTCAGCAGAGAGTTCCGGGCCAGCCTGCACAAAGGACTGGATTCTGCTGTGGAA GTGTGCGTGGTGTACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACCGGCTTCAACT GCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACCTGTACGTGAACCAGAC CGACATCTATTTCTGCAAGATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCC AACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTA GCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTAC AGT GGCC T T CAT CAT C T T TT GGG T C CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATG AACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAG ACTTCGCCGCCTACCGGTCCAGAG T G AAG T T C T C C AGAT C C G C C GAT G C T C C C G C C TAT AAG C A GGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATGTGCTGGAC AAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCC TGTACAA.TGAA.CTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGA G C G CAGAAGAGGCAAGG GAC AC GA T GGAC T G TAC CAG GGC C T GAG C AC C GC C AC CAAGGATAC C TATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 17061) CD28 Signal peptide: ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGACCGGC (SEQ ID NO: 17103) CD28 Extracellular Domain'. AACAAGATCCTGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACAACGCCGTGAACCTGAGCT GCAAGTACAGCTACAACCTGTTCAGCAGAGAGTTCCGGGCCAGCCTGCACAAAGGACTGGATTC TGCTGTGGAAGTGTGCGTGGTGTACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACC GGCTTCAACTGCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACCTGTACG T GAAC C AGAC C GAC AT C T AT T T C T G C AAGAT C GAAG T GAT G T AC C C G C C T C C T T AC C T G GAC AA C G AGAAG T C CAAG G G C AC CAT C AT C C AC G T GAAG G G CAAG C AC C T G T G T C C T T C T C GAC T G T T C CCCGGACCTAGCAAGCCT (SEQ ID NO: 17104) CD28 Transmembrane domain: TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACAGTGGCCT TCATCATCTTTTGGGTC (SEQ ID NO: 17105) CD28 Cytoplasmic Domain". CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGAC CAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCC (SEO ID NO: 17106) CD3z Intracellular Domain: AGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAGAACCAGCTGTACA ACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCC TGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTACAATGAACTGCAGAAA GACAAG AT G G C C GAG G C C TAG AG C GAG AT C G GAA T GAAG G G C GAG C G C AG AAGAG G CAAG G G AC ACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCA GGCCCTGCCTCCAAGA (SEQ ID NO: 17107)

[0205] A wildtype / natural human CD2 protein (NCBI: CD2__HUMAN; UniProt / Swiss-Prot: P06729.2) comprises or consists of the amino acid sequence of: MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSD KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKWLEKIFDLKIQE RVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKV SKESSVEPVSCPEKGLDIYLIIGICGGGSLLMVFVALLVFYITKRKKQRSRRNDEELETRAHRV ATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQV HQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEO ID NO: 17108)

[0206] A nucleotide sequence encoding wildtype / natural CD2 protein (NCBI: CCDS889.1) comprises or consists of the nucleotide sequence of: AT GAG C T T T C CAT G TAAA TT T GTAGCCAGC T T CC T T C T GAT T T T CAAT GTTTCTTCCAAAGGT G CAGTCTCCAAAGAGATTACGAATGCCTTGGAAACCTGGGGTGCCTTGGGTCAGGACATCAACTT G GAO AT T C C T AG T T T T C AAAT GAG T GAT GAT AT T GAG GAT AT AAAAT G G GAAAAAAC T T C AGAC AAGAAAAAG AT T G CAO AAT T C AGAAAAGAG7AAGAGAC T T T Cl AG G1AAAAGAT AC AT AT AAG C TAT T T AAAAAT G G AAC T C T G AAAAT T AAG C AT C T GAAGAC C GAT GAT C AG GA T AT C TAGAAG G T AvT C AAvT AT A,T GA T AC AAAAG G AAAAAAT G T G T T G GAAAAAAT AT T T GAT T T G AAGA T T C AAAAG AG G G T C T C AAAAC CAAAGAT C T C C T G GAC T T G TAT C AACACAACCOTGACC T GT GAGGTAAT GA ATGGAACTGACCCCGAATTAAACCTGTATCAAGATGGGAAACATCTAAAACTTTCTCAGAGGGT CATCACACACAAGTGGACCACCAGCCTGAGTGCAAAATTCAAGTGCACAGCAGGGAACAAAGTC AGCAAGGAATCCAGTGTCGAGCCTGTCAGCTGTCCAGAGAAAGGTCTGGACATCTATCTCATCA TTGGCATATGTGGAGGAGGCAGCCTCTTGATGGTCTTTGTGGCACTGCTCGTTTTCTATATCAC C AAAAG G AAAAAACAGAG GAG T C G GAG AAA T GAT GAG G AA C T G GAG AAAAG AG C C C AC AAAG TA GCTACTGAAGAAAGGGGCCGGAAACCCCACCAAATTCCAACTTCAACCCCTCAGAATCCAGCAA C T T C C C AACAT C C T C C T C CAC CAC C T G G T CAT C G T T C C CAG G CAC C TAG T CAT C G T C C C C C G C C TCCTGGACACCGTGTTCAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCGTCGGGCACACAAGTT CACCAGCAGAAAGGCCCGCCCCTCCCCAGACCTCGAGTTCAGCCAAAACCTCCCCATGGGGCAG CAGAAAACTCATTGTCCCCTTCCTCDAATDAA (SEQ ID NO: 17109)

[0207] An exemplar}' CSR CD2z protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular Domain, CD2 Transmembrane domain, CD2 Cytoplasmic Domain, CD3z Intracellular Domain): MSFPCKEVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSD KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQE RVSKPKISWTCINTELTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKV SKESSVEPVSCPEKGLDIYL11GICGGGS LLMVFVALLVFYITKRKKQRSRRNDEELETRAHRV HQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRNKEED.SARAPAYKQ.GQNQEYNELNLGRREEYE) vldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstat KDTYDALHMQALPPR (SEQ ID NO: 17062) CD2 Signal peptide: MSFPCKEVASFLLIFNVSSKGAVS (SEQ ID NO: 17110) CD2 Extracellular Domain'. KEITNALE TWGALGQDINLDIPS FQMS DDIDDIKWEKT S DKKKIAQFRKEKE T FKEKDTYKLFK NGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGT DPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (SEQ ID NO: 17111) CD2 Transmembrane domain: IYLIIGICGGGSLLMVWALLVFYIT (SEQ ID NO: 17112) CD2 Cytoplasmic Domain'. KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPP PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17113 ) CD3z Intracellular Domain : rVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1710 2 )

[0208] The present disclosure provides a non-naturally occurring CSR CD2 protein comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17062. The present disclosure provides a CD2 signal peptide comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17110. The present disclosure provides a CD2 extracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17111. The present disclosure provides a CD2 transmembrande domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17112. The present disclosure provides a CD2 cytoplasmic domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 68 100% identical to SEQ ID NO: 17113. The present disclosure provides a CD3z intracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17102.

[0209] An exemplary’ nucleotide sequence encoding a CSR CD2z protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular Domain, CD2 Transmembrane domain, CD2 Cytoplasmic Domain, CD3z Intracellular Domain): ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGGGCG CCGTGTCCAAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCT GGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGAC AAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTACAAGC TGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACATCTATAAGGT GTCCATCTACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAAGAG CGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGT GA± T CA C C CA CAA G T GGA CAA CA^A GCCT GA GCGC CAA GTT CAA. GT G CA CCGCCG GAAlA CAAlA GTG TCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGACATCTACCTGATCA TCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGfTCTACATCAC ft ft              ft ft ft ft ft              ft ft C* ft           71 2¾ ft ft / “» / •*« ft ft              ft ft ft / “* ft                        ft ft ft %2^j^00002^2&Oj^^00^00002^00^02&0002^x^002&002^00^^ x OO2^x^j^ 02^00000002¾. x.ri 0.23.0.1 O GCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCA CCTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACC ACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTG CACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCG CTGAGAACAGCCTGTCTCCAAGCAGCAACAGAGTGAADTTCAGCCGCAGCGCCGATGCTCCTGC C TAT AAG GAG G GACAGAAC GAG C T G TACAAC GAG C T GAATC T GGGGCGCAGAGAAGAGTAGGAT GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTC AAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAAT GAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACC AAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 17063) CD2 Signal peptide: ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGG1 CCGTGTCC (SEQ ID NO: 17114) CD2 Extracellular Domain’. AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCC :CAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAA GAT C G C C C AG T T C C G G A / iAGAGAAAGAGAC AT T C AAAGAG AA.G GAC AC C T AC7\AG C T G T T C AAG AAC G G CAC C C T GAAGAT CAAG CAC C T GAAAAC C GAC GAC CAG GACAT C TATAAGG T GT CCAT C T ACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA.GAGCGGGTGTC CAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACA GAC C C C GAG C T GAAC C T G TAG GAG GAT G G C AAAG AC C T G AAG C T GAG C GAG C G C G T GAT C AC C C ACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGA GTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGAC (SEQ ID NO: 17115) CD2 Transmembrane domain: ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGG TGTTCTACATCACC (SEQ ID NO: 17116) CD2 Cytoplasmic Domain-. AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTGG CCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCAC C T C T CAACAC C C T CCACC T CCACC T GGACACAGAT C T CAGGCCCCAT C T CACAGACC T CCACCA CCTGGTCATCGGGTGCAGCACCAGCCTCAGIAAAGACCTCCTGCTCCTAGCGGCACACAGGTGC AA C AG C A A AAAG GAG C T C GAC T G C C T C G G C C T AAAG T G GAG C C T AAAC C T C C T CAT G G C G C C G C TGAGAACAGCC T G T C T CCAAGCAGC AAC (S E Q ID N0: 17117) CD3z Intracellular Domain: AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACA ACGAGCTGAATCTGGGGCGCAGAAAAGAGTAAGATGTGCTGGAAAAGCGGAAAGGCAGAAATCC TGAAATGGGCGGCAAACCCAGACGGAAAAATCCTCAAGAAGGCCTGTATAATGAGCTGCAGAAA GACAAAATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGAC ACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCA GGCCCTGCCTCCAAGA (SEQ ID NO: 17107)

[0210] An exemplary mutant CSR CD2z-Dl 11H protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain, CD2 Transmembrane domain. CD2 Cytoplasmic domain, CD3z Intracellular domain): MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQD.TNLD.TPSFQMSDDIDDIKWEKTSD KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDLKIQE RVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKV SKESSVEPVSCPEKGLDIYL11GICGGGS LLMVFVALLVFYI TKRKKQRSRRNDEELETRAHRV ATEERGRKPHQIPASTPQNPATSQHPPPPPGimSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQV HQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRKKESRSAEAPAKKQGQNQLYNEEN'LGRREE'ID VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR (SEQ ID NO: 17118) CD2 Signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 17110) CD2 Extracellular domain with DI 11H mutation within the CD2 Extracellular domain'. KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFK NGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGT DPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGL (SEQ ID NO : 17119) CD2 Transmembrane domain: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 17112) CD2 Cytoplasmic domain". KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPP PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17113) CD3z Intracellular domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 17102)

[0211] The present disclosure provides a non-naturally occurring CSR CD2 protein comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast97%, atleast98%, atleast99% or 100% identical to SEQ IDNO:17118. The present disclosure provides a CD2 extracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: I7119.

[0212] An exemplaty nucleotide sequence encoding a mutant CSR CD2z-Dl 11H protein of the disclosure comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain , CD2 Transmembrane domain. CD2 Cytoplasmic domain, CD3z Intracellular domain): ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGGGCG CCGTGTCCAAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCT GGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGAC AAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTACAAGC TGTTC.AAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGAC.ATCTATAAGGT GTCCATCTACCACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAAGAG CGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGT GATCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTG TCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACAGGACATCTAGCTGATCA TCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACATCAC CAAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTG a a ha erm. a a errem rm r* 3 rerm rrm rm ma rrrmrh a rrrerrm HH^H^HA a r*a / ^ / ^ / ^^ / ^ / ^71 ^ / ^•TV'V'’^ / ^' / ^''T’ / ^ / ^'a / ^21 3 / ^21 'T / ^'T / ^S rrrrrm DTn^HA HA HA rrrrr^ HH LLI Cz2 L-^i-HO-HG-LzO 1 LzCz2iOLz2 OC-^iLzO2 CyCxAG^Cz^Lxrix LI O^L?CyOLzOL.-.ri2 LI C2uGu&CxHC,01 Cz O-HOLz ACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTG CACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCG CTGAGAACAGCCTGTCTCCAAGCAGCAACG'.GAGT GAAG 1 TCA.GuCGCAGCGuCGwTGC 1 uCTGu CTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGAT GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTC AAGAG G G C C T G T AT AA T GAG C T G C AGAAAGAC AAGA T G G C C GAG G C C T AC AG C GAG AT C G G.AAT GAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACC AAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEO ID NO: 17120) CD2 Signal peptide: ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGGGCG CCGTGTCC (SEQ ID NO: 17114) CD2 Extracelhdar domain with D11IH mutation within the CD2 Extracellular domain. AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCC C GAG C T T 0 C AGAT GAG C GAO GAG AT C GAT GAO AT C AAG T G G GAGAAAAC GAG C GAC AAGAAG7 A GAT C G C C C AG T T C C G G AAAG AGAAAGAG AC AT T C AAAGAG AAG G AC AC C T AC AAG C T G T T CAAG AAC G G CAC C C T GAAGAT CAAG CAC C T GAAAAC C GAC GAC CAG GAC AT C TATAAG GTGTCCATCT ACCACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAAGAGCGGGTGTC CAAGC C T AAGAT CAGC T GGAC C TGCATCAACAC CACAO T GAC CT GO GAAGT GAT GAACGGCACA GACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCC ACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGA GTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGAC (SEQ ID NO: 17121) CD2 Transmembrane domain: ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGG TGTTCTAGATCACC (SEQ ID NO: 17116) CD2 Cytoplasmic domain: AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTGG C C AC C G AG GAAAGAG G C AG.AAAG C C C C AC C AGAT T C C AG C C AG C AC AC C C C AGAAT C C T G C C AC C T C T CAACAC C C T C CAC C T C CAC C T G GACACAGATC T CAGGCCCCAT C T CACAGACC T CCACCA CCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGC ACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGC TGAGA / 1CAGCCTGTCTCCAAGCAGCAAC (SEQ ID NO: 17117) CD3z Intracellular domain: AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACA ACGAGCTGICITCTGGGGCGCAGAGIWIAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCC TGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAA GACAAGAT G G C C GAG G C C TAGAG C GAGAT C G GAAT GAAG G G C GAG C G CAG AAGAG G C AAA G GAC ACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAABGATACCTATGATGCCCTGCACATGCA GGCCCTGCCTCCAAGA (SEQ ID NO:17107) Endogenous TCR Knock-out

[0213] Gene editing compositions of the disclosure, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, may be used to target and decrease or eliminate expression of an endogenous T-cell receptor of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding an endogenous T-cell receptor of an allogeneic cell of the disclosure.

[0214] Nonlimiting examples of primers (including a T7 promoter, genome target sequence, and gRNA scaffold) for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-a) are provided in Table 10.

[0215] Table 10. Target sequences underlined Name Sequence SEQ ID NO: TCRa-gRNA-WT 1 TAATACGACTCACTATA GCTGGTACACGGCAGGGTCA GT T T TAGAGCTAGAAATAG 16821 TCRa-gRNA-WT TAATACGACTCACTATA GAGAATC AAAATCGGTGAAT 16822 TCRa-gRNA—'WT 4 TAATACGACTCACTATA GTGCTAGACATGAGGTCTA 168 2 3 TCRa-gRNA-WT 1-2G TAATACGACTCACTATAG GCTGGTACACGGCAGGGTCA 16824 TCRa-gRNA~WT 2 TAATACGACTCACTATA GAGAATC AAAATCGGTGAAT GT T T T AGAGC T AGAAAT AG 16825 TCRa-gRNA-WT 3 TAATACGACTCACTATA GG ATTTAGAGT CTCTCAGC GT T T TAGAGCTAGAAATAG 16826 TCRa-gRNA-WT 4 TAATACGACTCACTATA GTGCTAGACATGAGGTCTA GT T T TAGAGC TAGAAATAG 168 2 / TCRa-gRNA-WT TAATACGACTCACTATA GACACCTTCTTCCCCAGCCC GT T T TAGAGC TAGAAATAG 16828 TCRa-gRNA-NG1-L TAATACGACTCACTATA g t g gaa t a a t g c t g11 g11 g a GT TTT.AGAGCT AGAAA.T AG 168 2 9 TCRa-gRNA-NG2 -L T AAT AC GAO T C AC T AT A g c a t c a c a g g a a c 111 c t a a a GT T TTAGAGCT AGAAAT AG 16830 TCRa-gRNA-NG3-L TAAT AC GAC T CAC T AT A gtaaaaccaagaggccacag GT T T TAGAGC TAGAAATAG 168 31 TCRa-gRNA-NG4-L TAATACGACTCACTATA g acccggccac111caggagg GT T T TAGAGC TAGAAATAG 168 32 TCRa-gRNA-NG5-L TAATACGACTCACTATA gattaaacccggccactttc GT T T TAGAGC T AGAAAT AG 16833 TCRa-gRNA-NG1-R TAATACGACTCACTATA g agcccaggtaagggcagctt GT T T TAGAGC TAGAAATAG 16834 TCRa-gRNA-NG2-1-R TAATACGACTCACTATA g agctttgaaacaggtaagac GT T T TAGAGC TAGAAATAG 168 35 TCRa-gRNA-NG2-2-R TAATACGACTCACTATA gctttgaaacaggtaagaca GT T T T AGAGC TAGAAATAG 16836 TCRa-gRNA-NG3-R TAATACGACTCACTATA g 111 c a a a a c c t g t c a gtga t GT TT TAGAGC TAGAAATAG 168 3 / TCRa-gRNA-NG4-R TAATACGACTCACTATA g ctgcggctgtggtccagctg GT T TTAGAGCT AGAAAT AG 16838 TCRa-gRNA-NG5-1-R TA AT AC G AC T C AC T AT A g c t g_ t g g t c c a g c t g a g g t g_ GT T T TAGAGC TAGAAATAG 16839 TCRa-gRNA-NG5-2-R TAATACGACTCACTATA g ctgtggtccagctgaggtga GT T T TAGAGC TAGAAATAG 16840 TCRa-gRNA-NG5-3-R TAATACGACTCACTATA g tgtggtccagctgaggtgag GT T T T AGAGC TAGAAATAG 16841 TCRa-gRNA-NG5-3-Rb TAATACGACTCACTATA gtgtggtccagctgaggtgag GT TT TAGAGC TAGAAATAG 16842

[0216] Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-beta (TCR~P) are provided in Table 11.

[0217] Table 11. Target sequences underlined Name Sequence SEQ ID NO: TCRb-gRNA-WT T AAT ACG AC T C AC TATA GGC.TGCTCCTTGAGGGGCTG GTTTTAGAGCTAGAAATAG 16843 TCRb-gRNA-WT y TAATACGACTCACTATA GGCAGTATCTGGAGTCATTG GTTTTAGAGCTAGAAATAG 168 4 4 TCRb-gRNA-WT 3 TAATACGACTCACTATA GGCCTCGGCGCTGACGATCT 16845 TCRb- gRNA-WT TAATACGACTCACTATA GGCTCTCGGAGAATGACGAG 1 6 8 4 6> TCRb-gRNA-WT 3 TAlATA.CGACTCAiC.TATA GGCC.TCGGCGC.TGA1CGA1TCT GTTTTAiGAGCTAGAAATAG 16847 TCRb-gRNA-WT 4 TAATACGACTCACTATA GGAGAATGACGAGTGGACCC GTTTTAGAGCTAGAAATAG 16848 TCRb-gRNA-WT c; TAATACGACTCACTATA GGCTCTCGGAGAATGACGAG GTTTTAGAGCTAGAAATAG 16849 TCRb-gRNA-NG1-L TAATACGACTCACTATA G CAAACACAGCGACCTCGGGT GTTTTAGAGCTA.GAAATAG 16 8 5 0 TCRb-gRNA-NG2-L TAATACGACTCACTATA! G TGGCTCAiAAiCACAGCGACCT GTTTTAGAGCTAiGAiALATAG 16851 TCRb-gRNA-NG3-L TAATACGACTCACTATA G AGGGCGGGCTGCTCCTTGAG GTTTTAGAGCTAGAAATAG 16852 TCRb-gRNA-NG4-L TAATACGACTCACTATA GTATCTGGAGTCATTGAGGG GTTTTAGAGCTAGAAATAG 16 8 b 3 TCRb-gRNA-NG5-L TAAJTACGACTCAATATA G ACTGGA.CTTGACAGCGGAAG GTTTTAGAGCTAGAAATAG 1 68 5 4 TCRb-gRNA-N C" .1. ” R TA1ATA.CGACTCA1CTATA G AiGAGATCTC.CCACACC.CAAA GTTTTAiGAGCTAGAAATAG 16855 TCRb-gRNA-NG2 — R TAATACGACTCACTATA G CCACACCCAAAAGGCCACAC GTTTTAGAGCTAGAAATAG 16856 TCRb-gRNA-NG3-R TAATACGACTCACTATA G ACTGCCTGAGCAGCCGCCTG GTTTTAGAGCTAGAAATAG 16857 TCRb-gRNA-NG4-R TAATACGACTCACTATA G TGAGGGTCTCGGCCACCTTC GTTTTAGAGCTAGAAATAG 16858 TCRb-gRNA-NG 5 “R TAATACGACTCACTATA G ATGACGAGTGGACCCAGGAT GTTTTAGAGCTAGAAATAG 16859 TCRb-gRNA-NG6-L TAATACGACTCACTATA G TGGCTCAAACACAGCGACCT GTTTTAGAGCTAGAAATAG 168 6 0 TCRb-gRNA-N G 6 - R TAATACGACTCACTATA G CCACACCCAAAAGGCC.ACAC GTTTTAGAGCTAGAAATAG 168 61

[0218] Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting beta-2-microglobulin (P2M) are provided in Table 12.

[0219] Table 12. Target sequences underlined Primer No . Name Sequence SEQ ID NO: 1 B2-Prom-NG1-R TAATACGACTCACTATAG AGACAGGTGACGGTCCCTGC GT T T TAGAGCTAGAAATAG 168 62 2 B2-Prom-NG1-L TAATACGACTCACTATA GCAGTGCCAGGTTAGAGAGAl GT T T TAGAGCTAGAAATAG 16863 3 B2-Ex2-NG-R TAATACGACTCACTATA GAAGTTGACTTACTGAAGAA GT T T TAGAGC TAGAAATAG 16864 4 B2~ Ex2~ NG-L TAATACGACTCACTATA G ACCCAGACACATAGCAATTC GT T T TAGAGC TAGAAATAG 168 65 5 2 M w Q X M M M 1 ! 1 TAATACGACTCACTATA G T CAC GT CAT C CAGCAGAGAA GT T T TAGAGC TAGAAATAG 16866 6 B2 — Ex2~ NG2-L TAATACGACTCACTATA g a t a 11 cctcagGTACTCCA GT T T TAGAGCTAGAAATAG 168 6'7 7 b2MExl NG-left TAATACGACTCACTATA GGCCACGGAGCGAGACATCT GT T T TAGAGCTAGAAATAG 168 68 8 b2MExl NG-right TAA.TACGACTCACTATAG ACTCTCTCTTTCTGGCCTGG GT T T TAGAGC TAGAAATAG 168 6 9 9 El '-Q O’ H S [4 M > T ro TAATACGACTCACTATAG GAGAGAGAATT GAAAAAG GT T T TAGAGCTAGAAATAG 16870 Endogenous MHC Knock-out

[0220] Gene editing compositions of the disclosure, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, may be used to target and decrease or eliminate expression of an endogenous MHCI, MHCII, or MHC activator of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding one or more components of an endogenous MHCI, MHCII, or MHC activator of an allogeneic cell of the disclosure.

[0221] Nonlimiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are provided in Tables 13 and 14.

[0222] Table 13. Ge; ne Reagent / 7 Type Left Target Sequence SEQ ID NO: Right Target Sequence SEQ ID NO C2TA C2TA exon 4 NG CATCGCTGTTAAGAAGCTCC 16871 CTACCACTTCTATGACCAGA 16880 C2TA exon 6 NG GGCCCTCCAGCTGGGAGTCC 168 72 CAGTAAGTTTGTGGTGGGTG 16881 RFXANK RFXANK exonl NG1 GGGT CT GCTGGGT CT GGATG 16873 G GACC CT GAAGACCCCGGAG 16882 RFXANK exonl NG2 GTT CT GAGGCAGGGGTCT GC 16874 C C C G GAGA G GAG G C T G CAGA 16883 RFXAP RFXAP Exon 1 NG1 C CC G C C C CAACGCTGCCCCC 16875 CTGTGCGAAGGGGCCGGGGA 16884 RFXAP Exon 1 NG2 C C T T C G C A.C A_G GT AC C T AA.C 1687 6 AGAGGAGGCTGGGGAGGACG 16885 RFX5 RFX5 exon 1 NG1 GT CTT GGGGCT CTT AGCA.T C 168 7 7 CCCAGGT GGT GCT GAGGCT G 168 8 6 RFX5 exon 2 NG2 ACGGCCTTGCTGTGGGGAAG 16 8 7 8 GGGATCCTGGTAAGTGT GTT 16887 RFX5 exon5 NG3 TCTGATGATCTTGCCAAAGT 1687 9 AT CAAAGCT CGAAGGCTT GG 16888

[0223] Table 14. Gene Re aq en t / Type Exon or region NG-Left Target Sequence SEQ ID NO: NG-Right Target Sequence SEQ ID NO : Target sequence (if WT crispr) EQ ID O Beta2- MG B 2-P r omo t e r-NG1 promo ter GCAGTGCCAGGTTAGAGAGA 168 8 9 AGACAG GT GAC GGT C C C TGC 16913 B2-Promoter~ NG2 promoter C AAG C GAG C GA C G CAgT g C C 168 9 0 CCTGCGGGCCTTGTCCTGAT 16914 B2--Promoter- NG3 promoter C CAAT CAGGACAAG G C C C GC 168 91 TATAAGT GGAGGC GT C G C G C 16915 2—E x 2 — N G exon 2 ACC C AG7IC AC AT AG CAAT T C 16892 GAAGTT GACTTACT GAAGAA 16916 B2-Ex2~NG2 exon 2 gatattcctcagGTACTCCA 15893 TCACGTCATCCAGCAGAGAA 16917 B2-Exl-NG exon 1 GGCCAC GGAGC GAGACAT CT 16894 ACTCTCTCTTTCTGGCCTGG 16918 WT-B2MG- exon2 GGAGAGAGAAT T GAAAA^G 6937 WT-B2MG-promoter~4 cuts in promoter region Y u uG C C- T '1' GT C C T GAr ‘1' u u u 6938 WT-B2MG-promoter- 5 c u o s i n promoter region GGCACTGCGTCGCTGGCT 6939 C2TA C2TA exon 4 NG exon 4 CAT C GC T GT TAAGAAGC T C C 168 9 5 CTAC CACTTC TAT GAC CAGA 16919 C2TA exon 4 NG2 exon 4 GGTCCATCTGGTCATAGAAG 15896 AGATTGAGCTCTACTCAGGT 1692 0 C2TA exone NG exon6 GGCCCTCCAGCTGGGAGTCC 168 97 CAGTAAGTTT GT GGT GGGT G 16921 C2 TA exo n4 -WT exon 4 GGT CCAT CT GGTCATAGAAG 694 0 C2TA exon6-WT exon 6 G GAGT C C T G GAAGACATAC 6941 C2TA exon6 NG2 exon6 CCTTGCTCAGGCCCTCCAGC 16898 TGTGGTGGGTGGGGAGGTCT 16922 WO 2020 / 051374                                                 PCT / US2019 / 049816 RFXANK RFXANK exonl NG1 exon 1 GGGTCTGCTGGGTCTGGATG 168 9 9 GGACCCTGAAGACCCCGGAG 16923 RFXANK exonl NG2 exon 1 GT T C T GAGGCAGGGGT C T GC 1690 0 C C C GGAGAGGAGGC T GCAGA 16924 RFXANK exon2 NG1 exon 2 TGAGAGTGGTGGAGTGCTTC 16901 GAACGAGGTGTCAGCTCTGC 16925 RFXANK Exon2 NG2 exon 2 CTCGTTCCCTCGCTGCCGGT 16902 GGCCACCCTAGACTGTGAGT 16926 R FXANK~WT-exon1-3 exon 1 GGTCCCCAAGTTCTGAGGC 16942 RFXA.NK-WT-exonl-4 exonl GGCAGGGGTCTGCTGGGTC 16943 RFXAP RFXAP Exon 1 NG1 exon 1 CCCGCCCCAACGCTGCCCCC 16903 CT GT GC GAAGGGGCC GGGGA 16927 RFXAP Exon 1 NG2 exon 1 C C TT C G CAC AG GTAC CTAAC 16904 AGAGGAGGCTGGGGAGGACG 1692 8 RFXAP Exonl NG3 exon 1 CAGCCGGGGCTAGGGCCGCG 16905 CTTGGCGCCAGCCTCGGTGG 16929 RFXAP Exonl NG 4 exon 1 GCCGCGGCCGCCACCGAGGC 16906 CTAGTGATGCAACCCTGTGC 16930 RFXAP Exonl NG5 exon 1 GCCGCGCTCTCGCCTCCCCC 16907 GAGGACGAGGAGACTCACTC 16931 WT- RFXAP-exl--3 exon 1 GGCCCCCGGGGGCAGCGTT 16944 WT-- RFXAP--exl-- 4 exon 1 G GTAC C TGT GC GAAG GGGC 16945 RFX5 RFX5 exonl NG1 exon 1 GTCTTGGGGCTCTTAGCATC 1690 8 C C CAGGT GGT GC T GAGGCT G 16932 RFX5 exon2 NG2 exon 2 AC GGCCTTGCTGTGGGGAAG 16 9 0' 9 GGGATC CT GGTAAGT GT GTT 169 33 RFX5 exon5 NG3 exon5 TCTGATGATCTTGCCAAAGT 16910 AT C AAAGC T C GAAG G C T T G G 16934 RFX5 exonl NG2 GTCTTGGGGCTCTTAGCATC 16911 CCCCAGGTGGTGCTGAGGCT 16935 RFX5 exonl NG3 AGGCTCATCTTCTGCCATCC 16912 ACTGGGGGAAGGGCCCCCCC 16936 WO 2020 / 051374                                                 PCT / US2019 / 049816 Engineered HI^A-E compositions

[0224] MHCI knockout (KO) renders cells resistant to killing by T cells, but also makes them susceptible to natural killer (NK) cell-mediated cytotoxicity (‘"Missing-self hypothesis”) (see FIG. 30). It is hypothesized that NK rejection would reduce the in vivo efficacy and / or persistence of these KO cells in a therapeutic setting, such as allogeneic (allo) CAR-T therapy. Retention of MHCI on the surface of allo CAR-T ceils would render them susceptible to killing by host T cells, as observed in the classic mixed lymphocyte reaction (MLR) experiment. It is estimated that up to 10% of a person’s T cells are specific to foreign MHC, which would mediate the rejection of foreign, cells and tissues. A targeted KO of MHCI, specifically HLA-A, B and C, which can be achieved by targeted KO of B2M, results in a loss of additional HLA molecules including HLA-E. Loss of HLA-E, for example, renders the KO cells more susceptible to NK cell-mediated cytotoxicity due to the “Missingself Hypothesis”. NK-mediated cytotoxicity' against missing-self cells is a defense mechanism against pathogens that downregulate MHC on the surface of infected cells to evade detection and killing by cells of the adaptive immune system.

[0225] Two strategies are contemplated by the disclosure for engineering allo (MHCI-neg) T cells (including CAR-T cells) more resistant to NK cell-mediated cytotoxicity'. In some embodiments, a sequence encoding a molecule (such as single-chain HLA-E) that reduces or prevents NK killing is introduced or delivered to an allogeneic cell. Alternatively, or in addition, gene editing methods of the disclosure retain certain endogenous HLA molecules (such as endogenous HLA-E). For example, the first approach involves piggy Bae® (PB) delivery of a single-chain (sc)HLA-E molecule to B2M KO T cells.

[0226] The second approach uses a gene editing composition with guide RNAs selective for HLA-A, HL.A-B and HLA-C, but not, for example, HLA-E or other molecules that are protective against natural-killer cell mediated cytotoxicity for MHCI KO cells.

[0227] Alternative or additional molecules to HLA-E that are protective against NK cell-mediated cytotoxicity include, but are not limited to, CD47, interferon alpha / beta receptor I (IFNAR1), human IFNAR1, interferon alpha / beta receptor 2 (IFNAR2), human IFNAR2, HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, human carcino embryonic antigen-related cell adhesion molecule I (CEACAM1), viral hemoagglutinins, CD48, LLT1 (also referred to as C-type lectin domain family 2 member (CLC2D)), ULBP2, ULBP3, and sMICA or a variant thereof.

[0228] An exemplary CD47 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic')-. MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYV KWKFKGRDIYTFDGALNKSTVPTDFSSAKTEVSQLLKGDASLKMDKSDAVSHTGNY TCEVTELTREGETIIELKYRVVSWFSPNENI£ZriFWK4I££FWgF67A^ DEKTIALLVAGLVITViVIVGAJLFVPGEYSLKNATGLGLIVTSTGILrLLHYYVFSTAlG LTSFVIAILVIQVIAYILAWGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVAS NQKTIQPPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 17016)

[0229] An exemplary INF ARI protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic)-. MMVWLGATTLVLVAVAPWLSAAAGGPtNLKSPQKVEVDIIDDNFILRWNRSDESVGNVT WfDYQKTGMDNWI KLS GCQNIT S TKCNFS S LKLNVYEEIKLRIRAEKENT S S WYEVDS F TPFRKAQIGPPEVHLEAEDKAIVIHISPGTKDSWWALDGLSFTYSLVIWKNSSGVEERI ENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQ NQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQK GIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTP XTIQDY PLI YE 11FWENT SNAERKIIEKKT DVTVPNLKPLTVYCVPLAPA.HTMDEKLNKS SV F S DAVC E PCT Pt P G NT S KIWLIVGICIALFALPFVIYAAKVFLRCINYVFFPSLKPSSS IDE YFSEQPLKRLLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSGNYSN EDESESKTSEELQQDFV (SEQ ID NO: 17 017) .

[0230] An exemplary INFAR2 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide, Extracellular. TM, Cytoplasmic)'. MLLSQNAFIFRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISLRNFRSILSWELKNHS IVPTHYTLLYTiMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLF SCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEOSEGIVKK HKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAE SKRIGGITTVFLIAL VL TSTIVTLKWIG YICLRNSL PKVLNFHNFLA WPFPNL PPLEAMD MVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGGYTMHGLTVRPLGQASATSTESQLI DPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDYSSTEGSGGR ITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPT FPSPSSEGLWSEDAPSDQSDTSESDVDLGDGYIMR (SEQ ID NO: 17018).

[0231] An exemplary HLA-G1 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3): I4WMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSFTLQPvMZG CDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEG TCPVtVLVRYLEZvGKENLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQ tqdvelvetrpagdgtfqkwaavwpsgeeqrytchvqheglpepl'mlrwkqs S L PT I P IMG IVAGLWLAAWTGAAVAAVLWRKKSSD (SEQ ID NO: 17019) .

[0232] An exemplary HLA-G2 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3): MWMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWEQEGPE YWEEETRNTKAHAQTDRMNLQTLRGYYNQSEAD P P KT HVT HH PVFDYEATLR.CWALGFYPAEIILTWQR.DGEDQTQDVELVETR.PAGDGT FQKWAAVVVPSGEE QRYTCHVQHEGLPEPLMLRWKQS SL PT I p IMG IVAGLWLAAWTGAAVAAVLWRKKS S D (SEQ ID NO: 17020) .

[0233] An exemplary HLA-G3 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3): MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEAKQSS L PTIPI MGIVAGLVVLAAVVTGAAVAAVLWRKKSSD (SEQ ID NO: 17021) .

[0234] An exemplary HLA-G4 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3): MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSP / TLQPvWG CDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEG TCVEWLHRYLENGKEMLQRAKQSS L PTIPIMGIVAGLWLAAWTGAAVAAVLWRKKS S D (SEQ ID NO: 17022) .

[0235] An exemplary7 HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2., Alpha chain 3. intron 4): MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLGIVMTG CDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEG TCVEWLHRYLENGKEMLQRADPPATHVYHH PVFDYEATLRCWALGFYPAE11LTWQRDGEDQ TQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVR ESRSLSEDL (SEQ ID NO: 17023) .

[0236] An exemplary7 HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2., Alpha chain 3. intron 4): MVVMAPRTLFLLLSGALTLTEAWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPVWEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEApPPKTHyTHH PVFDY EAT LRCWALG FY PAE 11LTWQRDGEDQTQDV ELVETRPAGDGT FQKWAAVT> / APS GEE QPYTCHVQHEGJLP^^

[0237] An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1, Alpha chain 2, Alpha chain 3, intron 2): MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDS DSACPRMEPRAPWEQEGPEYWEETRNTKAHAQTDRMNLQTLRGYYNQSEASE (SEQ ID NO: 17 025) .

[0238] An exemplary CEACAM1 protein of the disclosure comprises or consists of the amino acid sequence of (Extracellular, TM, Cytoplasmic): MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQ QLFGYSWYKGERWGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFY TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWI NNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTP TISPSDTYYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCH ANNSVTGCNRTTVKTIIVTELSPWAKPQIKASKTTVTGDKDSWLTCSTNDTGISIRWF FKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALP QENGLS PGAIAGI VIG m L VALIA VALACFLHFGKTGRASDQRDLTEHKPSVSNHTQDH SNDPPNKMNEVTYSTLNFEAQQPTQPTSASPSLTATEIIYSEVKKQ (SEQ ID NO: 17 0 2 6) .

[0239] An exemplary viral hemagglutinin protein of the disclosure comprises or consists of the amino acid sequence of (HA for Influenza A virus(A / NewCaledonia / 20 / 1999(H!Nl); TM): MKAKdLVLdCT FT AT YADTICIGYHANN S T DT VDTVLEKNVTVT HSVN LLEDS HNGKLCL LKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELRE QLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQG AINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM VDGWYGAHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRM ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC FEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSL VLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: 17027).

[0240] An exemplary' CD48 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide, Chain, Pro peptide removed in mature form): MC S RGWD S C LAL E L L L L P L S L LVT SIQGHLVHMTWS GSNVTLNISE SLP ENYI^LTWYTFDQKIVEWSRKSKYFESKFKGRVRLDPQSGALYISKVQ KEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYL KLSCVIPGESWYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSN SVBSV^GTVCI.SPPCT'LARSFGVEWIASWLVVTVPTILGLLLT (SEQ ID NO: 17 0 2 8 ) ,

[0241] An exemplary’ LLT1 protein of the disclosure comprises or consists of the amino acid sequence of (Cytoplasmic, 77VY, Extracellular): MHDSNNVEKDITPSELPANPGCLHSKEHSIKATLIWRLFFLTMFLTTTVCGWAAASATR ANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELN FLLRYKGPSDHWIGLSREQGQPWKWINGTEWTRQFPILGAGECAYLNDKGASSARHYTER KWICSKSDIHV (SEQ ID NO: 1 / 029).

[0242] An exemplary ULBP2 protein of the disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. AAQ89028): 1 maaaaatkil Iclpllllls gwsragradp hslcyditvi pkfrpgprwc avqgqvdekt 61 flhydcgnkt vtpvsplgkk Invttawkaq npvlrewdi Iteqlrdiql enytpkeplt 121 Iqarmsceqk aeghssgswq fsfdgqifll fdsekrmwtt vhpgarkmke kwendkwam 181 sfhyfsmgdc igwledflmg mdstlepsag aplamssgtt qlratattli Icclliilpc 241 filpgi (SEQ ID NO: 1 / 030) .

[0243] An exemplary ULBP3 protein of the disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. NP_078794): 1 maaaaspail prlailpyll fdwsgtgrad ahslwynfti ihlprhgqqw cevqsqvdqk 61 nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl 121 tlqvrxnscec eadgyirgsw qfsfdgrkfl Ifdsnnrkwt vvhagarrmk ekwekdsglt 181 tffkmvsmrd ckswlrdflm hrkkrlepta pptmapglaq pkaiattlsp wsfliilcfi 241 Ipgi (SEQ ID NO: 17031).

[0244] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal Peptide. Portion of Extracellular domain, 7M and cytoplasmic domain) (Genbank Accession No. Q29983): 1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh Idgqpflrcd 61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce 121 ihednstrss qhfyydgelf Isqnletkew tmpqssraqt lamnvrnflk edamktkthy 181 hamhadclqe Irrylksgvv Irrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr 241 qdgvslshdt qqwgdvlpdg ngtyqtwvat. ricqgeeqrf tcymehsgnh sthpvpsgkv 301 lvlqshwgt.f hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr 361 datqlgfqpl msdlgstgst ega (SEQ ID NO: 17032) .

[0245] An exemplary7 sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Alpha-1, Alpha-2, Alpha-3): 1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh Idgqpflrcd 61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce 121 ihednstrss qhfyydgelf Isqnletkew tmpqssraqt lamnvrnflk edamktkthy 181 hamhadclqe Irrylksgvv Irrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr 241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv 301 Ivlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr 361 datqlgfqpl msdlgstgst ega (SEQ ID NO: 17033) .

[0246] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signalpeptide', Alpha-1. Alpha-2. Alpha-3): MGGVLLTQRTLLSLVLALLFPSMASM ephslry nltvlswdgs vqsgfltevh Idgqpflrcd 61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce 121 ihednstrss qhfyydgelf Isqnletkew tmpqssraqt 1                 thy 181 hamhadclqe Irrylksgvv Irrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr 241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv 301 Ivlqshw (SEQ ID NO:  I 1 034 ) .

[0247] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signalpeptide)'. M7GVZ,LTQRTLLSLVUlLLKPS2^aSMEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRC DRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKLDLRMTLAHIKLDQKEGLHSLQEIRVC EIHEDNSTRSSQHFYYNGELFLSQNLETKEWTMPQSSRAQTLTHYHAMHADCLQELRRYLKS GWLRRTVPPWDVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPD GNGTYQTWATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW (SEQ ID NO: 17 035) .

[0248] An exemplary bGBE Trimer (270G and 484S) protein of the disclosure comprises or consists of the amino acid sequence of: MSRSVALAVLALLSLSGLEAVMAPRTLILGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENG KSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC RVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFI SVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYY NQSEAGSHTLQ^HGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKS NDAS EAEHQRAYLE DTCVEWLHKY LE KGKET L L H L E PPKT HVTH H PISDHEAT L RCWALG FY PAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAWVPSGEEQRYTCHVQHEGLPEPVT LRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSE SHSL* (SEQ ID NO: 16972) .

[0249] An exemplary bGBE Trimer (270G and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of: atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggcctggaggccgt g a t g g c c c c c c g g a c c c t g a t c c t gqgagqaqgagqcage g qcqqaqqaqqc t c c g g a g g c g geggetetatc c a gc gc a c a cct a a gat cc a ggt gt a 11 ct cg g c a c cc a gc c gaga a cg g c aagagcaacttcctgaattgctacgtgagcggctttcacccttccgacatcgaggtggatct gctgaagaatggcgagagaatcgagaaggtggagcactccgacctgagcttctccaaggatt ggtctttttatctgctgtactataccgagtttacccctacagagaaggacgagtacgcctgt cgcgtgaaccacgtgacactgtcccagccaaagatcgtgaagtgggaccgggatatgggcgg c g g c g g c t c t g g c g g c g g c g g c a g c g g c g g c g g c g g c t c c g g a g g a g g c g g c t c t g g c a g c c a c t c c c t g a a g t a 111 c c a c a c c t c t g t g a g c c g g c c a g g c a g a g g a g a g c c a c g g 11 c a t c tctgtgggctacgtggacgatacacagttcgtgaggtttgacaatgatgccgccagcccaag a a t g g t g c c t a g g g c c c c a t g g a t g g a g c a g g a g g g c a g c g a g t a 11 g g g a c a g g g a g a c c c ggagcgccagagacacagcacagattttccgggtgaacctgagaaccctgaggggctactat aatcagtccgaggccggctctcacacactccagtggatgcacggatgcgagctgggaccaga tcjgccgcttcctgcggggctacgagcagtttgcctatgacggcaaggattacctgaccctga acgaggacctgagatcctggaccgccgtggatacagccgcccagatcagcgagcagaagtcc aatgacgcatctgaggcagagcaccagagggcatatctggaggatacctgcgtggagtggct g c a c a a g t a c c t g g a g a a g g g c a a g g a g a c a c t g c t g c a c c t g g a g c c c c c t a a g a c c c a c g t g a c a c a c c a c c c a a t c a g c g a c c a c g a g g c c a c c c t g a g g t g 11 g g g c a c t g g g c 11 c t a t cccgccgagatcaccctgacatggcagcaggacggagagggacacacccaggatacagagct g g t g g a g a c c a g g c c c g c c g g c g a t g g c a c a 111 c a g a a g t g g g c c g c c g t g g t g g t g c c 11 ccggagaggagcagagatacacctgtcacgtgcagcacgagggactgccagagccagtgacc ctgaggtggaagcctgccagccagccc.ac.aatccctatcgtgggaatc.atcgcaggcctggt gctgctgggctctgtggtgagcggagcagtggtggccgccgtgatctggcggaagaagagca gcggaggcaagggaggctcctactccaaggcagagtggagcgactccgcccagggctctgag agccactccctgtga (SEQ ID NO: 16973) .

[0250] An exemplary bCsBE Trimer (270R and 484S) protein of the disclosure comprises or consists of the amino acid sequence of: MS RSVALAVIjALLSLSGLEAVMAPRTIjIIjGGGGSGGGGSGGGGS 1QRT PKIQVYSRHPAENG KSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC RVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFI SVGYVDDTQFVRFDNDAASPRPRZPRAPW4EQEGSEYWDRETRSARDTAQIFRVNLRTLRGYY NQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKS NDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFY PAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVT LRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSE SHSL* (SEQ ID NO: 16974) .

[0251] An exemplary bCsBE Trimer (270R and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of: atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggcctggaggccgt gatggccococggaccctgatcctgggaggaggaggcagcggcggaggaggctccggaggcg gcggctctatccagcgcacacctaagatccaggtgtattctcggcacccagccgagaacggc aagagcaacttcctgaattgctacgtgagcggctttcacccttccgacatcgaggtggatct gctgaagaatggcgagagaatcgagaaggtggagcactccgacctgagcttctccaaggatt ggtctttttatctgctgtactataccgagtttacccctacagagaaggacgagtacgcctgt c g c g t g a a c c a c g t g a c a c t g t c c c a g c c a a a g a t c g t g a a g t g g g a c c g g g a t a t g g g c g g c g g c g g c t c t g g c g g c g g c g g c a g c g g c g g c g g c g g c t c c g g a g g a g g c g g c t c t g g c a g c c actccctgaagtatttccacacctctgtgagccggccaggcagaggagagccacggttcatc tctgtgggctacgtggacgatacacagttcgtgaggtttgacaatgatgccgccagcccaag a a t g g t g c c t a g g g c c c c a t g g a t g g a g c a g g a g g g c a g c g a g t a 11 g g g a c a g g g a g a c c c ggagcgccagagacacagcacagattttccgggtgaacctgagaaccctgaggggctactat a a t c a g t c c g a g g c c g g c t c t c a c a c a c t c c a g t g g a t g c a c g g a t g c g a g c t g g g a c c a g a tag c c g c 11 c c t g c g g g g c t a c g a g c a g 111 g c c t a t g a c g g c a a g g a 11 a c c t g a c c c t g a acgaggacctgagatcctggaccgccgtggatacagccgcccagatcagcgagcagaagtcc a a t g a c g c a t c t g a g g c a g a g c a c c a g a g g g c a t a t c t g g a g g a t a c c t g c g t g g a g t g g c t gcacaagtacctggagaagggcaaggagacactgctgcacctggagccccctaagacccacg t g a c a c a c c a c c c a a t c a g c g a c c a c g a g g c c a c c c t g a g g t g 11 g g g c a c t g g g c 11 c t a t c c c g c c g a g a t c a c c c t g a c a t g g c a g c a g g a c g g a g a g g g a c a c a c c c a g g a t a c a g a g c t g g t g g a g a c c a g g c c c g c c g g c g a t g g c a c a 111 c a g a a g t g g g c c g c c g t g g t g g t g c c 11 c c g g a g a g g a g c a g a g a t a c a c c t g t c a c g t g c a g c a c g a g g g a c t g c c a g a g c c a g t g a c c c t g a g g t g g a a g c c t g c c a g c c a g c c c a c a a t c c c t a t c g t g g g a a t c a t c g c a g g c c t g g t gctgctgggctctgtggtgagcggagcagtggtggccgccgtgatctggcggaagaagagca gcggaggcaagggaggctcctactccaaggcagagtggagcgactccgcccagggctctgag agccactccctgtga (SEQ ID NO: 16975).

[0252] An exemplary gBE Dimer (R and A) protein of the disclosure comprises or consists of the amino acid sequence of: MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEV DLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDN DAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWM HGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ QDGEGHTQDTELVETRPAGDGTFQKWAAvAYZPSGEEQRYTCHVQHEGLPEPVTLRWKPAS QPTI p I VG11AGLVLLGS WS GAWAAVIWRKKS S GGKGGS Y SKAEWS DS AQGS E S HS L (SEQ ID NO: 16976).

[0253] An exemplary gBE Dimer (R and <S) protein of the disclosure comprises or consists of the nucleic acid sequence of: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCCAT CCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACT TCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAAC GGCGAGCGGATCGAGAAGGTGGAA.CACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTA CCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACC ACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATGGGCGGAGGCGGATCT GGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCTGGATCTCACAGCCTGAA. GTACTTTCACACCTCCGTGTCCAGACCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCT ACGTGGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCT AGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGAAGCGCCAG AGACACAGCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGGGGCTACTACAATCAGTCTG AGGCCGGCTCTCACACCCTGCAGTGGATGCATGGATGTGAACTGGGCCCCGACAGACGGTTC CTGAGAGGCTATGAGCAGTTCGCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCT GAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCCA GCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATGGCTGCACAAGTAC CTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAGACACATGTGACCCACCA TCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGA TCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACA AGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCCAGCGGCGAGGA ACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCTGTGACTCTGAGATGGA AGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGAATCATTGCCGGCCTGGTGCTGCTGGGA TCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAA AGGCGGCTCCTACTCTAAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACAGCC TGTAGATAA (SEQ ID NO: 16977).

[0254] An exemplary gBE Dimer (G and 5) protein of the disclosure comprises or consists of the amino acid sequence of: DLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGS GGGGS GGGGS GGGGS GS H S LKY FHT SVS RPGRGE PRFISVGYVDDTQFVRFDN DAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWM HGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ QDGEGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKPAS QPT I PIVG11AGLVLLGSWSGAWAAVIWRKKSS GGKGGS YSKAEWS DS AQGS ESHSL (SEQ ID NO: 16978)

[0255] An exemplary gBE Dimer (G and A) protein of the disclosure comprises or consists of the amino acid sequence of: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCCAT CCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACT TCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAAC GGCGAGCGGATCGAGAAGGTGGAA.CACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTA CCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACC ACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATGGGCGGAGGCGGATCT GGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCTGGATCTCACAGCCTGAA. GTACTTTCACACCTCCGTGTCCAGACCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCT ACGTGGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCT AGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGAAGCGCCAG AGACACAGCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGGGGCTACTACAATCAGTCTG AGGCCGGCTCTCACACCCTGCAGTGGATGCATGGATGTGAACTGGGCCCCGACAGACAGTTC CTGAGAGGCTATGAGCAGTTCGCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCT GAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCCA GCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATGGCTGCACAAGTAC CTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAGACACATGTGACCCACCA TCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGA TCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACA AGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCCAGCGGCGAGGA ACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCTGTGACTCTGAGATGGA AGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGAATCATTGCCGGCCTGGTGCTGCTGGGA TCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAA AGGCGGCTCCTACTC2AAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACAGCC TGTAGATAA (SEQ ID NO: 16979)

[0256] A wildtype / natural human HLA-E protein (NCBI: HLAEHUMAN; UniProt / Swiss- Prot: P13747.4) comprises or consists of the amino acid sequence of: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTOFVRFDNDAA SPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCEL GPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCV EWLHKLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDT ELVETRPAGDGTFQKWAAAAA’PSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAG LVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 17122)

[0257] A nucleotide sequence encoding wildtype / natural HLA-E protein (NCBI: CCDS34379.1) comprises or consists of the nucleotide sequence of: ATGGTAGATGGAACCCTCCTTTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGC GGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCC GCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCG AGTCCGAGGATGGTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCG GGAGACACGGAGCGCCAGGGACACCGCACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCG GCTACTACAATCAGAGCGAGGCCGGGTCTCACACCCTGCAGTGGATGCATGGCTGCGAGCTG GGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTACGACGGCAAGGATTATCT CACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCGAGC AAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTG GAGTGGCTCCACAAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGAGCCCCCAAA. GACACACGTGACTCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGG GCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCAGGATGGGGAGGGCCATACCCAGGAC ACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCAGCTGTGGT ggtgccttctggagaggagcagagatacacgtgccatgtgcagcatgaggggctacccgagc CCGTCACCCTGAGATGGAAGCCGGCTTCCCAGCCCACCATCCCCATCGTGGGCATCATTGCT GGCCTGGTTCTCCTTGGATCTGTGGTCTCTGGAGCTGTGGTTGCTGCTGTGATATGGAGGAA GAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTAAGGCTGAGTGGAGCGACAGTGCCCAGG GGTCTGAGTCTCACAGCTTGTAA (SEQ ID NO: 17123)

[0258] An exemplary WT HLA-E Monomer (R and 61) protein of the disclosure comprises or consists of the amino acid sequence of: MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRF DNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQ WMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQ RAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLT WQQDGEGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKP AS QPTIPIVG11AGLVLLGS WSGAWAAVIWRKKS S GGKGGS Y SKAEWS DS AQGS ES H S L (SEQ ID NO: 16980)

[0259] An exemplary WT HLA-E Monomer (R and 5) protein of the disclosure comprises or consists of the nucleic acid sequence of: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGACTGGAAGCCGG CAGCCACAGCCTGAAGTACTTTCACACCAGCGTGTCCAGACCTGGCAGAGGCGAGCCTAGAT TCATCAGCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCTCT CCTCGGATGGTTCCTAGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGACAGAGA GACAAGAAGCGCCAGAGACACAGCCCAGATCTTCAGAGTGAACCTGCGGACCCTGCGGGGCT ACTACAATCAGTCTGAAGCCGGCTCTCACACCCTGCAGTGGATGCACGGATGTGAACTGGGC CCCGACAGAAGATTCCTGAGAGGCTACGAGCAGTTCGCCTACGACGGCAAGGACTACCTGAC ACTGAACGAGGACCTGAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGA AGTCTAACGACGCCTCTGAGGCCGAACACCAGAGAGCCTACCTGGAAGATACCTGCGTGGAA TGGCTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAGAC ACACGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCT TTTACCCCGCCGAGATCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATACA GAGCTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCCAGAA.ATGGGCTGCTGTGGTGGT TCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCTG TGACACTGAGGTGGAAGCCTGCCAGCCAGCCTACAATCCCCATCGTGGGAATCATTGCCGGC CTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCAGTGGTGGCCGCCGTGATCTGGCGGAAAAA AAGCTCAGGCGGCAAAGGCGGCTCCTACTCCAAAGCCGAGTGGAGCGATTCTGCCCAGGGCT CTGAAAGCCACTCTCTGTAGATAA (SEQ ID NO: 16981).

[0260] An exemplary WT HLA-E Monomer (G and 5) protein of the disclosure comprises or consists of the nucleic acid sequence of: MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRF DNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQ WMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQ RAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLT WQQDGEGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKP AS QPTIPIVG11AGLVLLGS WS GAWAAVIWRKKS S GGKGGS Y SKAEWS DS AQGS ES H S L (SEQ ID NO: 16982) .

[0261] An exemplaiy WT HLA-E Monomer (G and S) protein of the disclosure compri ses or consists of the nucleic acid sequence of: ATGAGCAGAT CT GT G GC C CT GGCT GTT CT GGCT CT GC T GT CT CT GT CT GGAC T G GAAG C C GG CAGCCACAGCCTGAAGTACTTTCACACCAGCGTGTCCAGACCTGGCAGAGGCGAGCCTAGAT TCA.TCAGCGTGGGCTACGTGGA.CGACACCCAGTTCGTCAGA.TTCGACAACGACGCCGCCTCT CCTCGGATGGTTCCTAGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGACAGAGA GACAAGAAGCGCCAGAGACACAGCCCAGATCTTCAGAGTGAACCTGCGGACCCTGCGGGGCT A.CTACAATCAGTCTGAAGCCGGCTCTCACACCCTGCAGTGGATGCACGGATGTGAACTGGGC CCCGACGGAAGATTCCTGAGAGGCTACGAGCAGTTCGCCTACGACGGCAAGGACTACCTGAC ACTGAACGAGGACCTGAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGA AGTCTAACGACGCCTCTGAGGCCGAACACCAGAGAGCCTACCTGGAAGATACCTGCGTGGAA TGGCTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAGAC ACACGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCT TTTACCCCGCCGAGATCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATACA GA.GCTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTGGT TCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACCTG TGACACTGAGGTGGAAGCCTGCCAGCCAGCCTACAATCCCCATCGTGGGAATCATTGCCGGC CTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCAGTGGTGGCCGCCGTGATCTGGCGGAAAAA AAGCTCAGGCGGCAAAGGCGGCTCCTACTCCAAAGCCGAGTGGAGCGATTCTGCCCAGGGCT CTGAAAGCCACTCTCTGTAGATAA (SEQ ID NO: 16983).

[0262] A wildApe / natural human B2M protein (NCBI: B2MG_HUMAN; UniProt / Swiss- Prot: P61769.1) comprises or consists of the amino acid sequence of MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17124)

[0263] A nucleotide sequence encoding wildApe / natural B2M protein (NCBI: CCDS10113.1) comprises or consists of the nucleotide sequence of: ATGTCTCGCTC CCAGCGTACTi GTGGCCTTAGCTGTGCTC AAAGATT 0AGGTTTACTCACGT CAT CCAGCAGAGAATGGAAAGT CAAATT TCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAAT GGAGAGAGAATTGAAAAAGTGGA.GCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTA TCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACC ATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA (SEQ ID NO: 17125)

[0264] An exemplary HLA-bCsBE (Single Chain Trimer) protein of the disclosure compri ses or consists of the amino acid sequence of (B2M Signal peptide, peptide, Linker, B2M domain, Linker, HLA-E peptide): MSRSVALAVLALLSLSGLEAWAPRTZZAGGGGSGGGGSGGGGSZQRTPKZQVYSRHPAENG KSNFLNCYVSGFHPSDIE VDLLKNGERIEK^HSDLSFSI^fSFYLL YYTEETP TEKDEYAC RVMHVTXSgPKZVKWZaWMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFI SVGYVDDTQFVRFDNDAASPRMVPRAPWEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYY NQSEAGSHTLQWHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKS NDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFY PAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVT LRWKPASQPTZPIVGIIAGLVLLGSWSGAVVAAVrWRKKSSGGKGGSYSKAEWSDSAQGSE SHSL (SEQ ID NO: 17064) B2M Signal peptide: MSRSVALAVLALLSLSGLEA. (SEQ ID NO: 17126) Peptide'. VMAPRTLIL (SEQ ID NO: 17127) Linker: GGGGSGGGGSGGGGS (SEO ID NO: 17128) B2M domain’. IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSF YLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17129) Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEOEGSEYWDR ETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYL TLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPK THVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAW VPSGEEQRYTCHVQHEGLPEPWLRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRK KSSGGKGGSYSKAEWSDSAQGSESHSL(SEQ ID NO: 17131)

[0265] An exemplary nucleotide sequence encoding a HLA-bGBE (Single Chain Trimer) protein of the disclosure comprises or consists of the nucleotide sequence of (B2M Signal peptide, peptide. Linker, B2M domain, Linker. HLA-E peptide): kj IU X         I kjkjUUU x UkjC-UU JL bL x       x bb IblbLLlblLl     1 bbAbbLC j. GATGGGG C C C CGgACC CTGA T G CTGGgAGGAGGAGGCAGCGgCgGAGGAGGCT CC GGAGGC G G .P< .1. G ATCCAGCGCACACCTAAGATCCAGG^ AAGAGGAAG^Ii^PGG^GAAtli^PGG^I,AGG^GAGGGGG^P^^PGAGGGtI!tI,GGGAGAtPGGAGGtliGGAtI,G^I, GCTGAAGAATGGCGAGAGAATCGAGAAGGTGGAGCACTCCGACCTGAGCTTCTCCAAGGATT GGTCTTTTTATCTGCTGTACTATACCGAGTTTACCCCTACAGAGAAGGACGAGTACGCCTGT CGCGTGAACCACGTGACACTGTCCCAGCCAAAGATCGTGAAGTGGGACCGGGATATGGGC G G CGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCAGCC ACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAGAGGAGAGCCACGGTTCATC 1G1G1GGGG    1GGAGGA1 ALALAb 1 1GG1GAGG 111 GAGA / aI GAI GGGGGGAGGGGAAG AATGGTGCCTAGGGCCCCATGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGGGAGACCC ill GL>oi?o 1 CyA / xGC* 1 Cy.A.Cy.A^.GUG 1 Cy-A-ol^oCrC- 1 / xG 1-A.l AATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGATGCGAGCTGGGACCAGA TGGCCGCTTCCTGCGGGGCTACGAGCAGTTTGCCTATGACGGCAAGGATTACCTGACCCTGA iAJ^ky.AlyUy.A.C.U 1          C.G J. (j^y / ^.C*GLyGL«(j 1                                   JL                           1GC* AATGACGCATCTGAGGCAGAGCACCAGAGGGCATATCTGGAGGATACCTGCGTGGAGTGGCT GCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGCCCCCTAAGACCCACG H7 / <X / <X / <X / i / 77K / ^ / 1 / ^¾ X rpn A C* / ’)'*4 A i-*1 / 1 7 / 7 / 1^ / 1 / ^ / 1 / ^^ / ^ / 7 / ^07 / ^^ / ^ / ^^1 / 701^ / ^ / 7 / 1 / ^^ / ^^ / ^ / 1 / ^ / 1^007 / ^077^07 1                            1                                         J. Cr / xlsrC? 1 v? 1 1            1 krkrkrC" 1 IC 1 J\1 CCCGCCGAGATCACCCTGACATGGCAGCAGGACGGAGAGGGACACACCCAGGATACAGAGCT GGTGGAGACCAGGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTGGTGCCTT CCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCAGAGCCAGTGACC CTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTATCGTGGGAATCATCGCAGGCCTGGT GCTGCTGGGCTCTGTGGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGGCGGAAGAAGAGCA GCGGAGGCAAGGGAGGCTCCTACTCCAAGGCAGAGTGGAGCGACTCCGCCCAGGGCTCTGAG AGCCACTCCCTGTGA (SEQ ID NO: 17065) B2M Signa? peptide: ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGGCCTGGAGGCC (SEQ 1D NO: 1 / :132 ) Peptide-. GTGATGGCCCCCCGGACCCTGATCCTG (SEQ ID NO: 17133) Linker: GGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT (SEQ ID NO: B2M domain’. ATCCAGCGCACACCTAAGATCCAGGTGTATTCTCGGCACCCAGCCGAGAACGGC.AAGAGCAA CTTCCTGAATTGCTACGTGAGCGGCTTTCACCCTTCCGACATCGAGGTGGATCTGCTGAAGA ATGGCGAGAGAATCGAGAAGGTGGAGCACTCCGACCTGAGCTTCTCCAAGGATTGGTCTTTT TATCTGCTGTACTATACCGAGTTTACCCCTACAGAGAAGGACGAGTACGCCTGTCGCGTGAA CCACGTGACACTGTCCCAGCCAAAGATCGTGAAGTGGGACCGGGATATG (SEQ ID NO: 1713 5 ) Linker: GGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCT (SEQ ID NO: 1 / 13 6) HLA-E peptide: GGCAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAGAGGAGAGCCACG GTTCATCTCTGTGGGCTACGTGGACGATACACAGTTCGTGAGGTTTGACAATGATGCCGCCA GCCCAAGAATGGTGCCTAGGGCCCCATGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGG GAGACCCGGAGCGCCAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACCCTGAGGGG CTACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGATGCGAGCTGG GACCAGATGGCCGCTTCCTGCGGGGCTACGAGCAGTTTGCCTATGACGGCAAGGATTACCTG ACCCTGAACGAGGACCTGAGATCCTGGACCGCCGTGGATACAGCCGCCCAGATCAGCGAGCA GAAGT C CAAT GACGCAT CT GAGGCAGAGCACCJ1GAGGGCATATCT GGAGGATAC CT GC GT GG AGTGGCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGCCCCCTAAG ACCCACGTGACACACCACCCAATCAGCGACCACGAGGCCACCCTGAGGTGTTGGGCACTGGG CTTCTA.TCCCGCCGAGATCACCCTGACATGGCAGCAGGACGGAGAGGGACACACCCAGGATA CAGAGCTGGTGGAGACCAGGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTG GTGCCTTCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCAGAGCC AGTGACCCTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTATCGTGGGAATCATCGCAG GCCTGGTGCTGCTGGGCTCTGTGGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGGCGGAAG AAGAGCAGCGGAGGCAAGGGAGGCTCCTACTCCAAGGCAGAGTGGAGCGACTCCGCCCAGGG CTCTGAGAGCCACTCCCTGTGA (SEQ ID NO: 17137)

[0266] An exemplary HLA-gBE (Single Chain Dimer) protein of the disclosure comprises or consists of the amino acid sequence of (B2M Signal peptide, B2M domain, Linker, HLA-E peptide): HSRSVALAVLAIiLShSG'LEA.IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGS GGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVP RAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRF LRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKY L E KG KE T L L H L E P P KT H V T H H PIS D H EAT L RC WAL G F Y PAE IT L TWQQ DGE GHT QDT E EVE T RPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLG SWSGAWAAVIWRKKSSGGKGGSYYKAEWSDSAQGSE,SHSL (SEQ ID NO: 17066) B2M Signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126) B2M domain'. IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSF YLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17129) Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDR ETRSARDTAQIFFtVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYL TLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPK THVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAW VPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRK KSSGGKGGSYYKAEWSDSAQGSESHSL (SEQ ID NO: 17131)

[0267] An exemplary’ nucleotide sequence encoding a HLA-gBE (Single Chain Dimer) protein of the disclosure comprises or consists of the nucleotide sequence of (B2M Signal peptide, B2Mdomain, Linker. HLA-E peptide): / xl            C* 11? 1       x IsrvyL. X « 1 1 1 bbb 1 1 bU ILylUlUlbylC*!. bbLU x CCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAACT TCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAAC GGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTA CCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACC ACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATGGGCGGAGGCGGATCT GGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCTGGATCTCACAGCCTGAA GTACTTTCACACCTCCGTGTCCAGACCTGGCAGAGGCGAGCCTAGATTCATCAGCGTGGGCT ACGTGGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCT AGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGCGAGACAAGAAGCGCCAG AGACACAGCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGGGGCTACTACAATCAGTCTG AGGCCGGCTCTCACACCCTGCAGTGGATGCATGGATGTGAACTGGGCCCCGACAGACGGTTC CTGAGAGGCTATGAGCAGTTCGCCTACGACGGCAAGGACTACCTGACACTGAACGAGGACCT GAGAA.GCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCAGAAGTCTAACGACGCCA GCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGGAATGGCTGCACAAGTAC CTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAGACACATGTGACCCACCA TCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGGCTTTTACCCTGCCGAGA TCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATACAGAGCTGGTGGAAACA AGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTTGTGCCCAGCGGCGAGGA ACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAAA.CTGTGACTCTGAGATGGA AGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGAATCATTGCCGGCCTGGTGCTGCTGGGA TCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTGGAGAAAGAAGTCCTCTGGCGGCAA AGGCGGCTCCTACTATAAGGCCGAGTGGAGCGATTCTGCCCAGGGCTCTGAAAGCCACAGCC TGTGA (SEQ ID NO: 17067) B2M Signal peptide: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 17132) B2M domain'. ATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACACCCCGCCGAGAACGGCAAGAGCAA CTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGA ACGGCGAGCGGATCGAGAAGGTGGAACACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTC TACCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAA CCACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATG (SEQ ID NO: 17135) Linker: GGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCT (SEQ ID NO: 1 / 13 6) HLA-E peptide: GGATCTCACAGCCTGAAGTACTTTCACACCTCCGTGTCCAGACCTGGCAGAGGCGAGCCTAG ATTCATCAGCGTGGGCTACGTGGACGACACCCAGTTCGTCAGATTCGACAACGACGCCGCCT CTCCTCGGATGGTTCCTAGAGCACCCTGGATGGAACAAGAGGGCAGCGAGTACTGGGATCGC GAGACAAGAAGCGCCAGAGACACAGCCCAGATCTTCCGCGTGAACCTGAGAACCCTGCGGGG CTACTACAATCAGTCTGAGGCCGGCTCTCACACCCTGCAGTGGATGCATGGATGTGAACTGG GCCCCGACAGACGGTTCCTGAGAGGCTATGAGCAGTTCGCCTACGACGGCAAGGACTACCTG ACACTGAACGAGGACCTGAGAAGCTGGACCGCCGTGGATACAGCCGCTCAGATCAGCGAGCA GAAGTCTAACGACGCCAGCGAGGCCGAACACCAGAGAGCCTATCTGGAAGATACCTGCGTGG AATGGCTGCACAAGTACCTGGAAAAGGGCAAAGAGACACTGCTGCACCTGGAACCTCCAAAG ACACATGTGACCCACCATCCTATCAGCGACCACGAGGCCACACTGAGATGTTGGGCCCTGGG CTTTTACCCTGCCGAGATCACACTGACATGGCAGCAGGATGGCGAGGGCCACACACAGGATA CAGAGCTGGTGGAAACAAGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTT GTGCCCAGCGGCGAGGAACAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCTGAACC TGTGACTCTGAGATGGAAGCCTGCCAGCCAGCCAACAATCCCCATCGTGGGAATCATTGCCG GCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTGGAGAAAG AAGTCCTCTGGCGGCAAAGGCGGCTCCTACTATAAGGCCGAGTGGAGCGATTCTGCCCAGGG CTCTGAAAGCCACAGCCTGTGA (SEQ ID NO: 17137)

[0268] An exemplary HLA-bE (Monomer) protein of the disclosure comprises or consists of the amino acid sequence of (B2M Signal peptide, HLA-E peptide): MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAAS PRWPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELG PDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVE WLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDT ELVETRPAGDGTFQKWAAWVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAG LVLLGSWSGAWAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL (SEQ ID NO: 17 0 6 8 ) B2M Signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1 / 126) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDR ETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYL TLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPK THVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAW VPSGEEQRYTCHVQHEGLPEPWLRWKPASQPTIPIVGIIAGLVLLGSWSGAWAAVIWRK KSSGGKGGSYYKAEWSDSAQGSESHSL (SEQ ID NO: 17131)

[0269] An exemplary nucleotide sequence encoding a HLA-bE (Monomer) protein of the disclosure comprises or consi sts of the nucleotide sequence of (B2M Signal peptide, HLA-E peptide): ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGGCCTGGAGGCCGG CAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAGAGGAGAGCCACGGT TCATCTCTGTGGGCTACGTGGACGAAACACAGTTCGTGAGGTTTGACAATGATGCCGCCAGC C CAAGAAT G G T G C C T AG G G C C C CAT G GAT G GAG C AG GAG G G CAG C GAG T AT T G G G AC AG G GA GACCCGGAGCGCCAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACCCTGAGGGGCT ACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGATGCGAGCTGGGA CCAGATCGCCGCTTCCTGCGGGGCTACGAGCAGTTTGCCTATGACGGCAAGGATTACCTGAC CCTGAACGAGGACCTGAGATCCTGGACCGCCGTGGATACAGCCGCCCAGATCAGCGAGCAGA AGTCCAATGACGCATCTGAGGCAGAGCACCAGAGGGCATATCTGGAGGATACCTGCGTGGAG TGGCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGCCCCCTAAGAC CCACGTGACACACCACCCAATCAGCGACCACGAGGCCACCCTGAGGTGTTGGGCACTGGGCT TCTATCCCGCCGAGATCACCCTGACATGGCAGCAGGACGGAGAGGGACACACCCAGGATACA GAGCTGGTGGAGACCAGGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTGGT GCCTTCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCAGAGCCAG TGACCCTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTATCGTGGGAATCATCGCAGGC CTGGTGCTGCTGGGCTCTGTGGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGGCGGAAGAA GAGCAGCGGAGGCAAGGGAGGCTCCTACTATAAGGCAGAGTGGAGCGACTCCGCCCAGGGCT CTGA (SEQ ID NO: 17069) B2M Signal peptide: ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGGCCTGGAGGCC (SEC / ID NO: 17132) HLA-E peptide: GGCAGCCACTCCCTGAAGTATTTCCACACCTCTGTGAGCCGGCCAGGCAGAGGAGAGCCACG GTTCATCTCTGTGGGCTACGTGGACGATACACAGTTCGTGAGGTTTGACAATGATGCCGCCA GCCCAAGAATGGTGCCTAGGGCCCCATGGATGGAGCAGGAGGGCAGCGAGTATTGGGACAGG GAGACCCGGAGCGCCAGAGACACAGCACAGATTTTCCGGGTGAACCTGAGAACCCTGAGGGG CTACTATAATCAGTCCGAGGCCGGCTCTCACACACTCCAGTGGATGCACGGATGCGAGCTGG GACCAGATCGCCGCTTCCTGCGGGGCTACGAGCAGTTTGCCTATGACGGCAAGGATTACCTG ACCCTGAACGAGGACCTGAGATCCTGGACCGCCGTGGATACAGCCGCCCAGATCAGCGAGCA GAAGT C CAAT GACGCATCT GAGGCAGAGCACCAGAGGGCATAT CT GGAGGATAC CT GCGT GG AGTGGCTGCACAAGTACCTGGAGAAGGGCAAGGAGACACTGCTGCACCTGGAGCCCCCTAAG ACCCACGTGACACACCACCCAATCAGCGACCACGAGGCCACCCTGAGGTGTTGGGCACTGGG CTTCTATCCCGCCGAGATCACCCTGACATGGCAGCAGGACGGAGAGGGACACACCCAGGATA CAGAGCTGGTGGAGACCAGGCCCGCCGGCGATGGCACATTTCAGAAGTGGGCCGCCGTGGTG GTGCCTTCCGGAGAGGAGCAGAGATACACCTGTCACGTGCAGCACGAGGGACTGCCAGAGCC AGTGACCCTGAGGTGGAAGCCTGCCAGCCAGCCCACAATCCCTATCGTGGGAATCATCGCAG GCCTGGTGCTGCTGGGCTCTGTGGTGAGCGGAGCAGTGGTGGCCGCCGTGATCTGGCGGAAG AAGAGCAGCGGAGGCAAGGGAGGCTCCTACTATAAGGCAGAGTGGAGCGACTCCGCCCAGGG CTCTGA (SEO ID NO: 17137) Immune and Immune Precursor Cells

[0270] In certain embodiments, immune cells of the disclosure comprise lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (Tscm cells), central memory T cells (Tcm), stem cell-like T cells, B lymphocytes (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[0271] In certain embodiments, immune precursor cells comprise any cells which can differentiate into one or more types of immune cells. In certain embodiments, immune precursor cells comprise multipotent stem cells that can self renew and develop into immune cells. In certain embodiments, immune precursor cells comprise hematopoietic stem cells (HSCs) or descendants thereof. In certain embodiments, immune precursor cells comprise precursor cells that can develop into immune cells. In certain embodiments, the immune precursor cells comprise hematopoietic progenitor cells (HPCs). Hematopoietic Stem Cells (HSCs)

[0272] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

[0273] HSCs of the disclosure may be isolated or derived from a primary or cultured stem cell. HSCs of the disclosure may be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

[0274] Immune precursor cells of the disclosure may comprise an HSC or an HSC descendent cell. Exemplary HSC descendent cells of the disclosure include, but are not limited to, multipotent stem cells, lymphoid progenitor cells, natural killer (XK} cells, T lymphocyte cells (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0275] HSCs produced by the methods of the disclosure may retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the “primitive” HSCs produced by the methods of the disclosure retain their “sternness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the “primitive” HSCs produced by the methods of the disclosure not only replenish their numbers, but expand in vivo. “Primitive” HSCs produced by the methods of the disclosure may be therapeutically-effective when administered as a single dose. In some embodiments, primitive HSCs of the disclosure are CD34+. In some embodiments, primitive HSCs of the di sclosure are CD34+ and CD38-. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38- and CD90+. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38-, CD90+ and CD45RA-. In some embodiments, primitive HSCs of the disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+. In some embodiments, the most primitive HSCs of the disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49fh

[0276] In some embodiments of the disclosure, primitive HSCs, HSCs, and'or HSC descendent cells may be modified according to the methods of the disclosure to express an exogenous sequence (e.g. a chimeric antigen receptor or therapeutic protein). In some embodiments of the disclosure, modified primitive HSCs, modified HSCs, and / or modified HSC descendent cells may be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and / or a modified B-cell of the disclosure. T Cells

[0277] Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0278] Unlike traditional biologies and chemotherapeutics, modified-T cells of the disclosure possess the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, in some embodiments, modified-T cells of the disclosure not only drive an initial response, but also persist in the patient as a stable population of viable memory' T cells to prevent potential relapses. Alternatively, in some embodiments, when it is not desired, modified-T cells of the disclosure do not persist in the patient.

[0279] Intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory' T cells, especially stem cell memory' (Tscm) or stem cell-like T cells. Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (Tcm) T cells or Tcm like cells, effector memory (Tem) and effector T cells (Te), thereby producing better tumor eradication and long-term modified-T cell engraftment. A linear pathway' of differentiation may be responsible for generating these cells: Naive T cells (Tn) > Tscm > Tcm > Tem > Te > Tie, whereby Tn is the parent precursor cell that directly' gives rise to Tscm, which then, in turn, directly gives rise to Tcm, etc. Compositions of T cells of the disclosure may' comprise one or more of each parental T cell subset with Tscm cells being the most abundant (e.g. Tscm > Tcm > Tem > Te > Tte).

[0280] In some embodiments of the methods of the disclosure, the immune cell precursor is differentiated into or is capable of differentiating into an early' memory' T cell, a stem cell like T-cell, a Naive T cells (Tn), a Tscm, a Tcm, a Tem, a Te, or a Tte. In some embodiments, the immune cell precursor is a primitive HSC, an HSC, or a HSC descendent cell of the disclosure.

[0281] In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell, a stem cell like T-cell, a Naive T cells (Tn), a Tscm, a Tcm, a Tem, a Te, or a Tte.

[0282] In some embodiments of the methods of the disclosure, the immune cell is an early memory T cell.

[0283] In some embodiments of the methods of the disclosure, the immune cell is a stem cell like T-cell.

[0284] In some embodiments of the methods of the disclosure, the immune cell is a Tscm.

[0285] In some embodiments of the methods of the disclosure, the immune cell is a Tcm

[0286] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of an early memory T cell. In certain embodiments, the plurality of modified early memory' T cells comprises at least one modified stem cell-like T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified Tscm. In certain embodiments, the plurality of modified early memory' T cells comprises at least one modified Tcm.

[0287] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of a stem cell-like T cell. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified Tscm. In certain embodiments, the plurality of modified stem cell-like T cells comprises at least one modified Tcm.

[0288] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, w'herein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of a stem memory' T cell (Tscm). In certain embodiments, the cell-surface markers comprise CD62L and CD45RA. In certain embodiments, the cell-surface markers comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CD95, IL-2RP, CCR7, and CD62L.

[0289] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of a central memory T cell (Tcm). In certain embodiments, the cell-surface markers comprise one or more of CD45RO, CD95, IL-2R0, CCR7, and CD62L.

[0290] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T ceils, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of a naive T cell (Tn). In certain embodiments, the cell-surface markers comprise one or more of CD45RA, CCR7 and CD62L.

[0291] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at leas t 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality’ of modified T cells expresses one or more cellsurface marker(s) of an effector T-cell (modified Teff). In certain embodiments, the cellsurface markers comprise one or more of CD45RA, CD95, and IL-2RP.

[0292] In some embodiments of the methods of the disclosure, the methods modify and / or the methods produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cellsurface marker(s) of a stem cell-like T cell, a stem memory T cell (Tscm) or a central memory' T cell (Tcm).

[0293] In some embodiments of the methods of the disclosure, a buffer comprises the immune cell or precursor thereof. The buffer maintains or enhances a level of cell viability’ and / or a stem-like phenoty pe of the immune cell or precursor thereof, including T-cells. In certain embodiments, the buffer maintains or enhances a level of cell viability and / or a stemlike phenoty pe of the primary7 human T cells prior to the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability7 and / or a stem-like phenotype of the primary human T cells during the nucleofection. In certain embodiments, the buffer maintains or enhances a level of cell viability and / or a stem-like phenotype of the primary' human T cells foll owing the nucleofection. In certain embodiments, the buffer comprises one or more of KC1, MgCh, CINa, Glucose and Ca(NCh)2 in any absolute or relative abundance or concentration, and, optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and a phosphate buffer. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM CalNOyh. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM CafNChh and a supplement 102 comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KC1, 15 mM MgCh, 90 mM CINa, 10 mM Glucose and 0.4 mM Ca(NOs)2 and a supplement comprising 40 mM Na2HPO4 / NaH2PO4 at pH 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 pl of the buffer and between 5xKO and 25xl06 cells. In certain embodiments, the composition comprises a scalable ratio of 250x10° primary human T cells per milliliter of buffer or other media during the introduction step.

[0294] In some embodiments of the methods of the disclosure, the methods comprise contacting an immune cell of the disclosure, including a T cell of the disclosure, and a T-cell expansion composition. In some embodiments of the methods of the disclosure, the step of introducing a transposon and / or transposase of the disclosure into an immune cell of the disclosure may further comprise contacting the immune cell and a T-cell expansion composition. In some embodiments, including those in which the introducing step of the methods comprises an electroporation or a nucleofection step, the electroporation or a nucleofection step may be performed with the immune cell contacting T-cell expansion composition of the disclosure.

[0295] In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises, consists essentially of or consists of phosphorus; one or more of an octanoic acid, a palmitic acid, a linoleic acid, and an oleic acid; a sterol; and an alkane.

[0296] In certain embodiments of the methods of producing a modified T cell of the disclosure, the expansion supplement comprises one or more cytokine(s). The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplaty lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFy). The one or more cytokine(s) may comprise IL-2.

[0297] In some embodiments of the methods of the disclosure, the T-cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic 103 acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 0.9 mg / kg to 90 mg / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg and a sterol at a concentration of about 1 mg / kg. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of between 6.4 pmol / kg and 640 pmol / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol / kg and 70 pmol / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol / kg and 25 pmol / kg, inclusive of the endpoints. In certain embodiments of this method, the T-cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 pmol / kg, palmitic acid at a concentration of about 7 pmol / kg, linoleic acid at a concentration of about 7.5 pmol / kg, oleic acid at a concentration of about 7.5 pmol / kg and a sterol at a concentration of about 2.5 pmol / kg.

[0298] In certain embodiments, the T-cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement to produce a plurality of expanded modified T-cells, wherein at least 2% of the plurality of modified T-cells expresses one or more cell-surface marker(s) of an early memory T cell, a stem cell-like T cell, a stem memory T cell (Tscm) and / or a central memory T cell (Tcm). In certain embodiments, the T-cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, 104 stearic acid hydrazide, oleamide, a sterol and an alkane. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 0.9 mg / kg to 90 mg / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive of the endpoints (wherein mg / kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and a sterol at a concentration of about 1 mg / kg (wherein mg / kg = parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of about 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and a sterol at a concentration of about 1.01 mg / kg (wherein mg / kg = parts per million). In certain embodiments, the T-cell expansion composition comprises octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and a sterol at a concentration of 1.01 mg / kg (wherein mg / kg :=: parts per million). In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of between 6.4 pmol / kg and 640 pmol / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol / kg and 70 pmol / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol / kg and 25 pmol / kg, inclusive of the endpoints. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 pmol / kg, palmitic acid at a concentration of about 7 pmol / kg, linoleic acid at a concentration of about 7.5 pmol / kg, oleic acid at a concentration of about 7.5 pmol / kg and a sterol at a concentration of about 2.5 pmol / kg. In certain embodiments, the T-cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 pmol / kg, palmitic acid at a concentration of about 7.27 105 pmol / kg, linoleic acid at a concentration of about 7.57 pmol / kg, oleic acid at a concentration of about 7.56 umol / kg and a sterol at a concentration of about 2.61 pmol / kg. In certain embodiments, the T-celi expansion composition comprises octanoic acid at a concentration of about 63.75 pmol / kg, palmitic acid at a concentration of about 7.27 pmol / kg, linoleic acid at a concentration of about 7.57 pmol / kg, oleic acid at a concentration of 7.56 pmol / kg and a sterol at a concentration of 2.61 pmol / kg.

[0299] As used herein, the terms ‘"supplemented T-cell expansion composition” or '‘T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid. In certain embodiments, the media comprises an amount of phosphorus that is 10-fold higher than may be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as Catalog number 12440053).

[0300] As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” mas7 be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove’s MDM, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following elements present in the corresponding average concentrations: boron at 3.7 mg / L, sodium at 3000 mg / L, magnesium at 18 mg / L, phosphorus at 29 mg / L, potassium at 15 mg / L and calcium at 4 mg / L.

[0301] As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used 106 interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g., nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following components: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No. 3322-62-1), phenol red (CAS No. 14374-8) and lanolin alcohol.

[0302] In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate and phosphate.

[0303] As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, isoleucine, leucine, phenylalanine and tryptophan. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free ammo acids in the corresponding average mole percentages: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamic acid (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about 1%), valine (about 3.5%), isoleucine (about 3%), leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan (about 0.5%). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of the following free amino acids in the corresponding average mole percentages: histidine (about .78%), asparagine (about 0.4%), serine (about 1.6%), glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine (about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).

[0304] As used herein, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-Mercaptoethanol, Iscove’s MDM, and an expansion supplement at 37°C. Alternatively, or in addition, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of phosphorus, an octanoic fatty acid, a palmitic fatty acid, a linoleic fatty acid and an oleic acid. In certain embodiments, the 108 media comprises an amount of phosphorus that is 1 O-fold higher than may be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as Catalog number 12440053).

[0305] In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol (e.g. cholesterol). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 0.9 mg / kg to 90 mg / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg, inclusive of the endpoints; and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg, inclusive of the endpoints (wherein mg / kg = parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and a sterol at a concentration of about 1 mg / kg (wherein mg / kg =: parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of about 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and a sterol at a concentration of about 1.01 mg / kg (wherein mg / kg = parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of 9.19 mg / kg, palmitic acid at a concentration of 1.86 mg / kg, linoleic acid at a concentration of 2.12 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and a sterol at a concentration of 1.01 mg / kg (wherein mg / kg == parts per million). In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of between 6.4 pmol / kg and 640 pmol / kg, inclusive of the endpoints; palmitic acid at a concentration of between 0.7 pmol / kg and 70 109 pmol / kg, inclusive of the endpoints; linoleic acid at a concentration of between 0.75 pmol / kg and 75 pmol / kg, inclusive of the endpoints; oleic acid at a concentration of between 0.75 pmol / kg and 75 umol / kg, inclusive of the endpoints; and a sterol at a concentration of between 0.25 pmol / kg and 25 pmol / kg, inclusive of the endpoints. In certain embodiments, the terms ‘"supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 64 pmol / kg, palmitic acid at a concentration of about 7 umol / kg, linoleic acid at a concentration of about 7.5 pmol / kg, oleic acid at a concentration of about 7.5 umol / kg and a sterol at a concentration of about 2.5 pmol / kg.

[0306] in certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 pmol / kg, palmitic acid at a concentration of about 7.27 umol / kg, linoleic acid at a concentration of about 7.57 pmol / kg, oleic acid at a concentration of about 7.56 pmol / kg and a sterol at a concentration of about 2.61 umol / kg. In certain embodiments, the terms “supplemented T-cell expansion composition” or “T-cell expansion composition” may be used interchangeably with a media comprising one or more of octanoic acid at a concentration of about 63.75 umol / kg, palmitic acid at a concentration of about 7.27 pmol / kg, linoleic acid at a concentration of about 7.57 pmol / kg, oleic acid at a concentration of 7.56 pmol / kg and a sterol at a concentration of 2.61 pmol / kg.

[0307] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of the P13K-Akt-mTOR pathway. Modified T-cells of the disclosure, including modified stem cell-like T cells, Tscm and / or Tcm of the disclosure, may be incubated, cultured, grown, stored, or otherwise, combined at any step in the methods of the procedure with a growth medium comprising one or more inhibitors a component of a PI3K pathway. Exemplary inhibitors a component of a PI3K pathway include, but are not limited to, an inhibitor of GSK3p such as TWS119 (also known as GSK 3B inhibitor XII; CAS Number 601514-19-6 having a chemical formula C18H14N4O2). Exemplary inhibitors of a component of a PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO ™). Additional Exemplary’ inhibitors of a component of a P13K pathway include, but are not limited to, an allosteric Akt inhibitor VIII (also referred to as Akti-1 / 2 having Compound number 10196499), ATP competitive inhibitors (Orthosteric inhibitors targeting the ATP-110 binding pocket of the protein kinase B (Akt)), Isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), Azepane derivatives (A series of structures derived from (-)-balanol), Aminofurazans (GSK690693), Heterocyclic rings (7-azaindole, 6-phenylpurine derivatives, pyrrolo|2,3-d] pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, ipatasertib (GDC-0068, RG7440), A-674563, and A-443654), Phenylpyrazole derivatives (AT7867 and ATI3148), Thiophenecarboxamide derivatives (Afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivative (DC 120), uprosertib (GSK2141795)), Allosteric inhibitors (Superior to orthosteric inhibitors providing greater specificity, reduced side-effects and less toxicity), 2,3-diphenylquinoxaline analogues (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f]|l,6]naphthyridin-3(2H)-one derivative (MK-2206)), Alkylphospholipids (Edelfosine (l-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-I8-OCH3) ilmofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphohomocholine), Indole-3-carbinol analogues (Indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindolo [2,3-b]carbazole-2,10-dicarboxylate (SRI3668), OSU-A9), Sulfonamide derivatives (PH-316 and PHT-427), Thiourea derivatives (PIT-1, PIT-2, DM-PIT-1, N-|(l-methyl-lH-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), Purine derivatives (Triciribine (TCN, NSC 154020), triciribine mono-phosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivatives, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-l,3-dien-l-yl)-lH-pyrazol-3-yl]phenol, 3-oxo-tirucallic acid, 3a- and 3P-acetoxy-tirucallic acids, acetoxy-tirucallic acid, and irreversible inhibitors (antibiotics, Lactoquinomycin, Frenolicin B, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-l,2-pyridine derivatives).

[0308] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T cell and an inhibitor of T cell effector differentiation. Exemplary inhibitors of T cell effector differentiation include, but are not limited to, a BET inhibitor (e.g. JQ1, a hienotnazolodiazepine) and / or an inhibitor of the BET family of proteins (e.g. BRD2, BRD3, BRD4, and BRDT).

[0309] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a Tscm and / or a Tcm) of the disclosure, the method comprises contacting a modified T ceil and an agent that reduces nucleo-cytoplasmic Acetyl-CoA. Exemplary agents that reduce nucleo-cytoplasmic Acetyl-CoA include, but are not limited to, 2-hydroxy-citrate (2-HC) as well as agents that increase expression of Acssl.

[0310] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a TSCM and / or a TCM) of the disclosure, the method comprises contacting a modified T cell and a composition comprising ahistone deacetylase (HDAC) inhibitor. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of valproic acid, Sodium Phenylbutyrate (NaPB) or a combination thereof. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising an HD AC inhibitor comprises or consists of Sodium Phenylbutyrate (NaPB).

[0311] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a Tscm and / or a Tcm) of the disclosure, the activation supplement may comprise one or more cytokine(s). The one or more cytokine(s) may comprise any cytokine, including but not limited to, lymphokines. Exemplar}' lympokines include, but are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (INFy). The one or more cytokine(s) may comprise IL-2.

[0312] In certain embodiments of the methods of producing a modified T cell (e.g. a stem cell-like T cell, a Tscm and / or a Tcm) of the disclosure, the activation supplement may comprise one or more activator complexes. Exemplar}' and nonlimiting activator complexes may comprise a monomeric, dimeric, trimeric or tetrameric antibody complex that binds one or more of CD3, CD28, and CD2. In some embodiments, the activation supplement comprises or consists of an activator complex that comprises a human, a humanized or a recombinant or a chimeric antibody . In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3 and CD28. In some embodiments, the activation supplement comprises or consists of an activator complex that binds CD3, CD28 and CD2. Natural Killer (NK) cells

[0313] In certain embodiments, the modified immune or immune precursor cells of the disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes that differentiate from lymphoid progenitor cells.

[0314] Modified NK cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0315] In certain embodiments, non-activated NK cells are derived from CD3-depleted leukopheresis (containing CD14 / CD19 / CD56+ cells).

[0316] In certain embodiments, NK cells are electroporated using a Lonza 4D nucleofector or BTX ECM 830 (500V, 700 usee pulse length, 0.2 mm electrode gap, one pulse). All Lonza 4D nucleofector programs are contemplated as within the scope of the methods of the disclosure.

[0317] In certain embodiments, 5xlOE6 cells were electroporated per electroporation in 100 pL P3 buffer in cuvettes. However, this ratio of cells per volume is scalable for commercial manufacturing methods.

[0318] In certain embodiments, NK cells were stimulated by co-culture with an additional cell line. In certain embodiments, the additional cell line comprises artificial antigen presenting cells (aAPCs). In certain embodiments, stimulation occurs at day 1, 2, 3, 4, 5, 6, or 7 following electroporation. In certain embodiments, stimulation occurs at day 2 following electroporation.

[0319] In certain embodiments, NK cells express CD56. B cells

[0320] In certain embodiments, the modified immune or immune precursor cells of the disclosure are B cells. B cells are a type of lymphocyte that express B cell receptors on the cell surface. B cell receptors bind to specific antigens.

[0321] Modified B cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0322] In certain embodiments, HSPCs are modified using the methods of the di sclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days. In certain embodiments, HSPCs are modified using the methods of the disclosure, and then primed for B cell differentiation in presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for 5 days.

[0323] In certain embodiments, following priming, modified HSPC cells are transferred to a layer of feeder cells and fed bi-weekly, along with transfer to a fresh layer of feeders once per week. In certain embodiments, the feeder cells are MS-5 feeder cells.

[0324] In certain embodiments, modified HSPC cells are cultured with MS-5 feeder cells for at least 7, 14, 21, 28, 30, 33, 35, 42 or 48 days. In certain embodiments, modified HSPC cells were cultured with MS-5 feeder cells for 33 days. Inducible Proapoptotic Polypeptides

[0325] Inducible proapoptotic polypeptides of the disclosure are superior to existing inducible polypeptides because the inducible proapoptotic polypeptides of the disclosure are far less immunogenic. While inducible proapoptotic polypeptides of the disclosure are recombinant polypeptides, and, therefore, non-naturally occurring, the sequences that are recombined to produce the inducible proapoptotic polypeptides of the disclosure do not comprise non-human sequences that the host human immune system could recognize as ■‘non-self' and, consequently, induce an immune response in the subject receiving an inducible proapoptotic polypeptide of the disclosure, a cell comprising the inducible proapoptotic polypeptide or a composition comprising the inducible proapoptotic polypeptide or the cell comprising the inducible proapoptotic polypeptide.

[0326] The disclosure provides inducible proapoptotic polypeptides comprising a ligand binding region, a linker, and a proapoptotic peptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the nonhuman sequence comprises a restriction site. In certain embodiments, the proapoptotic peptide is a caspase polypeptide. In certain embodiments, the caspase polypeptide is a caspase 9 polypeptide. In certain embodiments, the caspase 9 polypeptide is a truncated caspase 9 polypeptide. Inducible proapoptotic polypeptides of the disclosure may be non-naturally occurring.

[0327] Caspase polypeptides of the disclosure include, but are not limited to, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides associated with apoptosis including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides that initiate apoptosis, including caspase 2, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the disclosure include, but are not limited to, those caspase polypeptides that execute apoptosis, including caspase 3, caspase 6, and caspase 7.

[0328] Caspase polypeptides of the disclosure may be encoded by an ammo acid or a nucleic acid sequence having one or more modifications compared to a wild type amino acid or a nucleic acid sequence. The nucleic acid sequence encoding a caspase polypeptide of the disclosure may be codon optimized. The one or more modifications to an amino acid and / or nucleic acid sequence of a caspase polypeptide of the disclosure may increase an interaction, a cross-linking, a cross-activation, or an activation of the caspase polypeptide of the disclosure compared to a wild type amino acid or a nucleic acid sequence. Alternatively, or in addition, the one or more modifications to an amino acid and / or nucleic acid sequence of a caspase polypeptide of the disclosure may decrease the immunogenicity of the caspase polypeptide of the disclosure compared to a wild type amino acid or a nucleic acid sequence.

[0329] Caspase polypeptides of the disclosure may be truncated compared to a wild type caspase polypeptide. For example, a caspase polypeptide may be truncated to eliminate a sequence encoding a Caspase Activation and Recruitment Domain (CARD) to eliminate or minimize the possibility of activating a local inflammatory' response in addition to initiating apoptosis in the cell comprising an inducible caspase polypeptide of the disclosure. The nucleic acid sequence encoding a caspase poly peptide of the disclosure may be spliced to form a. variant amino acid sequence of the caspase polypeptide of the disclosure compared to a wild type caspase polypeptide. Caspase polypeptides of the disclosure may be encoded by recombinant and / or chimeric sequences. Recombinant and / or chimeric caspase polypeptides of the disclosure may include sequences from one or more different caspase polypeptides. Alternatively, or in addition, recombinant and / or chimeric caspase polypeptides of the disclosure may include sequences from one or more species (e.g. a human sequence and a non-human sequence). Caspase polypeptides of the disclosure may be non-naturally occurring.

[0330] The ligand binding region of an inducible proapoptotic polypeptide of the disclosure mas7 include any polypeptide sequence that facilitates or promotes the dimerization of a first inducible proapoptotic polypeptide of the disclosure with a second inducible proapoptotic polypeptide of the disclosure, the dimerization of which activates or induces cross-linking of the proapoptotic polypeptides and initiation of apoptosis in the cell.

[0331] The ligand-binding (“dimerization”) region may comprise any polypeptide or functional domain thereof that will allow' for induction using an endogenous or non-naturally 115 occurring ligand (i.e. and induction agent), for example, a non-naturally occurring synthetic ligand. The ligand-binding region may be internal or external to the cellular membrane, depending upon the nature of the inducible proapoptotic polypeptide and the choice of ligand (i.e. induction agent). A wide variety of ligand-binding polypeptides and functional domains thereof, including receptors, are known. Ligand-binding regions of the disclosure may include one or more sequences from a receptor. Of particular interest are ligand-binding regions for which ligands (for example, small organic ligands) are known or may be readily produced. These ligand-binding regions or receptors may include, but are not limited to, the FKBPs and cyclophilin receptors, the steroid receptors, the tetracycline receptor, and the like, as well as “non-naturally occurring” receptors, which can be obtained from antibodies, particularly the heavy or light chain subunit, mutated sequences thereof, random ammo acid sequences obtained by stochastic procedures, combinatorial syntheses, and the like. In certain embodiments, the ligand-binding region is selected from the group consisting of a FKBP ligand-binding region, a cyclophilin receptor ligand-binding region, a steroid receptor ligandbinding region, a. cyclophilin receptors ligand-binding region, and a tetracycline receptor ligand-binding region. [03321 The ligand-binding regions comprising one or more receptor domain(s) may be at least about 50 amino acids, and fewer than about 350 amino acids, usually fewer than 200 amino acids, either as the endogenous domain or truncated active portion thereof. The binding region may, for example, be small (< 25 kDa, to allow efficient transfection in viral vectors), monomeric, nonimmunogenic, have synthetically accessible, cell permeable, nontoxic ligands that can be configured for dimerization.

[0333] The ligand-binding regions comprising one or more receptor domain(s) may be intracellular or extracellular depending upon the design of the inducible proapoptotic polypeptide and the availability of an appropriate ligand (i.e. induction agent). For hydrophobic ligands, the binding region can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region will usually be external to the cell membrane, unless there is a transport system for internalizing the ligand in a form in which it is available for binding. For an intracellular receptor, the inducible proapoptotic polypeptide or a transposon or vector comprising the inducible proapoptotic polypeptide may encode a signal peptide and transmembrane domain 5’ or 3' of the receptor domain sequence or may have a lipid attachment signal sequence 5' of the receptor domain sequence. Where the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular. 103341 Antibodies and antibody subunits, e.g., heavy or light chain, particularly fragments, more particularly all or part of the variable region, or fusions of heavy and light chain to create high-affinity binding, can be used as a ligand binding region of the disclosure. Antibodies that are contemplated include ones that are an ectopically expressed human product, such as an extracellular domain that would not trigger an immune response and generally not expressed in the periphery (i.e,, outside the CNS / brain area). Such examples, include, but are not limited to low affinity nerve growth factor receptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryomc antigen). Yet further, antibodies can be prepared against haptenic molecules, which are physiologically acceptable, and the individual antibody subunits screened for binding affinity. The cDNA encoding the subunits can be isolated and modified by deletion of the constant region, portions of the variable region, mutagenesis of the variable region, or the like, to obtain a binding protein domain that has the appropriate affinity for the ligand. In this way, almost any physiologically acceptable haptenic compound can be employed as the ligand or to provide an epitope for the ligand. Instead of antibody units, endogenous receptors can be employed, where the binding region or domain is known and there is a useful or known ligand for binding.

[0335] For multimerizing the receptor, the ligand for the ligand-binding region'receptor domains of the inducible proapoptotic polypeptides may be multimeric in the sense that the ligand can have at least two binding sites, with each of the binding sites capable of binding to a ligand receptor region (i.e. a ligand having a first binding site capable of binding the ligandbinding region of a first inducible proapoptotic polypeptide and a second binding site capable of binding the ligand-binding region of a second inducible proapoptotic polypeptide, wherein the ligand-binding regions of the first and the second inducible proapoptotic polypeptides are either identical or distinct). Thus, as used herein, the term “multimeric ligand binding region” refers to a ligan d-binding region of an inducible proapoptotic polypeptide of the disclosure that binds to a multimeric ligand. Multimeric ligands of the disclosure include dimeric ligands. A dimeric ligand of the disclosure may have two binding sites capable of binding to the ligand receptor domain. In certain embodiments, multimeric l igands of the discl osure are a dimer or higher order oligomer, usually not greater than about tetrameric, of small synthetic organic molecules, the individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. A variety of pairs of synthetic ligands and 117 receptors can be employed. For example, in embodiments involving endogenous receptors, dimeric FK506 can be used with an FKBP12 receptor, dimerized cyclosporin A can be used with the cyclophilin receptor, dimerized estrogen with an estrogen receptor, dimerized glucocorticoids with a glucocorticoid receptor, dimerized tetracycline with the tetracycline receptor, dimerized vitamin D with the vitamin D receptor, and the like. Alternatively, higher orders of the ligands, e.g., trimeric can be used. For embodiments involving non-naturally occurring receptors, e.g., antibody subunits, modified antibody subunits, single chain antibodies comprised of heavy and light chain variable regions in tandem, separated by a flexible linker, or modified receptors, and mutated sequences thereof, and the like, any of a large variety of compounds can be used. A significant characteristic of the units comprising a multimeric ligand of the disclosure is that each binding site is able to bind the receptor with high affinity, and preferably, that they are able to be dimerized chemically. Also, methods are available to balance the hydrophobicity / hydrophilicity of the ligands so that they are able to dissolve in serum at functional levels, yet diffuse across plasma membranes for most applications.

[0336] Activation of inducible proapoptotic polypeptides of the disclosure may be accomplished through, for example, chemically induced dimerization (CID) mediated by an induction agent to produce a conditionally controlled protein or polypeptide. Proapoptotic polypeptides of the disclosure not only inducible, but the induction of these polypeptides is also reversible, due to the degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor.

[0337] In certain embodiments, the ligand binding region comprises a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the ligand binding region comprises a FKBP 12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In certain embodiments, in which the ligand binding region comprises a FKBP12 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), the induction agent may comprise API903, a synthetic drug (CAS Index Name: 2-Piperidinecarboxylic acid, l-[(2S)-l-oxo-2-(3,4,5-trimethoxyphenyl)butyl|-, 1,2-ethanediy lbis[imino(2-oxo-2,1 -ethanediyl)oxy-3,1 -phenylene] (1 R)-3-(3,4-dimethoxyphenyl)propylidene]]ester, [2S-[1(R*),2R*[S*[S*[1(R*),2R*]]]]]-(9C1) CAS Registry' Number: 195514-63-7; Molecular Formula: C78H98N4O20; Molecular Weight: 1411.65)). In certain embodiments, in which the ligand binding region comprises a FKBPI2 polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), 118 the induction agent may comprise AP20187 (CAS Registry Number: 195514-80-8 and Molecular Formula: C82H107N5O20). In certain embodiments, the induction agent is an AP20187 analog, such as, for example, API510. As used herein, the induction agents AP20187, AP1903 and AP1510 may be used interchangeably.

[0338] API 903 API is manufactured by Alphora Research Inc. and AP1903 Drug Product for Injection is made by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of the non-ionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, this formulation is a clear, slightly yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in a milky solution. This phase transition is reversed upon re-warming to room temperature. The fill is 2.33 mL in a 3 mL glass vial (approximately 10 mg AP1903 for Injection total per vial). Upon determining a need to administer API 903, patients may be, for example, administered a single fixed dose of AP1903 for Injection (0.4 mg / kg) via IV infusion over 2 hours, using a non-DEHP, nonethylene oxide sterilized infusion set. The dose of AP I 903 is calculated individually for all patients, and is not be recalculated unless body weight fluctuates by ^10%. The calculated dose is diluted in 100 mL in 0.9% normal saline before infusion. In a previous Phase 1 study of AP1903, 24 healthy volunteers were treated with single doses of AP1903 for Injection at dose levels of 0.01, 0.05, 0.1, 0.5 and 1.0 mg / kg infused IV over 2 hours. AP1903 plasma levels were directly proportional to dose, with mean Cmax values ranging from approximately 10-1275 ng / mL over the 0.01-1.0 mg / kg dose range. Following the initial infusion period, blood concentrations demonstrated a rapid distribution phase, with plasma levels reduced to approximately 18, 7, and 1% of maximal concentration at 0.5, 2 and 10 hours post-dose, respectively. API903 for Injection was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile. luliucci J D, et al., J Clin Pharmacol. 41: 870-9, 2001.

[0339] The fixed dose of AP1903 for injection used, for example, may be 0.4 mg / kg intravenously infused over 2 hours. The amount of API 903 needed in vitro for effective signaling of cells is 10-100 nM (1600 Da MW). This equates to 16-160 pg / L or ~0.016-1.6 gg / kg (1.6-160 gg / kg). Doses up to 1 mg / kg were well-tolerated in the Phase I study of AP1903 described above. Therefore, 0.4 mg / kg may be a safe and effective dose of AP1903 for this Phase I study in combination with the therapeutic cells.

[0340] The amino acid and / or nucleic acid sequence encoding ligand binding of the disclosure may contain sequence one or more modifications compared to a wild type amino 119 acid or nucleic acid sequence. For example, the amino acid and / or nucleic acid sequence encoding ligand binding region of the disclosure may be a codon-optimized sequence. The one or more modifications may increase the binding affinity’ of a ligand (e.g. an induction agent) for the ligand binding region of the disclosure compared to a wild type polypeptide. Alternatively, or in addition, the one or more modifications may decrease the immunogenicity of the ligand binding region of the disclosure compared to a wild type polypeptide. Ligand binding regions of the disclosure and / or induction agents of the disclosure may be non-naturally occurring. [0341 ] Modified cells, transposons and / or vectors of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise anon-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an “iC9 safety switch”. In certain embodiments, modified cells and / or transposons of the discl osure may comprise an inducibl e caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, modified cells and / or transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible proapoptotic poly peptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise aFK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide mas7 comprise a modification at position 36 of the sequence. The modifi...

Claims

1. A non-naturally occurring chimeric stimulatory receptor (CSR) comprising:(a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds and wherein the CD2 extracellular domain or a portion thereof to which an agonist binds comprises a D111H mutation;(b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and(c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3Z protein or a portion thereof.

2. The CSR of claim 1, wherein the CD2 extracellular domain comprises an amino acidsequence at least 90% identical to SEQ ID NO: 17119.

3. The CSR of claim 1, wherein the CD2 extracellular domain comprises the amino acidsequence of SEQ ID NO: 17119.

4. The CSR of any one of claims 1 to 3, wherein the CSR comprises an amino acidsequence at least 90% identical to SEQ ID NO: 17118.

5. The CSR of claim 4, wherein the CSR comprises the amino acid sequence of SEQ IDNO: 17118.

6. A nucleic acid sequence encoding the CSR of any one of claims 1 to 5.

7. A vector comprising the nucleic acid sequence of claim 6.

8. A transposon comprising the nucleic acid sequence of claim 6.2019335014   21 May 20269.     A cell comprising the CSR of any one of claims 1 to 5, the nucleic acid of claim 6, thevector of claim 7, or the transposon of claim 8.

10. The cell of claim 9, wherein the cell is an allogeneic cell or the cell is an autologous cell.

11. A composition comprising a plurality of cells comprising the cell of claim 9 or 10.

12. A modified T lymphocyte (T-cell), comprising:(a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and(b) the chimeric stimulatory receptor (CSR) of any one of claims 1 to 5.

13. The modified T-cell of claim 12, further comprising an inducible proapoptotic polypeptide, or further comprising a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

14. The modified T-cell of claim 12 or 13, further comprising a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

15. The modified T-cell of claim 14, wherein the non-naturally occurring polypeptide comprising a HLA-E further comprises: a B2M signal peptide, and / or a B2M polypeptide, and / or a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide, and / or a peptide and a B2M polypeptide.

16. The modified T-cell of claim 15, wherein the non-naturally occurring polypeptide comprising an HLA-E further comprises:a first linker positioned between the B2M signal peptide and the peptide, and2019335014   21 May 2026a second linker positioned between the B2M polypeptide and the peptide encoding the HLA-E.

17. The modified T-cell of claim 12, further comprising a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof.

18. The modified T-cell of claim 17, wherein the non-naturally occurring antigen receptor comprises a chimeric antigen receptor (CAR).

19. The modified T-cell of any one of claims 12 to 18, wherein the CSR is transiently expressed in the modified T-cell or wherein the CSR is stably expressed in the modified T-cell.

20. The modified T-cell of claim 14, wherein the polypeptide comprising the HLA-E polypeptide is transiently expressed in the modified T-cell or wherein the polypeptide comprising the HLA-E polypeptide is stably expressed in the modified T-cell.

21. The modified T-cell of claim 13, wherein the inducible proapoptotic polypeptide is stably expressed in the modified T-cell.

22. The modified T-cell of claim 17, wherein the non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in the modified T-cell.

23. The modified T-cell of any one of claims 12 to 22, wherein the modified T-cell is an allogeneic cell, an autologous cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (TSCM), a central memory T cell (TCM) or a stem cell-like T cell.

24. A composition comprising a population of modified T-cells, wherein a plurality of the modified T-cells of the population comprise the CSR of any one of claims 1 to 5 or wherein a plurality of the modified T-cells of the population comprise the modified T-cell according to any one of claims 12 to 23.2019335014   21 May 202625. A method of treating a cancer comprising administering to a subject in need thereof a therapeutically-effective amount of the composition according to claim 24.

26. Use of the composition according to claim 24 in the manufacture of a medicament for treating a cancer in a subject in need thereof.

27. The method of claim 25, wherein the method further comprises administering to the subject at least one molecule that binds the CSR; or the use of claim 26, wherein the medicament is to be administered with at least one molecule that binds the CSR.

28. A method of producing a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of any one of claims 1 to 5 or a sequence encoding the same, to produce a plurality of modified T-cells under conditions that stably or transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells.

29. A method of expanding a population of modified T-cells comprising introducing into a plurality of primary human T-cells a composition comprising the CSR of any one of claims 1 to 5 or a sequence encoding the same, to produce a plurality of modified T-cells under conditions that stably or transiently express the CSR within the plurality of modified T-cells and preserve desirable stem-like properties of the plurality of modified T-cells and contacting the cells with an activator composition to produce a plurality of activated modified T-cells, wherein expansion of the plurality of modified T-cells is at least two fold higher than the expansion of a plurality of wild-type T-cells not expressing the CSR under the same conditions.

30. A composition comprising a population of modified T-cells expanded by the method of claim 29.

31. A method of treating cancer comprising administering to a subject in need thereof a therapeutically-effective amount of the composition of claim 30.2019335014   21 May 202632. Use of the composition of claim 30 in the manufacture of a medicament for treating cancer comprising administering to a subject in need thereof.

33. The method of claim 31 or the use of claim 32, wherein the modified T-cells within the population of modified T-cells administered to the subject no longer express the CSR.

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