Allogeneic cell compositions and methods of using the same

Abstract

To provide chimeric stimulatory receptors (CSRs), cell compositions comprising CSRs, methods of making and methods of using the same for the treatment of a disease or disorder in a subject.SOLUTION: A non-naturally occurring chimeric stimulatory receptor (CSR) comprises: (a) an ectodomain comprising an 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 727,498, filed September 5, 2018, U.S. Provisional Patent Application No. 62 / 744,073, filed October 10, 2018, U.S. Provisional Patent Application No. 62 / 815,334, filed March 7, 2019, and U.S. Provisional Patent Application No. 62 / 815,880, filed March 8, 2019. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] The present disclosure is directed to molecular biology, and more specifically to chimeric receptors, allogeneic cell compositions, methods for making same, and methods for using same.

[0003] Incorporation by reference of sequence listing The contents of the file named "POTH-046_001WO_SequenceListing.txt", created on September 5, 2019, and having a size of 55.7 MB, are incorporated herein by reference in their entirety. [Background technology]

[0004] There is a long-felt but unmet need in the art for allogeneic cell compositions that overcome the challenges presented by eliminating genes involved in graft-versus-host response and host-versus-graft response. The present disclosure provides allogeneic cell compositions, methods of making and using these compositions, that include improvements in non-naturally occurring structures to restore responsiveness of allogeneic cells to environmental stimuli and reduce or prevent rejection due to natural killer cell-mediated cytotoxicity. Summary of the Invention

[0005] The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising an activating component, wherein the activating component is isolated from 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 from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0006] The activating component can include 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 inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activating component binds. The activating component can include a CD2 extracellular domain or a portion thereof to which an agonist binds.

[0007] The signal transduction domain can include one or more of a human signal transduction domain, 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 inhibitory protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain can include a CD3 protein or a portion thereof. The CD3 protein can include a CD3 zeta protein or a portion thereof.

[0008] The endodomain can further comprise a cytoplasmic domain. The cytoplasmic domain can be isolated from or derived from a third protein. The first protein and the third protein can be the same. 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 the same. The transmembrane domain can be isolated from or derived from a fifth protein. The first protein and the fifth protein can be the same.

[0009] In some embodiments, the activating component does not bind to a naturally occurring molecule. In some embodiments, the activating component binds to a naturally occurring molecule, but the CSR does not transduce a signal upon binding of the activating component to a naturally occurring molecule. In some embodiments, the activating component binds to a non-naturally occurring molecule. In some embodiments, the activating component does not bind to a naturally occurring molecule, but does bind to a non-naturally occurring molecule. The CSR can selectively transduce a signal upon binding of the activating component to a non-naturally occurring molecule. In a preferred embodiment, the present disclosure provides a non-naturally occurring chimeric stimulating 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 the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising 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 the at least one signal transduction domain comprises a CD3 zeta protein or a portion thereof. In some embodiments, the non-naturally occurring 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 embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 17062.

[0010] The present disclosure also provides non-naturally occurring chimeric stimulating receptors (CSRs) in which the ectodomain comprises a modification. The modification can include a mutation or truncation of the amino acid sequence of the activating component or first protein when compared to the wild-type sequence of the activating component or first protein. The mutation or truncation of the amino acid sequence of the activating component can include a mutation or truncation of the 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 to naturally occurring CD58. In some embodiments, 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 embodiment, the CD2 extracellular domain comprising the mutation or truncation comprises the amino acid sequence of SEQ ID NO: 17119.

[0011] In a preferred embodiment, the present disclosure provides a non-naturally occurring chimeric stimulating 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, the activation component comprises an agonist-binding CD2 extracellular domain or a portion thereof, and the agonist-binding CD2 extracellular domain or a portion thereof comprises a mutation or truncation; (b) a transmembrane domain comprising 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 the at least one signal transduction domain comprises a CD3 zeta protein or a portion thereof. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 17118. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 17118.

[0012] The present disclosure provides a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0013] The present disclosure provides a cell comprising any of the CSRs disclosed herein.The present disclosure provides a cell comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0014] The modified cells disclosed herein can be allogeneic cells or autologous cells.In some preferred embodiments, the modified cells are allogeneic cells.In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0015] The present disclosure provides a composition comprising any of the CSRs disclosed herein.The present disclosure provides a composition comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs 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.

[0016] The present disclosure provides modified T lymphocytes (T cells) comprising: (a) a modification in an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; and (b) a chimeric stimulating receptor (CSR) that comprises: (i) an ectodomain that includes an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain that includes at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, where the first protein and the second protein are not identical.

[0017] The modified T cells can further comprise an inducible pro-apoptotic polypeptide. The modified T cells can further comprise a modification of the endogenous sequence encoding beta-2-microglobulin (B2M), which reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0018] The engineered T cells 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 an HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, the linker being 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 HLA-E can further comprise a first linker positioned between the B2M polypeptide and the peptide and a second linker positioned between the B2M polypeptide encoding HLA-E and the peptide.

[0019] The engineered T cells 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).

[0020] The CSR may be transiently expressed in the modified T cells. The CSR may be stably expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be transiently expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be stably expressed in the modified T cells. An inducible pro-apoptotic polypeptide may be transiently expressed in the modified T cells. An inducible pro-apoptotic polypeptide may be stably expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein may be transiently expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein may be stably expressed in the modified T cells.

[0021] The modified T cells can be autologous cells. The modified T cells can be allogeneic cells. The modified T cells can be early memory T cells, stem cell-like T cells, stem memory T cells (T SCM ), central memory T cells (T CM ), or stem cell-like T cells.

[0022] The present disclosure provides compositions comprising any of the modified T cells disclosed herein. The present disclosure also provides compositions comprising a population of modified T lymphocytes (T cells), wherein a plurality of the population of modified T cells comprises a CSR disclosed herein. The present disclosure also provides compositions comprising a population of T lymphocytes (T cells), wherein a plurality of the population of T cells comprises a modified T cell disclosed herein.

[0023] The present disclosure provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of any of the compositions disclosed herein, or a composition for use in treating a disease or disorder. In one embodiment, the composition is a modified T cell or a population of modified T cells disclosed herein. The present disclosure also provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds to CSR.

[0024] The present disclosure provides methods for producing a population of modified T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a CSR of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, to produce a plurality of modified T cells. The present disclosure provides compositions comprising the population of modified T cells produced by this method. In some embodiments, 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 are stem memory T cells (T SCM ) or T SCM In some embodiments, 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 are central memory T cells (T CM ) or T CMThe composition expresses one or more cell surface markers of T-cell-like cells, and the one or more cell surface markers include CD45RO and CD62L. The composition can be for use in treating a disease or disorder. The present disclosure also provides uses of the compositions produced by the methods for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition produced by the methods. The treatment method can further comprise administering to the subject an activator composition to activate the population of in vivo modified T cells, to induce cell division of the population of in vivo modified T cells, or a combination thereof.

[0025] The present disclosure provides methods for producing a population of engineered T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a CSR of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express the CSR in the plurality of engineered T cells and maintain desirable stem-like properties of the plurality of engineered T cells, to produce a plurality of engineered T cells. The present disclosure provides compositions comprising the population of engineered T cells produced by this method. In some embodiments, 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 are stem memory T cells (T SCM ) or T SCMIn some embodiments, 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 are central memory T cells (T CM ) or T CM The modified T cells express one or more cell surface markers of CD45-like cells, and the one or more cell surface markers include CD45RO and CD62L. The composition can be for use in treating a disease or disorder. The present disclosure also provides uses of the compositions produced by the method for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition produced by the method. In some embodiments, the modified T cells within the population of modified T cells administered to the subject no longer express CSR.

[0026] The present disclosure provides a method of expanding a population of modified T cells, comprising: introducing a composition comprising a CSR of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells to produce a plurality of modified T cells; and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the CSR under the same conditions. In some embodiments, 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 CSRs are stem memory T cells (T SCM ) or T SCM In some embodiments, 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 are central memory T cells (T CM ) or T CMThe modified T cells express one or more cell surface markers of T cell-like cells, and the one or more cell surface markers include CD45RO and CD62L. The present disclosure provides a composition comprising a population of modified T cells expanded by the present method. The composition can be for use in treating a disease or disorder. The present disclosure also provides use of a composition expanded by the method for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition expanded by the present method. The treatment method can further comprise administering to the subject an activator composition 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.

[0027] The present disclosure provides a method for expanding a population of modified T cells, the method comprising: introducing a composition comprising a CSR of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express a CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells to produce a plurality of modified T cells; and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not transiently express the CSR under the same conditions. The present disclosure provides a composition comprising the population of modified T cells expanded by the method. In some embodiments, 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 CSRs are stem memory T cells (T SCM ) or T SCMIn some embodiments, 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 are central memory T cells (T CM ) or T CM The composition expresses one or more cell surface markers of CD45-like cells, and the one or more cell surface markers include CD45RO and CD62L. The composition can be for use in treating a disease or disorder. The present disclosure also provides uses of the composition expanded by the method for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of the composition expanded by the method. In some embodiments, the engineered T cells within the population of engineered T cells administered to the subject no longer express CSR.

[0028] Any of the above aspects can be combined with any other aspect.

[0029] 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 invention belongs. As used herein, unless the context clearly dictates otherwise, the singular can also include the plural; for example, 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 elements. Throughout this specification, the word "comprising," or variations such as "comprises" or "comprising," should be understood to mean 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 may be understood to be 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."

[0030] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this 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. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the following detailed description, and from the claims. [Brief explanation of the drawings]

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent and patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] [Figure 1] FIG. 1 is a schematic diagram showing the T cell receptor (TCR) and co-receptors CD28 and CD2. [Figure 2]

[0023] Figure 1 is a schematic diagram showing that primary and secondary costimulation is delivered to T cells via binding of agonist mAbs (anti-CD3, anti-CD28, and anti-CD2). Full T cell activation is highly dependent on TCR engagement in combination with a second signal from a costimulatory receptor that promotes the immune response. Primary and secondary costimulation can be delivered to T cells via treatment with and engagement of surface receptors with agonist mAbs (e.g., anti-CD3, anti-CD28, and anti-CD2). [Figure 3] Schematic diagram showing that in the absence of a TCR, only secondary costimulation is delivered to T cells via binding of an agonist mAb. Because full T cell activation is highly dependent on primary stimulation via CD3ζ in conjunction with a second signal via a costimulatory receptor, T cell activation and expansion are suboptimal and therefore reduced. [Figure 4]

[0023] Figure 1 is a schematic diagram showing that stimulation in the absence of TCR is enhanced by expression of chimeric stimulating receptors (CSRs). In the absence of TCRs but in the presence of surface-expressed CSRs / s, primary and secondary costimulatory signals are delivered when T cells are treated with standard agonist mAbs. More complete T cell activation is achieved via CSR-mediated stimulatory signals, thus enhancing T cell activation and expansion. [Figure 5] FIG. 1 is a schematic diagram showing an exemplary CSR CD28z of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram showing an exemplary CSR CD2z of the present disclosure. [Figure 7]Figure 1 shows a schematic diagram of a strategy for mutating CSR CD2z to eliminate binding of its natural ligand (CD58). 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 sensitivity to binding by anti-CD2 activator reagents. This may be desirable for two main reasons: 1) CD58 expression by activated T cells may interact with wild-type (WT) CD2z CSR, potentially preventing optimal performance of CSR, and 2) WT CD2z CSR may act as a natural ligand, CAR, such that CSR-expressing T cells may mediate cytotoxic activity against cells expressing CD58, including activated T cells. Therefore, mutant CD2z CSRs that are unable to interact with CD58 but retain the ability to bind activating anti-CD2 reagents for optimal cell expansion are desired. [Figure 8] 1 is a schematic diagram showing an exemplary CSR CD2z-D111H of the present disclosure. The D111H mutation is within the CD2 extracellular domain of the CSR CD2z-D111H construct. [Figure 9A]

[0023] Figure 1 shows a series of plots showing that piggyBac® delivery of CSR enhances the expansion of TCRb / b2M double knockout CAR-T cells. Pan-T cells isolated from the blood of a normal donor were genetically modified using the piggyBac® DNA modification system in combination with the CAS-CLOVER™ gene editing system. Cells were electroporated in a single reaction with a transposon encoding the CAR, a selection gene, and CSR (either CD28z or CD2z), mRNA encoding the super piggyBac™ transposase enzyme, mRNA encoding Cas-CLOVER™, and multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out the TCR and MHCI (double knockout; DKO). Cells were then stimulated with agonist mAbs, anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification over a 16-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). A greater expansion of DKO cells was observed in samples expressing either CD2z or CD28z CSR, as was a higher frequency of CAR-only DKO cells (Figures 9A and 9B). At least a two-fold expansion of cells was observed in DKO CAR-T cell samples expressing either CD2z or CD28z CSR compared to DKO CAR-T cells alone. [Figure 9B] Same as the explanation for Figure 9A. [Figure 10A](A) and (B) are a series of plots showing that CSR CD2z or CD28z of purified DKO CAR-T cells results in enhanced expansion upon restimulation. After the initial genetic modification and 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-MHCI- cells using magnetic beads. The purified cells were then restimulated using anti-CD2, anti-CD3, and anti-CD28 agonist mAbs. At the end of the 14-day culture period, TCR and MHCI expression (A) and the magnitude of expansion of the cell population (B) were determined. After this secondary expansion, all purified DKO cells, including cells expressing either CD2z or CD28z CSR, remained highly pure relative to DKO cells (>98.8% DKO). DKO CAR-T cells expressing either CD2z or CD28z CSR resulted in enhanced expansion when compared with cells expressing neither CSR. [Figure 10B] Same as the explanation for Figure 10A. [Figure 11]Figure 1 shows a graph demonstrating that cytokine supplementation can further expand purified DKO CAR-T cells expressing CSR upon restimulation. After initial genetic modification and a first round of stimulation and expansion, CSR-expressing cells were purified for DKO cells using magnetic beads. The purified cells were then restimulated using anti-CD2, anti-CD3, and anti-CD28 agonist mAbs in the presence of exogenous purified recombinant IL7 and IL15. At the end of the 14-day culture period, the magnitude of expansion of the cell population was determined. After secondary expansion, all purified DKO cells, including those expressing either CD2z CSR or CD28z CSR, remained highly pure relative to TCR-MHCI- cells (>98.8% double knockout (data not shown)). In addition, cells robustly proliferated in the presence of IL7 and IL15, which was greater than without supplementation. These data indicate that exogenous cytokines can be added to further expand WT CAR-T cells expressing CSR. [Figure 12] 1 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 surface expression of CAR and compared to control WT CAR-T cells and mock T cells. Expression of CD2z or CD28z CSR is not significantly affected by expression of CAR molecules on the T cell surface. [Figure 13]

[0023] Figure 1 is a graph showing that CSR expression does not significantly affect the cytotoxicity of DKO CAR-T cells 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 K562-PSMA-luciferase (eK562-Luc.PSMA) at an E:T ratio of 10:1, 3:1, or 1:1 for 48 hours. Luciferase signal was measured to determine cytotoxicity. eK562-Luc.PSMA kill is indicated by a dotted line, while eK562-Luc.BCMA kill is indicated by a solid line. All CAR+ T cells expressed an anti-BCMA-specific CAR. DKO CAR-T cells exhibited in vitro cytotoxicity similar to that of WT CAR-TCR cells. This activity was not significantly affected by co-expression of CD2z or CD28z CSR. [Figure 14]

[0023] Figure 1 is a graph showing that CSR expression does not significantly affect DKO CAR-T cell secretion of IFNg in vitro. Supernatants from 48-hour killing assays were assayed for secreted IFNg as a measure of antigen-specific function of BCMA CAR-T cells. All CAR-T cells, regardless of CD2z or CD28z CSR expression, secrete IFNg in response to coculture with BCMA-expressing target cells (eK562-Luc.BCMA), whereas cells expressing an irrelevant target (eK562-Luc.PSMA) do not secrete IFNg. [Figure 15]This is a series of plots showing that CSR expression 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 labeled with Cell Trace Violet (CTV) and diluted as cells expanded. Cells were cocultured with eK562-Luc.PSMA or eK562-Luc.BCMA cells at an E:T ratio of 1:2 for 5 days. Regardless of the presence or absence of CD2z or CD28z, all CAR-T cells proliferated in response to BCMA-expressing target cells (eK562-Luc.BCMA) but not to cells expressing an irrelevant antigen (eK562-Luc.PSMA). [Figure 16] This pair of graphs shows that the memory phenotype of DKO CAR-T cells is not significantly affected by 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 surface CD45RA, CD45RO, and CD62L expression to define Tscm, Tcm, Tem, and Teff cells: Tscm (CD45RA+CD45RO-CD62L+), Tcm (CD45RA-CD45RO+CD62L+), Tem (CD45RA-CD45RO+CD62L-), and Teff (CD45RA+CD45RO-CD62L-). Regardless of the presence or absence of CD2z, WT and DKO CAR-T cells consist primarily of highly favored Tscm and Tcm cells. However, when CD28z is expressed in DKO CAR-T cells, the phenotype becomes significantly more differentiated, favoring Tcm and Tem cells, which may adversely affect the in vivo function of these CAR T cells because they appear more differentiated. [Figure 17]This is a series of graphs showing that the expression of activation / exhaustion markers in DKO CAR-Ts is not significantly affected by 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 key depletion molecules Lag3, PD1, and Tim3. Regardless of the presence or absence of CD2z, WT and DKO CAR-T cells show little to no expression of depletion molecules compared to mock T cells. However, expression of CD28z CSR in DKO CAR-Ts during the expansion process results in significant upregulation of the exhaustion markers Lag3, PD1, and Tim3. This phenotype may negatively impact the in vivo function of these CAR T cells, as they appear more exhausted. In contrast, expression of CD2z has little or no effect on the exhaustion phenotype of DKO CAR-T cells, but significantly enhances the expansion capacity of the cells. [Figure 18]10 is a graph showing that CSR delivery enhances CAR-T cell expansion. CSR was delivered to CAR-T cells either transiently via mRNA or stably via piggyBac®. Pan-T cells isolated from the blood of normal donors were genetically modified using the piggyBac® DNA modification system and standard Poseida process. Cells were electroporated in a single reaction with mRNA encoding the Super piggyBac™ transposase enzyme (SPB), a transposon encoding a BCMA CAR, and a selection gene, along with additional mRNA encoding CSR (either CD28z or CD2z; resulting in transient expression), or a CD19 mRNA control, or a transposon encoding a BCMA CAR, a selection gene, and a CSR (either CD28z or CD2z; resulting in stable expression). Cells were then stimulated with agonist mAbs, anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification over a 19-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). Bars represent the total live CAR-T cells in the well, and numbers indicate the fold enhancement of expansion over CAR-T cells produced in the absence of CSR or CD19 mRNA control. Samples expressing either CD2z or CD28z CSR, either transiently or stably, exhibited a greater degree of expansion of CAR-T cells. [Figure 19]1 is a bar graph showing that CSR expression is not significantly affected by CAR-T cell cytotoxicity. CSR was delivered to CAR-T cells either transiently via mRNA or stably via piggyBac®. Pan-T cells isolated from the blood of normal donors were genetically modified using the piggyBac® DNA modification system and 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 a selection gene, along with additional mRNA encoding CSR (either CD28z or CD2z; resulting in transient expression), or a transposon encoding a BCMA CAR, a selection gene, and a CSR (either CD28z or CD2z; resulting in stable expression). Cells were then stimulated with agonist mAbs, anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification over a 19-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). To evaluate the killing ability of CAR-T cells, cells were cocultured with engineered K562-BCMA-luciferase (eK562-Luc.BCMA) or the negative control strain K562-luciferase (eK562-Luc) at an E:T ratio of 10:1, 3:1, or 1:1 for 48 hours. Luciferase signal was measured to determine cytotoxicity. eK562-Luc killing is shown in the left bar graph, and eK562-Luc.BCMA killing is shown in the right bar graph. All CAR+ T cells expressed anti-BCMA-specific CARs and exhibited similar in vitro cytotoxicity against BCMA+ target cells. In summary, this activity was not significantly affected by transient or stable CSR coexpression. [Figure 20]

[0023] Figure 1 is a schematic diagram showing that stimulation in the presence of TCR is enhanced by expression of chimeric stimulating receptors (CSRs). In the presence of surface-expressed CSRs, either transiently or stably expressed, enhanced primary and secondary costimulatory signals are delivered when T cells are treated with a reagent that displays an agonist mAb. In one embodiment, this schematic diagram represents autologous cells. More complete T cell activation is achieved through stimulatory signals mediated by CSRs, thereby enhancing T cell activation and expansion. [Figure 21] This is a series of graphs showing that CSR is expressed on the surface of T cells and does not cause cell activation in the absence of exogenous stimuli. Pan-T cells from normal blood donors were stimulated with anti-CD3 / anti-CD28 beads in standard T cell culture medium and then rested. These cells were then electroporated (BTX ECM830 electroporator at 500V, 700µs) with 10µg of mRNA encoding either CD28 CSR, CD2 CSR, or wild-type CD19 control. Two days later, electroporated cells were examined for surface expression of each molecule by flow cytometry, and the data are displayed as stacked histograms. In addition, cell size (FSC-A) and CD69 expression were assessed as possible indicators of cell activation over mock-electroporated control cells. Increased surface expression of CD28, CD2, and CD19 was detected in T cells electroporated with either 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 stimuli. [Figure 22]

[0023] Figure 1 is a series of line graphs showing that CSR molecules can be delivered transiently during manufacturing for enhanced CAR-T cell expansion. Pan-T cells isolated from the blood of healthy donors 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. Autologous CAR-T cells were produced by nucleofection of mRNA encoding the super piggyBac® transposase enzyme (SPB), and a transposon encoding the CAR, a selection gene, and a safety switch. For the production of AlloCAR-T, cells were electroporated (EP) in a single reaction with mRNA encoding the SPB enzyme, mRNA encoding CC, multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out TCR and MHCI, and a transposon encoding either the CAR, a selection gene, and the CSR CD2z, or a transposon encoding a safety switch that did not encode the CAR, selection gene, and CSR. For CAR-T cells that did not receive the transposon-encoded CSR for stable integration, the CD2z CSR was transiently provided to cells as mRNA only once in the first EP reaction, at varying amounts of 5 μg, 10 μg, and 20 μg in a 100 μl EP reaction. After EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification using the selection gene over a 19-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). Each data point is displayed as a line graph for various production dates. A greater degree of CAR-T cell expansion was observed in samples in which CD2z CSR was provided stably (transposon-encoded) or transiently (mRNA-encoded) compared to CAR-T cells generated without CSR.These data indicate that CSR can be transiently delivered as mRNA during manufacturing to enhance the expansion of both autologous and allogeneic CAR-T products. [Figure 23A] Figure 23B shows bar graphs depicting 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 and subcloned into an in-house mRNA generation vector, and high-quality mRNA was then generated for each. K562 cells were electroporated with 9 μg of mRNA, and surface expression of each molecule was analyzed by flow cytometry the following day. The data are shown in bar graphs. Each molecule was stained with an anti-CD2 activator reagent, an anti-CD2 monoclonal antibody (clone TS1 / 8), or an anti-CD2 polyclonal antibody reagent (goat anti-human CD2). Variable binding was observed for each construct, and the data are summarized in Figure 23C. [Figure 23B]This is a series of bar graphs showing CSR CD2z mutant degranulation data. A panel of CSR CD2z mutants was tested for their ability to mediate degranulation against CD58-positive cell targets. T cell degranulation, a surrogate for T cell death, can be measured by FACS staining of intracellular CD107a expression after co-culture with target cell lines expressing the target antigen. Specifically, pan-T cells from normal blood donors were stimulated with anti-CD3 / anti-CD28 beads in standard T cell culture medium and then rested. These cells were then electroporated (BTX ECM830 electroporator at 500V, 700µs) with 9µg of mRNA expressing the CSR CD2z mutants and cultured overnight. The following day, the cells were co-cultured for 4-6 hours in the presence of various target cell lines. Positive target cell lines included K562 or Rat2 cells electroporated or lipofected with mRNA encoding human CD58, respectively; negative controls were either non-electroporated Rat2 cells or T cells expressing CSR CD2z mutants alone. Only T cells expressing CSR CD2z mutants that recognized surface-expressed human CD58 were able to degranulate at levels above background. Little reactivity was observed with D111H, K67R / Y110D, K67R / Q70K / Y110D / D111H, deltaK106-120, CD3z deletion, and sham controls; the data are summarized in Figure 23C. [Figure 23C] Summary of staining and degranulation data. Data from surface expression and binding studies, as well as data from degranulation experiments for each CSR CD2z mutant, are summarized in the table. Two candidates that retain binding to anti-CD2 activator reagents that are expressed on the surface and / or do not mediate anti-CD58 degranulation activity are the D111H and K67R / Y110D CSR CD2z mutants. Only the D111H mutant strongly binds all staining reagents on the cell surface and completely abolishes anti-CD58 degranulation activity. [Figure 23D]A series of flow cytometry plots showing the expression of CD48, CD58, or CD59 in 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, was tested. Degranulation of engineered T cells was assessed against cell lines K562 and Rat2 engineered to overexpress the target ligands, and expression was confirmed by FACS staining. Red histograms represent unstained cells; blue histograms represent cells electroporated / lipofected with mRNA and stained for expression of the respective markers by FACS. [Figure 23E]This bar graph shows that CSR CD2z recognizes human CD58 but not CD48 or CD59. To confirm possible ligands for the CSR WT CD2z molecule, a panel of known and suspected ligands, including human CD48, CD58, and CD59, was tested. Degranulation of engineered T cells was assessed against cell lines K562 and Rat2 engineered to overexpress the target ligands, and expression was confirmed by FACS staining. Cells were electroporated / lipofected with mRNA and stained for expression of the respective markers by FACS. As a control, a BCMA CAR was included, as was a K562 cell line overexpressing BCMA. In addition, GFP-transfected T cells were also included as a control. T cell degranulation, a surrogate for T cell death, can be measured by FACS staining for intracellular CD107a expression after coculture with a target cell line expressing the target antigen. Pan-T cells from normal blood donors were stimulated with anti-CD3 / anti-CD28 beads in standard T cell culture medium and then rested. These T cells were then electroporated with mRNA expressing CSR WT CD2z, BCMA CAR, or GFP and cultured overnight. The following day, the cells were co-cultured for 4–6 hours in the presence of various target cell lines electroporated / lipofected with mRNA encoding human CD48, CD58, or CD59. Negative controls included either non-electroporated K562 or Rat2 cells, or electroporated T cells alone. T cells expressing either CSR WT CD2z or BCMA CAR were able to degranulate at levels above background when co-cultured with cell lines overexpressing human CD58 or BCMA, respectively, but were unable to degranulate to human CD48 or CD59. Little reactivity was observed with GFP-expressing T cells. [Figure 24A]1 is a bar graph showing that delivery of the CSR CD2z-D111H mutant enhances the expansion of Allo CAR-T cells. Pan-T cells isolated from the blood of healthy donors 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 CSR (no CSR) for the production of autologous (Auto) CAR-T cells, without CC gene editing as a control. Auto CAR-T cells were produced by nucleofection of mRNA encoding the super piggyBac® transposase enzyme (SPB), and a transposon encoding the CAR, a selection gene, and a safety switch. For the production of Allo CAR-T cells, cells were electroporated (EP) in a single reaction with mRNA encoding the SPB enzyme, mRNA encoding CC, multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out TCR and MHCI, and a transposon encoding either the CAR, selection gene, and CSR CD2z, as well as either WT or mutant (D111H) CSR CD2z, or a transposon encoding a safety switch that did not encode the CAR, selection gene, and CSR. For the latter Allo CAR-T cells that did not receive the transposon-encoded CSR for stable integration, WT or mutant (D111H) CSR CD2z was transiently provided to cells as mRNA only once in the first EP reaction. After EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification using the selection gene over a culture period of up to 15 days. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown), and then all unedited TCR-positive cells were depleted via negative selection to obtain a population of Allo CAR-T cells that were >99% TCR-negative (data not shown).All samples were run in duplicate, except for the Auto (no CSR) control, and the peak expansion data for each (indicating the day of peak expansion) are shown in bar graphs with error bars representing standard deviation. A greater degree of expansion of Allo CAR-T cells was observed in samples provided with WT or mutant (D111H) CD2z stably (transposon-encoded) or transiently (mRNA-encoded) compared to Allo CAR-T cells generated without CSR. [Figure 24B]This is a series of bar graphs showing that delivery of the CSR CD2z-D111H 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 generate allogeneic (Allo) CAR-T cells. Cells were electroporated (EP) in a single reaction with mRNA encoding the SPB enzyme, mRNA encoding the CC, multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out TCR and MHCI, and a transposon encoding either a CAR, a selection gene, and either WT or mutant (D111H) CSR CD2z, or a transposon encoding a safety switch that did not encode a CAR, selection gene, and CSR. For the latter cells that did not receive the transposon-encoded CSR for stable integration, WT or mutant (D111H) CSR CD2z was transiently provided as mRNA only once in the first EP reaction. After EP, all cells were subsequently stimulated with a cocktail of agonist mAbs anti-CD2, anti-CD3, and anti-CD28 and then selected for gene modification using a selection gene over a culture period of up to 15 days. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). All samples were run in duplicate, and data are shown in bar graphs with error bars representing standard deviation. Similar or greater degrees of gene editing of allo-CAR-T cells were observed in samples receiving WT or mutant (D111H) CD2z stably (as transposon-encoded) or transiently (as mRNA-encoded) compared to allo-CAR-T cells generated without CSR. [Figure 24C]This bar graph demonstrates that the memory phenotype of Allo CAR-T cells is not significantly affected by delivery of CD2z CSR. Allo CAR-T cells without CSR and Allo CAR-T cells with CSR delivered either stably or transiently were stained for surface CD45RA, CD45RO, and CD62L expression to define Tscm, Tcm, Tem, and Teff cells: TSCM (CD45RA+CD45RO-CD62L+), Tcm (CD45RA-CD45RO+CD62L+), Tem (CD45RA-CD45RO+CD62L-), and Teff (CD45RA+CD45RO-CD62L-). All samples were run in duplicate, and the data are shown in bar graphs with error bars representing standard deviation. Delivery of CSR did not dramatically affect the levels of favorable Tscm and Tcm cells within the product. [Figure 25] FIG. 1 is a schematic diagram illustrating an exemplary HLA-bGBE composition of the present disclosure. [Figure 26] FIG. 1 is a schematic diagram showing an exemplary HLA-gBE composition of the present disclosure. [Figure 27] This pair of graphs shows that expression of single-chain HLA-E reduces NK cell-mediated cytotoxicity against HLA-deficient T cells. B2M and TCRαβ were knocked out from Jurkat T cells using CRISPR. B2M / TCRαβ double knockout (DKO) T cells were electroporated with mRNA encoding the HLA-E molecule (HLA-bGBE) and single-chain expressed with B2M and the peptide VMAPRETLIL (SEQ ID NO: 17127) (B2M / peptide / HLA-E). DKO T cells electroporated with various amounts of mRNA encoding single-chain HLA-E were used as targets for NK cell expansion in a 3-hour coculture with artificial antigen-presenting cells (aAPCs). The percentage of cytotoxicity was calculated based on the number of target cells remaining after 3 hours compared to target cells alone. These data show that surface expression of HLA-E on DKO T cells reduces the total level of cell killing by NK cells in a dose-dependent manner. [Figure 28]List of gRNA sequences (top to bottom) and primer sequences (top to bottom). [Figure 29] This is a series of flow cytometry plots showing targeted knockout of endogenous HLA-ABC, but not HLA-E. Having demonstrated that surface expression of HLA-E in MHCI KO T cells can enhance resistance to NK cell-mediated cytotoxicity, we explored further strategies beyond the introduction of a single HLA-E gene. To do so, multiple guide RNAs (gRNAs) were designed to disrupt expression of the primary host-versus-graft (HvG) targets, HLA-A, HLA-B, and HLA-C, while minimizing disruption of endogenous HLA-E. Specifically, the guides were designed to target conserved regions that occur in all 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 the 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 one round of T cell expansion, and the data are presented below. These data demonstrate that gene editing techniques can be used to target the destruction of MHCI while maintaining the levels of endogenous HLA-E on the surface of gene-edited T cells. [Figure 30] FIG. 1 is a schematic diagram of the loss-of-self hypothesis of natural killer cell-mediated toxicity against MHCI-KO cells. [Figure 31] Schematic representation of the Csy4-T2A-Clo051-G4Slinker-dCas9 construct map (Embodiment 2). [Figure 32] 1 is a schematic representation of the pRT1-Clo051-dCas9 double NLS construct map (Embodiment 1). [Figure 33] FIG. 1 is a schematic diagram showing an exemplary method for producing an allogeneic CAR-T of the present disclosure. [Figure 34A]1 is a graph showing highly efficient gene incorporation of endogenous TCRa in proliferating Jurkat cells and resting primary human pan-T cells as an exemplary method for the production of allogeneic and universal CAR-T using Cas-CLOVER™ (an RNA-guided fusion protein comprising dCas9-Clo051). The Cas-CLOVER system disrupted the expression of TCRα at relatively high levels in rapidly proliferating Jurkat T cells and non-dividing resting T cells. [Figure 34B]

[0023] Figure 1 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™. TCRa, TCRB, and B2M, key targets mediating alloreactivity, were efficiently edited by Cas-CLOVER in resting human T cells. [Figure 35] A series of flow cytometry plots showing the ability of Cas-CLOVER to multiplex by co-delivering TCRβ and β2M reagents to primary human T cells. TCRβ / β2M double knockout (DKO) cells were further enriched using antibody bead-based purification, and purified cells were analyzed by FACS for downregulation of surface-expressed CD3 and β2M. [Figure 36]This is a series of graphs showing the reduction in alloreactivity after TCR and MHCI knockout. The alloreactivity of WT or DKO (TCR and MHCI) CAR-T cells was analyzed by mixed lymphocyte reaction (MLR) and IFNγ by ELISpot assay. On the left, WT or gene-edited DKO CAR-T cells were labeled with celltrace violet (CTV) and mixed at a 1:1 ratio with irradiated peripheral blood mononuclear cells (PBMCs) and incubated for 12 days or 20 hours, respectively, before analyzing proliferation- or activation-induced secretion of IFNγ by ELISpot assay. WT or DKO CAR-T cells were incubated at a 1:1 ratio with PBMCs from either allogeneic (donor #1 PBMCs and donor #2 PBMCs) or autologous (autologous PBMCs) donors. After 12 days, CTV dye dilution was assessed by FACS. These results demonstrated significant proliferation of WT CAR-T cells when incubated with allogeneic PBMCs. Proliferation rates of 40% and 39% were observed when WT CAR-T cells were cultured with allogeneic PBMCs from two different donors, compared to only 2% when WT CAR-T cells were incubated with autologous PBMCs. Meanwhile, DKO CAR-T cells did not proliferate when incubated with allogeneic PBMCs, indicating that knockout of the TCR and MHCI resulted in the elimination of graft-versus-host alloreactivity. This was also true for short-term IFNγ assays (bottom left), which showed that only WT CAR-T cells were activated and secreted IFNγ when incubated with allogeneic PBMCs, whereas DKO CAR-T cells did not. On the right, irradiated WT or DKO CAR-T cells were mixed with CFSE-labeled PBMCs at a 1:1 ratio and incubated for 12 days or 20 hours, respectively, before analysis of proliferation- or activation-induced secretion of IFNγ by ELISpot assay. After 12 days, CFSE dye dilution was assessed by FACS. These results show significant proliferation of PBMCs (mostly T cells) when incubated with allogeneic CAR-T cells, with 37% and 9% of PBMCs proliferating compared to only 2% when incubated with autologous CAR-T cells.On the other hand, PBMCs did not proliferate above background when incubated with allogeneic CAR-T cells, indicating that TCR and MHCI knockout resulted in the elimination of host-versus-graft alloreactivity. This was also true for short-term IFNγ by ELISpot assay (bottom left), which showed that only WT CAR-T cells, but not DKO CAR-T cells, were activated and secreted IFNγ by PBMCs when incubated with allogeneic CAR-T. [Figure 37] This is a series of graphs showing that DKO and WT CAR-T have similar CAR expression and stem-like phenotype. Gene editing does not affect CAR-T cell phenotype. TCRβ / β2M DKO and WT T cells expressing a BCMA CAR 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 of T stem cell memory (TSCM). These data indicate that gene editing of allogeneic CAR-T does not significantly reduce the composition of memory CAR-T cells, which remain highly elevated and predominantly retain a Tscm phenotype. [Figure 38] This is a series of graphs demonstrating that DKO CAR-Ts are highly functional. Gene editing does not affect CAR-T cell functionality. TCRβ / β2M DKO and WT T cells expressing a BCMA CAR were analyzed for function. Proliferation against the H929 (BCMA+) tumor line was assessed by mixing CAR-T cells with H929 cells, incubating for 7 days, and analyzing for tumor-specific proliferation by FACS. Cytotoxicity and IFNg secretion against the H929 (BCMA+) tumor line were assessed by mixing CAR-T cells with H929 cells at various ratios, incubating for 24 hours, and analyzing for tumor-specific killing by FACS. Cytotoxicity data are normalized to tumor cell-only samples. These data demonstrate that gene editing to generate DKO CAR-T cells does not significantly affect their functional capacity. [Figure 39A]Figure 1 shows a schematic diagram illustrating the preclinical evaluation of the P-PSMA-101 transposon when delivered by full-length plasmid (FLP) versus nanotransposon (NT) at a "stress" dose using a mouse xenograft model. A mouse xenograft model using the luciferase-expressing LNCaP cell line (LNCaP.luc) subcutaneously (SC) injected into nonsteroidal anti-cancer (NSG) mice was utilized to evaluate the in vivo antitumor efficacy of the P-PSMA-101 transposon, as delivered by full-length plasmid (FLP) or nanotransposon (NT), at two different "stress" doses (2.5 x 10^6 or 4 x 10^6) of total CAR-T cells from two different normal donors. All CAR-T cells were generated using piggyBac® (PB) delivery of the P-PSMA-101 transposon using either FLP or NT delivery. Mice were injected with LNCaP into the axilla and treated when tumors developed (100-200 mm3 by caliper measurement). Mice were treated with two different "stress" doses (2.5x10^6 or 4x10^6) of P-PSMA-101 CAR-T by IV injection for more resolution in detecting possible functional differences in efficacy between transposon delivery by FLP and NT. [Figure 39B]

[0033] Figure 34A is a series of graphs showing tumor volume assessments of mice treated as described in Figure 34A. Tumor volume assessments 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) are displayed as group means with error bars (top) and individual mice (bottom). The y-axis shows tumor volume (mm3) assessed by caliper measurement. The x-axis shows days after T cell treatment. A "stress" dose of P-PSMA-101 transposon delivered by NT showed enhanced antitumor efficacy, as measured by caliper, compared to FLP and control mice against established SC LNCaP.luc solid tumors. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, and where the first protein and the second protein are not identical.

[0034] The activating component can comprise, consist essentially of, or consist of one or more of a human transmembrane receptor, a human cell surface receptor, a component of the T cell receptor (TCR), a component of the 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 activating component can comprise, consist essentially of, or consist of a portion of one or more of a component of the T cell receptor (TCR), a component of the 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 agonist of the activating component binds.

[0035] The ectodomain can comprise, consist essentially of, or consist of an agonist-binding CD2 extracellular domain or a portion thereof, or the ectodomain can comprise, consist essentially of, or consist of an agonist-binding CD28 extracellular domain or a portion thereof. The activating component can comprise, consist essentially of, or consist of an agonist-binding CD2 extracellular domain or a portion thereof, or the activating component can comprise, consist essentially of, or consist of an agonist-binding CD28 extracellular domain or a portion thereof. The agonist-binding CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17111. The agonist-binding CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17111. The CD2 extracellular domain to which the agonist binds comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17111. The CD28 extracellular domain to which the agonist binds comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which the agonist binds comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17099. The CD28 extracellular domain to which the agonist binds comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17099.

[0036] The signal transduction domain can comprise, consist essentially of, or consist of one or more of a human signal transduction domain, a component of a T cell receptor (TCR), a component of a TCR complex, a component of a TCR coreceptor, 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 essentially of, or consist of a CD3 protein or a portion thereof. The signal transduction domain can comprise, consist essentially of, or consist of a CD3 protein or a portion thereof. The CD3 protein can comprise, consist essentially of, or consist of a CD3 zeta protein or a portion thereof. The CD3 zeta protein comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3 zeta protein comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17102. The CD3ζ protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:17102.

[0037] The endodomain of the CSR of the present disclosure can further comprise, consist essentially of, or consist of a cytoplasmic domain. The cytoplasmic domain can be isolated from or derived from a third protein. In some embodiments, the first protein and the third protein of the CSR of the present disclosure are identical. The cytoplasmic domain can comprise, consist essentially of, or consist of a CD2 cytoplasmic domain or a portion thereof, or the cytoplasmic domain can comprise, consist essentially of, or consist of a CD28 cytoplasmic domain or a portion thereof.

[0038] The CD2 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17113. The CD2 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 17113. The CD28 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17101. The CD28 cytoplasmic domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17101.

[0039] The endodomain of the CSR of the present disclosure can further comprise, consist essentially of, or consist of a signal peptide. The signal peptide can be isolated from or derived from a fourth protein. In some embodiments, the first protein and the fourth protein of the CSR of the present disclosure are identical. The signal peptide can comprise, consist essentially of, or consist of a CD2 signal peptide or a portion thereof; the signal peptide can comprise, consist essentially of, or consist of a CD28 signal peptide or a portion thereof; or the signal peptide can comprise, consist essentially of, or consist of a CD8a signal peptide or a portion thereof. The CD2 signal peptide comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17110. The CD28 signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17098. The CD28 signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17098. The CD8a signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17037. The CD8a signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17037.

[0040] The transmembrane domain of the CSR of the present disclosure can be isolated from or derived from the fifth protein. In some embodiments, the first protein and the fifth protein of the CSR of the present disclosure are identical. The transmembrane domain can comprise, consist essentially of, or consist of the CD2 transmembrane domain or a portion thereof, or the transmembrane domain can comprise, consist essentially of, or consist of the CD28 transmembrane domain or a portion thereof. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17112. The CD28 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17100. The CD28 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17100.

[0041] In some embodiments, the activating component of the CSR of the present disclosure does not bind or is unable to bind to a naturally occurring molecule. In some embodiments, the activating component of the CSR of the present disclosure binds or is capable of binding to a naturally occurring molecule, and the CSR transduces a signal upon binding of the activating component to the naturally occurring molecule. In other embodiments, the activating component of the CSR of the present disclosure can bind to a naturally occurring molecule, but the CSR does not transduce a signal upon binding of the activating component to the naturally occurring molecule. In a preferred embodiment, the activating component of the CSR of the present disclosure binds or is capable of binding to a non-naturally occurring molecule. The activating component of the CSR of the present disclosure selectively transduces a signal upon binding of a non-naturally occurring molecule to the activating component. In one embodiment, the naturally occurring molecule is a naturally occurring agonist / activator for the activating component of the CSR. The naturally occurring agonist / activator capable of binding to the CSR activating 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 embodiments, the naturally occurring agonist / activator capable of binding to a CSR activating 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 one embodiment, the non-naturally occurring molecule is a non-naturally occurring agonist / activator for the CSR activating component. The non-naturally occurring agonist / activator capable of binding to a CSR activating 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 embodiments, the non-naturally occurring agonist / activator capable of binding to a CSR activating 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 embodiments, the non-naturally occurring agonist / activator capable of binding to a CSR activating component may be selected from the group consisting of the anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998), and the humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (siplizumab) (Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009).

[0042] In some embodiments, the ectodomain of the CSR of the present disclosure can include a modification. The modification can include a mutation or truncation of the amino acid sequence of the activating component or first protein compared to the wild-type sequence of the activating component or first protein. The mutation or truncation of the amino acid sequence of the activating component or first protein can include a mutation or truncation of the CD2 extracellular domain or a portion thereof to which the agonist binds. The mutation or truncation of the CD2 extracellular domain reduces or eliminates binding to naturally occurring CD58.

[0043] Reduced binding is a reduction in the binding ability of the mutated or truncated CD2 extracellular domain by 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% when compared to a naturally occurring wild-type counterpart. Elimination of binding is a reduction in the binding ability of the mutated or truncated CD2 extracellular domain by 100% when compared to a naturally occurring wild-type CD2 extracellular domain.

[0044] The mutated or truncated CD2 extracellular domain binds to anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind to naturally occurring CD58. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17119. The mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising a mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising a mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising a mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17118.

[0045] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein, and where the activating component binds to a non-naturally occurring molecule but not to a naturally occurring molecule; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, and where the first protein and the second protein are not identical.

[0046] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating moiety, where the activating moiety is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, where the first protein and the second protein are not identical, and where the CSR does not transduce a signal upon binding of a naturally occurring molecule to the activating moiety.

[0047] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, where the first protein and the second protein are not identical, and where the CSR transduces a signal upon binding of a non-naturally occurring molecule to the activating component.

[0048] The present disclosure also provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising a signal peptide and an activating component, wherein the signal peptide comprises the CD2 signal peptide or a portion thereof, and the activating component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the 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 the CD2 cytoplasmic domain or a portion thereof, and the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

[0049] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activating 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 essentially 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 essentially of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO: 17062. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 90% identical to SEQ ID NO: 17062. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 95% identical to SEQ ID NO: 17062. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 99% identical to SEQ ID NO: 17062. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17062.

[0050] The present disclosure further provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising, consisting essentially 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 the activation component comprises a wild-type CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising 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 the at least one signal transduction domain comprises a CD3 zeta protein or a portion thereof. In one embodiment, mutation or truncation of the CD2 extracellular domain reduces or eliminates binding to naturally occurring CD58. In another embodiment, the mutated or truncated CD2 extracellular domain binds to anti-CD2 activating agonists and anti-CD2 activating molecules but does not bind to naturally occurring CD58.

[0051] The present disclosure further provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activating 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 stimulatory receptor (CSR) can comprise, consist essentially 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 essentially of, or consist of an amino acid sequence at least 85% identical to SEQ ID NO: 17118. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 90% identical to SEQ ID NO: 17118. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 95% identical to SEQ ID NO: 17118. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 99% identical to SEQ ID NO: 17118. A non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17118.

[0052] The present disclosure also provides a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides a transposon, vector, donor sequence, or donor plasmid comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. In one embodiment, the vector can be a viral vector. In one embodiment, the viral vector can be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector.

[0053] The present disclosure also provides cells comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides cells comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides cells comprising, consisting essentially of, or consisting of a transposon, vector, donor sequence, or donor plasmid comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. In one aspect, the vector can be a viral vector. In one aspect, the viral vector can be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector. The cells of the present disclosure comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein can be allogeneic or autologous cells. In some preferred embodiments, the cells are allogeneic cells.

[0054] The present disclosure also provides compositions comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a transposon, vector, donor sequence, or donor plasmid comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. In one embodiment, the vector can be a viral vector. In one embodiment, the viral vector can be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a cell or a plurality of cells comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein.

[0055] The present disclosure provides modified cells comprising, consisting essentially of, or consisting of a chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, and where the first protein and the second protein are not identical.

[0056] The present disclosure also provides a modified cell comprising, consisting essentially of, or consisting of: (a) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, wherein the first protein and the second protein are not identical; and (b) an inducible pro-apoptotic polypeptide.

[0057] The present disclosure also provides a modified cell comprising, consisting essentially of, or consisting of: (a) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, wherein the first and second proteins are not identical; (b) a sequence encoding an inducible pro-apoptotic polypeptide, and the cell is a T cell; and (c) a modification in an endogenous sequence encoding a T cell receptor (TCR), where the modification reduces or eliminates the level of TCR expression or activity.

[0058] The present disclosure provides modified cells that comprise, consist essentially of, or consist of: (a) a modification of an endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity; and (b) a non-naturally occurring sequence that includes an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

[0059] The present disclosure provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; and (b) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0060] The present disclosure provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, wherein the first protein and the second protein are not identical; and (c) a non-naturally occurring chimeric antibody receptor.

[0061] The present disclosure provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) a modification of an endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity; and (c) a chimeric stimulating receptor (CSR) that comprises: (i) an ectodomain that includes an activating component, wherein the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain that includes at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0062] The present disclosure provides a modified T lymphocyte (T cell) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) a modification of an endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity; and (c) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated from a first protein. (ii) an ectodomain isolated from or derived from a first protein; (iii) a transmembrane domain; and (iv) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein, and the first protein and the second protein are not identical.

[0063] The present disclosure also provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) a modification of an endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity; (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulating receptor (CSR) that comprises (i) an activating component. Also provided is a modified T lymphocyte (T cell) comprising, consisting essentially of, or consisting of a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising a first protein, wherein the activating component is isolated from 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 from or derived from a second protein, and wherein the first protein and the second protein are not identical.

[0064] The present disclosure also provides a modified T lymphocyte (T cell) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) a modification of an endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity; (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain that comprises an activating component; Also provided is an engineered T lymphocyte (T cell) comprising, consisting essentially of, or consisting of: a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain isolated from 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 from or derived from a second protein, and the first and second proteins are not identical; and (e) a non-naturally occurring chimeric antigen receptor.

[0065] The present disclosure also provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; (b) a modification that reduces or eliminates the level of expression or activity of 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 stimulating receptor (CSR) that comprises: (i) an endogenous sequence encoding an activating component; Also provided is a modified T lymphocyte (T cell) comprising, consisting essentially of, or consisting of a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, and where the first protein and the second protein are not identical.

[0066] The present disclosure also provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; (b) a modification that reduces or eliminates the level of expression or activity of 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 that includes HLA class I histocompatibility antigen alpha chain E (HLA-E); and (d) a chimeric stimulating receptor. Also provided are modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, where the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, where the at least one signal transduction domain is isolated from or derived from a second protein, and where the first protein and the second protein are not identical.

[0067] The modified cells of the disclosure (preferably, the modified T cells of the disclosure) can further comprise, consist essentially of, or consist of an inducible pro-apoptotic polypeptide. The inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 14641. The inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14641.

[0068] The modified cells of the present disclosure (preferably, the modified T cells of the present disclosure) can further comprise, consist essentially of, or consist of a modification of the endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity. A reduced level of expression or activity is when the expression of MHC-I in the cell or the functional activity of MHC-I in the cell is reduced by 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% when compared to the naturally occurring wild-type counterpart of the cell. A reduced level of expression or activity is when the expression of MHC-I in the T cell or the functional activity of MHC-I in the T cell is reduced by 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% when compared to a naturally occurring wild-type T cell of the cell. An elimination of the level of expression or activity is when the expression of MHC-I in the cell or the functional activity of MHC-I in the cell is reduced by 100% when compared to a naturally occurring wild-type counterpart of the cell. An elimination of the level of expression or activity is when the expression of MHC-I in the T cell or the functional activity of MHC-I in the T cell is reduced by 100% when compared to a naturally occurring wild-type T cell.

[0069] The modified cells of the present disclosure (preferably, the modified T cells of the present disclosure) can further comprise, consist essentially of, or consist of a non-naturally occurring polypeptide comprising HLA class I histocompatibility antigen alpha chain E (HLA-E). The HLA-E polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The HLA-E polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17131.

[0070] The non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a B2M signal peptide. The B2M signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17126. The B2M signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17131. The B2M signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17131.

[0071] A non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a B2M polypeptide. The B2M polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17129.

[0072] The non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a linker molecule (herein referred to as a linker). The non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a linker, wherein the linker is located between the B2M polypeptide and the HLA-E polypeptide. The linker comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17130.

[0073] The non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a peptide and a B2M polypeptide. The peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17127.

[0074] The non-naturally occurring polypeptide comprising HLA-E can further comprise, consist essentially of, or consist of a first linker located between the B2M signal peptide and the peptide, and a second linker located between the B2M polypeptide and the HLA-E polypeptide. The first linker comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17128. The second linker comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:17130.

[0075] In one embodiment, the non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially 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 located between the B2M signal peptide and the first linker, the B2M polypeptide can be located between the first linker and the second linker, and the second linker can be located between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17064. A non-naturally occurring polypeptide comprising HLA-E can be encoded by a nucleic acid having the sequence of SEQ ID NO:17065.

[0076] In one embodiment, the non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of a B2M signal peptide, a B2M polypeptide, a linker, and an HLA-E polypeptide. The B2M polypeptide can be located between the B2M signal peptide and the linker, and the linker can be located between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising HLA-E can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17066. The non-naturally occurring polypeptide comprising HLA-E can be encoded by a nucleic acid having the sequence of SEQ ID NO: 17067.

[0077] In one embodiment, a non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of a B2M signal peptide and an HLA-E polypeptide. The B2M signal peptide can be located before the HLA-E polypeptide (e.g., 5' in the context of the nucleic acid sequence, or amino-terminal in the context of the amino acid sequence). A non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17068. A non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17068. A non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17068. A non-naturally occurring polypeptide comprising HLA-E can be encoded by a nucleic acid having the sequence of SEQ ID NO: 17069.

[0078] The modified cells of the present disclosure (preferably, the modified T cells of the present disclosure) can further comprise, consist essentially of, or consist of a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In a preferred embodiment, the non-naturally occurring antigen receptor comprises, consists essentially of, or consists of a chimeric antigen receptor (CAR). The CAR comprises, consists essentially of, or consists 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 essentially of, or consist of a signal peptide. The ectodomain of the CAR can further comprise, consist essentially of, or consist of a hinge between the antigen recognition region and the transmembrane domain. The endodomain of the CAR can further comprise, consist essentially of, or consist of a human CD3ζ endodomain. At least one costimulatory domain of the CAR can further comprise, consist essentially of, or consist of human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In a preferred embodiment, at least one costimulatory domain comprises a human CD28 and / or 4-1BB costimulatory domain.

[0079] The modified cells of the present disclosure can be immune cells or immune cell precursors. The immune cells can be 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). In a preferred embodiment, the immune cells are T cells, early memory T cells, stem cell-like T cells, stem memory T cells (T SCM ), central memory T cells (T CM), or stem cell-like T cells. The immune cell precursor can be 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 embodiment, the cell is a modified allogeneic T cell. In another embodiment, the cell is a modified allogeneic T cell expressing a chimeric antigen receptor (CAR), a CAR T cell.

[0080] The modified cells of the present disclosure (preferably, modified T cells of the present disclosure) can transiently or stably express the CSR of the present disclosure. In one embodiment, the CSR of the present disclosure is transiently expressed in the modified cells of the present disclosure (preferably, modified T cells of the present disclosure). In one embodiment, the CSR of the present disclosure is stably expressed in the modified cells of the present disclosure (preferably, modified T cells of the present disclosure).

[0081] The modified cells of this disclosure (preferably, modified T cells of this disclosure) can transiently or stably express a non-naturally occurring polypeptide comprising an HLA-E of this disclosure. In one embodiment, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is transiently expressed in the modified cells of this disclosure (preferably, modified T cells of this disclosure). In one embodiment, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is stably expressed in the modified cells of this disclosure (preferably, modified T cells of this disclosure).

[0082] The modified cells of the present disclosure (preferably, modified T cells of the present disclosure) can transiently or stably express the inducible pro-apoptotic polypeptides of the present disclosure. In one embodiment, the inducible pro-apoptotic polypeptides of the present disclosure are transiently expressed in the modified cells of the present disclosure (preferably, modified T cells of the present disclosure). In a preferred embodiment, the inducible pro-apoptotic polypeptides of the present disclosure are stably expressed in the modified cells of the present disclosure (preferably, modified T cells of the present disclosure).

[0083] The engineered cells of the present disclosure (preferably, engineered T cells of the present disclosure) can transiently or stably express a sequence encoding a non-naturally occurring antigen receptor or a therapeutic protein of the present disclosure. In one embodiment, the sequence encoding a non-naturally occurring antigen receptor or a therapeutic protein of the present disclosure is transiently expressed in the engineered cells of the present disclosure (preferably, engineered T cells of the present disclosure). In a preferred embodiment, the sequence encoding a non-naturally occurring antigen receptor or a therapeutic protein of the present disclosure is stably expressed in the engineered cells of the present disclosure (preferably, engineered T cells of the present disclosure).

[0084] In one embodiment, a CSR of this disclosure is stably expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0085] In one embodiment, a CSR of this disclosure is stably expressed, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is stably expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0086] In one embodiment, a CSR of this disclosure is stably expressed, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0087] In one embodiment, a CSR of this disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0088] In one embodiment, a CSR of this disclosure is transiently expressed, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0089] In one embodiment, a CSR of this disclosure is transiently expressed, a non-naturally occurring polypeptide comprising an HLA-E of this disclosure is stably expressed, an inducible pro-apoptotic polypeptide of this disclosure is stably expressed, and a sequence encoding a non-naturally occurring antigen receptor or therapeutic protein is stably expressed in an engineered cell of this disclosure (preferably, an engineered T cell of this disclosure).

[0090] The present disclosure provides modified cells, preferably modified T cells, comprising, consisting essentially of, or consisting of: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the level of TCR expression or activity; and (b) a sequence encoding a chimeric stimulating receptor (CSR), wherein the CSR comprises: (i) an ectodomain comprising an activating component, wherein the activating component is isolated from 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 from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0091] The modified cells can further comprise, consist essentially of, or consist of a sequence encoding an inducible pro-apoptotic polypeptide. The modified cells can further comprise, consist essentially 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 essentially of, or consist of a chimeric antigen receptor (CAR).

[0092] The transposon, vector, donor sequence, or donor plasmid can comprise, consist essentially of, or consist of a sequence encoding CSR, a sequence encoding an inducible pro-apoptotic polypeptide, or a combination thereof. The transposon, vector, donor sequence, or donor plasmid can further comprise, consist essentially of, or consist of a sequence encoding a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein. The transposon, vector, donor sequence, or donor plasmid can further comprise, consist essentially of, or consist of a sequence encoding a selectable marker. The transposon can be a piggyBac® transposon, piggy-Bac®-like transposon, Sleeping Beauty transposon, Helraiser transposon, Tol2 transposon, or TcBuster transposon. The CSR-encoding sequence can be transiently expressed in cells. The CSR-encoding sequence can be stably expressed in cells. The inducible pro-apoptotic polypeptide-encoding sequence can be stably expressed in cells. The sequence encoding a non-naturally occurring antigen receptor or the sequence encoding a therapeutic protein is stably expressed in the cell. In some embodiments, the sequence encoding a CSR can be transiently expressed in the cell, and the sequence encoding an inducible pro-apoptotic polypeptide can be stably expressed in the cell. In some embodiments, the sequence encoding a CSR can be stably expressed in the cell, and the sequence encoding an inducible pro-apoptotic polypeptide can be stably expressed in the cell. In some embodiments, the sequence encoding a CSR can be transiently expressed in the cell, and the sequence encoding an inducible pro-apoptotic polypeptide can be stably expressed in the cell, and the sequence encoding a non-naturally occurring antigen receptor or the sequence encoding a therapeutic protein is stably expressed in the cell.In some embodiments, the sequence encoding CSR can be stably expressed in cells, the sequence encoding an inducible pro-apoptotic polypeptide can be stably expressed in cells, and the sequence encoding a non-naturally occurring antigen receptor or the sequence encoding a therapeutic protein can be stably expressed in cells. In one embodiment, the vector can be a viral vector. In one embodiment, the viral vector can be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector.

[0093] The first transposon, first vector, first donor sequence, or first donor plasmid can comprise, consist essentially of, or consist of a sequence encoding a CSR. The first transposon, first vector, first donor sequence, or first donor plasmid can further comprise, consist essentially of, or consist of a sequence encoding a first selectable marker.

[0094] The second transposon, second vector, second donor sequence, or second donor plasmid can comprise, consist essentially of, or consist of one or more of a sequence encoding an inducible pro-apoptotic polypeptide, a sequence encoding a non-naturally occurring antigen receptor, and a sequence encoding a therapeutic protein. The second transposon, second vector, second donor sequence, or second donor plasmid can further comprise, consist essentially 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 essentially of, or consist of a cell surface marker. The selection marker can comprise, consist essentially of, or consist of a protein that is active in dividing cells but not in non-dividing cells. The selection marker can comprise, consist essentially of, or consist of a metabolic marker.

[0095] In one embodiment, the selectable marker can comprise, consist essentially of, or consist of a dihydrofolate reductase (DHFR) mutein enzyme. The DHFR mutein enzyme can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17012.

[0096] The DHFR mutein enzyme of SEQ ID NO: 17012 can further comprise, consist essentially 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 essentially of, or consist of one or more of a phenylalanine (F) or leucine (L) substitution at position 80, a leucine (L) or valine (V) substitution at position 113, and a valine (V) or aspartic acid (D) substitution at position 153.

[0097] The modified cells of the present disclosure (preferably, the modified T cells of the present disclosure) can further comprise, consist essentially of, or consist of a gene editing composition. The gene editing composition can comprise, consist essentially of, or consist of a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The gene editing composition can be transiently expressed by the modified cells. The gene editing composition can be stably expressed by the modified cells.

[0098] The gene editing composition can comprise, consist essentially 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 a sequence encoding a nuclease domain thereof can comprise, consist essentially of, or consist of a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain can comprise, consist essentially 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 essentially of, or consist of a nuclease-inactivated Cas (dCas) protein. The nuclease or its nuclease domain can comprise, consist essentially of, or consist of a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise, consist essentially of, or consist of a fusion protein. The gene editing composition can comprise, consist essentially of, or consist of a fusion protein. The fusion protein can comprise, consist essentially 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 essentially of, or consist of the amino acid sequence of SEQ ID NO: 17013. The fusion protein is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 17014. The fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17058. The fusion protein is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 17059.

[0099] The gene editing composition can further comprise, consist essentially of, or consist of a guide sequence.The guide sequence can comprise, consist essentially of, or consist of an RNA sequence.In an embodiment where the modified cell is a T cell, the guide RNA can comprise, consist essentially of, or consist of a sequence complementary to a target sequence encoding an endogenous TCR.The guide RNA can comprise, consist essentially of, or consist of a sequence complementary to a target sequence encoding a B2M polypeptide.The guide RNA can comprise, consist essentially of, or consist of a sequence complementary to a target sequence within a safe harbor site of a genomic DNA sequence.

[0100] The transposon, vector, donor sequence, or donor plasmid can further comprise, consist essentially of, or consist of a guide sequence and a gene editing composition comprising a sequence encoding a fusion protein comprising a sequence encoding inactivated Cas9 (dCas9) and a sequence encoding Clo051 nuclease or a nuclease domain thereof.

[0101] The first transposon, first vector, first donor sequence, or first donor plasmid can further comprise, consist essentially of, or consist of a guide sequence and a gene editing composition comprising a sequence encoding a fusion protein comprising a sequence encoding inactivated Cas9 (dCas9) and a sequence encoding Clo051 nuclease or a nuclease domain thereof.

[0102] The second transposon, second vector, second donor sequence, or second donor plasmid can further comprise, consist essentially of, or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding inactivated Cas9 (dCas9) and a sequence encoding Clo051 nuclease or a nuclease domain thereof.

[0103] The third transposon, third vector, third donor sequence, or third donor plasmid can comprise, consist essentially of, or consist of a gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding inactivated Cas9 (dCas9) and a sequence encoding Clo051 nuclease or a nuclease domain thereof.

[0104] The Clo051 nuclease or its nuclease domain can induce a single- or double-strand break in the target sequence. The donor sequence or donor plasmid can be integrated at the location of the single- or double-strand break, or at the location of cellular repair within the target sequence, or a combination thereof.

[0105] The present disclosure provides compositions comprising, consisting essentially of, or consisting of modified cells of the present disclosure (preferably, modified T cells of the present disclosure).

[0106] The present disclosure provides a plurality of modified cells comprising any of the non-naturally occurring chimeric stimulating receptors (CSRs) disclosed herein, and a plurality of modified cells comprising any of the modified cells disclosed herein. The plurality of modified cells can comprise, consist essentially of, or consist of immune cells or immune cell precursors. The plurality of immune cells can comprise, consist essentially 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).

[0107] The present disclosure provides a composition comprising a population of modified cells, wherein a plurality of the modified cells comprises any of the non-naturally occurring chimeric stimulatory receptors (CSRs) disclosed herein, and a composition comprising a population of modified cells, wherein a plurality of the modified cells comprises any of the modified cells disclosed herein. The modified cell population can comprise, consist essentially of, or consist of immune cells or immune cell precursors. The immune cell population can comprise, consist essentially 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 include a pharmaceutically acceptable carrier.

[0108] The present disclosure provides a composition comprising a population of modified T lymphocytes (T cells), wherein the population of a plurality of modified T cells comprises any of the non-naturally occurring chimeric stimulating receptors (CSRs) disclosed herein, and provides a composition comprising a population of T lymphocytes (T cells), wherein the population of a plurality of T cells comprises any of the modified T cells disclosed herein. The composition can include a pharmaceutically acceptable carrier.

[0109] Preferably, the present disclosure provides a composition comprising a population of T lymphocytes (T cells), wherein the population of a plurality of T cells comprises a non-naturally occurring chimeric stimulating receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating component, wherein the activating component is isolated from 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 from or derived from a second protein, and wherein the first protein and the second protein are not identical. The composition may comprise a pharmaceutically acceptable carrier. In some embodiments, 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 comprises CSRs.

[0110] The population of multiple T cells can further comprise an inducible pro-apoptotic polypeptide. In some embodiments, 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 comprises an inducible pro-apoptotic polypeptide.

[0111] The population of multiple T cells can further comprise a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the level of expression or activity of the TCR. In some embodiments, 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 a modification of an endogenous sequence encoding a TCR, wherein the modification reduces or eliminates the level of expression or activity of the TCR.

[0112] The population of multiple T cells can further comprise a modification of an endogenous sequence encoding beta-2-microglobulin (B2M), wherein the modification reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity. In some embodiments, 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 a modification of an endogenous sequence encoding B2M, wherein the modification reduces or eliminates the level of MHC-I expression or activity.

[0113] The population of multiple T cells can further comprise a modification in the endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity, as well as a modification in the endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0114] In some embodiments, 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 a modification in the endogenous sequence encoding the TCR that reduces or eliminates the level of TCR expression or activity, and a modification in the endogenous sequence encoding B2M that reduces or eliminates the level of MHC-I expression or activity.

[0115] The population of multiple T cells can further comprise a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. In some embodiments, 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 comprises a non-naturally occurring sequence comprising an HLA-E polypeptide.

[0116] The population of multiple T cells can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In some embodiments, 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 comprises a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In a preferred embodiment, the non-naturally occurring antigen receptor is a chimeric antigen receptor (CAR).

[0117] Multiple populations of T cells are identified: early memory T cells, stem cell-like T cells, and stem memory T cells (T SCM ), central memory T cells (T CM ), or stem cell-like T cells. In some embodiments, stem cell-like T cells, stem cell memory T cells (T SCM ), and central memory T cells (T CM ) 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%, 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 engineered T cells.

[0118] In some embodiments, 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 CSRs are stem memory T cells (T SCM ) or T SCM The IL-16-16 and IL-16-16 cells express one or more cell surface markers of CD45RA and CD62L.

[0119] In some embodiments, 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 are central memory T cells (T CM ) or T CM The cells express one or more cell surface markers of the CD45R1 and CD62L-like population, and the one or more cell surface markers include CD45R1 and CD62L.

[0120] In some embodiments, 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 express one or more of CD127, CD45RO, CD95, and IL-2Rβ cell surface markers.

[0121] The present disclosure provides a composition for use in treating a disease or disorder disclosed herein, or the use of a composition for treating any disease or disorder disclosed herein. The present disclosure also provides a method for treating a disease or disorder, comprising, consisting essentially of, or consisting of administering to a subject in need of treatment a therapeutically effective amount of a composition disclosed herein. The composition can comprise, consist essentially 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.

[0122] The present disclosure provides a method for producing modified T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into primary human T cells under conditions that stably express the CSR in the modified T cells and maintain desirable stem-like properties of the modified T cells, to produce modified T cells. The primary human T cells can be resting primary human T cells. The present disclosure provides modified T cells produced by the disclosed methods. The present disclosure provides methods for administering modified T cells comprising a stably expressed CSR produced by the disclosed methods. The present disclosure provides methods for administering modified T cells comprising a stably expressed CSR produced by the disclosed methods to treat a disease or disorder.

[0123] The present disclosure provides a method for producing a population of modified T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, to produce a plurality of modified T cells. The primary human T cells can be 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 for administering a population of modified T cells comprising a stably expressed CSR produced by the disclosed method. The present disclosure provides a method for administering a population of modified T cells comprising a stably expressed CSR produced by the disclosed method to treat a disease or disorder.

[0124] The present disclosure provides a method for producing modified T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into primary human T cells under conditions that transiently express the CSR in the modified T cells and maintain desirable stem-like properties of the modified T cells, to produce modified T cells. The primary human T cells can be resting primary human T cells. The present disclosure provides modified T cells produced by the disclosed methods. The present disclosure provides methods for administering modified T cells produced by the disclosed methods that contain a transiently expressed CSR. In one embodiment, the present disclosure provides methods for administering modified T cells produced by the disclosed methods that contain a transiently expressed CSR after the modified T cells no longer express the CSR. The present disclosure provides methods for administering modified T cells produced by the disclosed methods that contain a transiently expressed CSR to treat a disease or disorder. In one embodiment, the present disclosure provides methods for administering modified T cells produced by the disclosed methods that contain a transiently expressed CSR to treat a disease or disorder.

[0125] The present disclosure provides a method for producing a population of modified T cells, the method comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, to produce a plurality of modified T cells. The primary human T cells can be 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 for administering a population of modified T cells comprising a transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method for administering a population of modified T cells comprising a transiently expressed CSR produced by the disclosed method after the plurality of T cells no longer express the CSR. The present disclosure provides a method for administering a population of modified T cells comprising a transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one aspect, the disclosure provides a method of administering a population of modified T cells produced by the disclosed methods after a plurality of the T cells no longer express CSR to treat a disease or disorder.

[0126] The method of producing modified T cells or producing a population of modified T cells can further include introducing a modification in an endogenous sequence encoding a T cell receptor (TCR), where the modification reduces or eliminates the level of TCR expression or activity. The method of producing modified T cells or producing a population of modified T cells can further include introducing a modification in an endogenous sequence encoding beta-2-microglobulin (B2M), where the modification reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-1) expression or activity. In some embodiments, the method of producing modified T cells or producing a population of modified T cells can further include both introducing a modification in an endogenous sequence encoding a TCR, where the modification reduces or eliminates the level of TCR expression or activity, and introducing a modification in an endogenous sequence encoding B2M, where the modification reduces or eliminates the level of MHC-1 expression or activity.

[0127] The method for producing modified T cells or a population of modified T cells can further comprise introducing into a primary human T cell or a plurality of primary human T cells a composition comprising an antigen receptor, a therapeutic protein, or a sequence encoding the same. In one embodiment, the antigen receptor is a non-naturally occurring antigen receptor. In a preferred embodiment, the method for producing modified T cells or a population of modified T cells can further comprise introducing into a primary human T cell or a plurality of primary human T cells a composition comprising a chimeric antigen receptor (CAR) or a sequence encoding the same. The method can further comprise introducing into a primary human T cell or a plurality of primary human T cells a composition comprising an inducible pro-apoptotic polypeptide or a sequence encoding the same. The method for producing modified T cells or a population of modified T cells can further comprise introducing into a primary human T cell or a 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 pro-apoptotic polypeptide or a sequence encoding the same.

[0128] The method for producing modified T cells or a population of modified T cells can further include contacting the modified T cells or the population of modified T cells with an activator composition. The activator composition can comprise, consist essentially of, or consist of one or more agonists or activators capable of binding to the CSR activation component of the modified T cells or a plurality of modified T cells. The agonists / activators can be naturally occurring or non-naturally occurring. In preferred embodiments, the agonist / activator is an antibody or antibody fragment. The agonist / activator 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 embodiments, the agonist / activator 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. The agonist / activator can be contacted with the modified T cells or population of modified T cells in vitro, ex vivo, or in vivo. In preferred embodiments, the agonist / activator activates the modified T cells or population of modified T cells, induces cell division in the modified T cells or population of modified T cells, increases cell division (e.g., cell doubling time) in the modified T cells or population of modified T cells, increases the fold expansion in the modified T cells or population of modified T cells, or any combination thereof.

[0129] The present disclosure provides a method of expanding a population of modified T cells, comprising: introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells to produce a plurality of modified T cells; and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the 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 ten-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the CSR of the present disclosure under the same conditions.

[0130] The present disclosure provides a method of expanding a population of engineered T cells, comprising: introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express the CSR in the plurality of engineered T cells and maintain desirable stem-like properties of the plurality of engineered T cells to produce a plurality of engineered T cells; and contacting the cells with an activator composition to produce a plurality of activated engineered T cells, wherein the expansion of the plurality of engineered T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not transiently express the CSR of the present disclosure under the same conditions. The method wherein the expansion of the plurality of engineered 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 ten-fold greater than the expansion of a plurality of wild-type T cells that do not transiently express the CSR of the present disclosure under the same conditions.

[0131] The activator composition of the population expansion method can comprise, consist essentially of, or consist of one or more agonists or activators capable of binding to the CSR activation component of the engineered T cell or plurality of engineered T cells. The agonist / activator can be naturally occurring or non-naturally occurring. In preferred embodiments, the agonist / activator is an antibody or antibody fragment. The agonist / activator 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 embodiments, the agonist / activator 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.

[0132] The conditions can include culturing the modified T cell or a plurality of modified T cells in a medium containing a sterol; an alkane; phosphorus, and one or more of octanoic acid, palmitic acid, linoleic acid, and oleic acid. The culturing can be in vivo or ex vivo. The modified T cell can be an allogeneic cell, or the plurality of modified T cells can be allogeneic cells. The modified T cell can be an autologous T cell, or the plurality of modified T cells can be autologous T cells.

[0133] In some embodiments, the medium can include one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg inclusive of the endpoint, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive of the endpoint, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive of the endpoint, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive of the endpoint, and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg inclusive of the endpoint.

[0134] In some embodiments, the medium can include 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 sterols at a concentration of about 1 mg / kg.

[0135] In some embodiments, the medium may include one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg, including the endpoint; palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg, including the endpoint; linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, including the endpoint; oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg, including the endpoint; and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg, including the endpoint.

[0136] In some embodiments, the medium can include one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and sterols at a concentration of about 2.5 μmol / kg.

[0137] The present disclosure provides compositions comprising any modified T cells produced by the methods disclosed herein.The present disclosure provides compositions comprising any population of modified T cells produced by the methods disclosed herein.The present disclosure provides compositions comprising any modified T cells expanded by the methods disclosed herein.The present disclosure provides compositions comprising any population of modified T cells expanded by the methods disclosed herein.

[0138] The present disclosure provides compositions for use in treating a disease or disorder disclosed herein, or the use of a composition for treating any of the diseases or disorders disclosed herein. The present disclosure also provides methods for treating a disease or disorder, comprising, consisting essentially of, or consisting of administering to a subject in need of treatment a therapeutically effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds to the activating component of CSR disclosed herein. The composition can comprise, consist essentially of, or consist of any of the engineered cells or populations of engineered cells disclosed herein. Preferably, any of the engineered T cells or CAR T cells disclosed herein. Any non-naturally occurring molecule that can bind to the activating component of CSR disclosed herein and selectively transduce a signal upon binding can be administered. Preferably, the non-naturally occurring molecule is a non-naturally occurring CSR agonist / activator for the activating component. The non-naturally occurring agonist / activator that can bind to the CSR activating 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 embodiments, the non-naturally occurring agonist / activator capable of binding to a CSR activating 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 embodiments, the non-naturally occurring agonist / activator capable of binding to an activating component may be selected from the group consisting of the anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998), and the humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (siplizumab) (Bissonnette et al. Arch. Dermatol. Res. 301(6):429-442, 2009).In some embodiments, administration of a non-naturally occurring molecule capable of binding to the activating component of CSR stimulates cell division of modified cells in vivo. Accordingly, the present disclosure provides methods of stimulating cell division of modified cells of the present disclosure in vivo by administering to a subject harboring modified cells of the present disclosure a non-naturally occurring CSR agonist / activator for the activating component.

[0139] In some embodiments, the disease or disorder is a cell proliferation disease or disorder. In some embodiments, the cell proliferation disease or disorder is cancer. The cancer can be a solid tumor cancer or a hematological cancer. In some embodiments, the solid tumor is prostate cancer or breast cancer. In a preferred embodiment, the prostate cancer is castration-resistant prostate cancer. In some embodiments, the hematological cancer is multiple myeloma.

[0140] The modified cells or populations of modified cells contained within the disclosed compositions can be cultured in vitro or ex vivo before administration to a subject in need thereof. The modified cells can be allogeneic modified cells or autologous modified cells. In some embodiments, the cells are allogeneic modified T cells or autologous modified T cells. In some embodiments, the cells are allogeneic modified CAR T cells or autologous modified CAR T cells. In some embodiments, the cells are allogeneic modified CAR T cells comprising the CSR of the present disclosure, or autologous modified CAR T cells comprising the CSR of the present disclosure.

[0141] The modified cell composition or a composition comprising a population of modified cells can be administered to a patient by any means known in the art. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered intravenously. Intravenous administration can be by intravenous injection or infusion. In some embodiments, the composition is administered locally. In some embodiments, the composition is administered by intrathecal, intracerebroventricular, intraocular, or intraosseous injection or infusion.

[0142] A therapeutically effective amount can be a single dose or multiple doses of a composition comprising a modified cell composition or a population of modified cells. In some embodiments, a therapeutically effective dose is a single dose, and the allogeneic cells of the composition engraft and / or persist for a sufficient time to treat a disease or disorder. In some embodiments, a 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 produced simultaneously.

[0143] In some embodiments, the uses and methods for treating a disease or disorder further provide that after administration of a modified cell composition disclosed herein or a composition comprising a population of modified cells disclosed herein, the subject does not develop graft-versus-host (GvH) disease, host-versus-graft (HvG) disease, or a combination thereof.

[0144] The allogeneic cells of the present disclosure are engineered to prevent adverse reactions to engraftment after administration to a subject. The allogeneic cells can be any type of cell.

[0145] In some embodiments of the compositions and methods of the present disclosure, the allogeneic cells are stem cells. In some embodiments, the 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), pluripotent stem cells, multipotent stem cells, and hematopoietic stem cells (HSCs).

[0146] In some embodiments of the compositions and methods of the present disclosure, the allogeneic cells are differentiated somatic cells.

[0147] In some embodiments of the compositions and methods of the present disclosure, the allogeneic cells are immune cells. In some embodiments, the allogeneic cells are T lymphocytes (T cells). In some embodiments, the allogeneic cells are T cells that do not express one or more components of a naturally occurring T cell receptor (TCR). In some embodiments, the allogeneic cells are T cells that express a non-naturally occurring antigen receptor. Alternatively, or in addition, in some embodiments, the allogeneic cells are T cells that express a non-naturally occurring chimeric stimulating 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 costimulatory molecule and transduces a signal into the intracellular space of the allogeneic cell that recapitulates TCR signaling or TCR costimulatory signaling. Chimeric stimulatory receptor (CSR)

[0148] An adoptive cell composition that is "universally" safe for administration to any patient requires a significant reduction or elimination of alloreactivity.

[0149] To this end, the allogeneic cells of the present disclosure are modified to disrupt the expression or function of T cell receptors (TCRs) and / or major histocompatibility complex (MHC) classes. TCRs mediate graft-versus-host (GvH) reactions, while MHCs mediate host-versus-graft (HvG) reactions. In preferred embodiments, any expression and / or function of TCRs is reduced in the allogeneic cells of the present disclosure to prevent T cell-mediated GvH, which can cause death in the subject. Thus, in particularly preferred embodiments, the present disclosure provides pure TCR-negative allogeneic T cell compositions (e.g., each cell of the composition expresses TCRs at such low levels that they are either undetectable or absent).

[0150] In a preferred embodiment, the expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated in the allogeneic cells of the present disclosure to prevent HvG and, consequently, improve engraftment of the allogeneic cells of the present disclosure in a subject. Improved engraftment of the allogeneic cells of the present disclosure results in longer persistence of the cells and, therefore, a larger therapeutic window for the subject. Specifically, in the allogeneic cells of the present disclosure, the expression and / or function of beta-2-microglobulin (B2M), a structural component of MHC-I, is reduced or eliminated in the allogeneic cells of the present disclosure.

[0151] The above-described strategy for generating allogeneic cells of the present disclosure poses additional challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), a part of the TCR complex. Loss of CD3ζ in TCR-KO T cells dramatically reduces the ability of these cells to be optimally activated and expanded using standard stimulatory / activator reagents, including, but not limited to, agonist anti-CD3 mAbs. Disruption of the expression or function of any one component of the TCR complex results in the loss of all components of the complex, including TCR-alpha (TCRα), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ). Both CD3ε and CD3ζ are required for T cell activation and expansion. Agonistic anti-CD3 mAbs typically recognize CD3ε and possibly another protein within the complex that also signals to CD3ζ. CD3ζ provides the primary stimulus for T cell activation (along with a secondary costimulatory signal) for optimal activation and expansion. Under normal conditions, full T cell activation depends on TCR engagement in combination with a second signal from one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL, etc.) that promote the immune response. However, in the absence of the TCR, T cell expansion is significantly reduced when stimulated using standard activation / stimulation reagents containing agonistic anti-CD3 mAbs. In fact, T cell expansion is reduced to only 20–40% of normal expansion levels when stimulated using standard activation / stimulation reagents containing agonistic anti-CD3 mAbs.

[0152] The present disclosure provides chimeric stimulating receptors (CSRs) that deliver a CD3z primary stimulus to allogeneic T cells in the absence of endogenous TCR (and consequently endogenous CD3ζ) when stimulated using standard activation / stimulation reagents, including agonist anti-CD3 mAbs.

[0153] In the absence of endogenous TCRs, the chimeric stimulating receptors (CSRs) of the present disclosure provide CD3ζ stimulation to enhance the activation and expansion of allogeneic T cells. In other words, in the absence of endogenous TCRs, the chimeric stimulating receptors (CSRs) of the present disclosure rescue allogeneic cells from activation-based disadvantages compared to non-allogeneic T cells expressing endogenous TCRs. In some embodiments, the CSRs of the present disclosure contain an agonist mAb epitope extracellularly and a CD3ζ stimulating domain intracellularly, functionally converting surface anti-CD28 or anti-CD2 binding events into CD3z signaling events in allogeneic T cells engineered to express the CSR. In some embodiments, the CSRs contain wild-type CD28 or CD2 proteins and a CD3z intracellular stimulating domain, generating CD28z CSRs and CD2z CSRs, respectively. In preferred embodiments, the CD28z CSRs and / or CD2z CSRs further express a non-naturally occurring antigen receptor and / or a therapeutic protein. In a preferred embodiment, the non-naturally occurring antigen receptor comprises a chimeric antigen receptor.

[0154] The data provided herein demonstrate that modified allogeneic T cells of the present disclosure that contain / express a CSR of the present disclosure improve or aid in the expansion of allogeneic T cells that no longer express an endogenous TCR when compared to cells that do not contain / express a CSR of the present disclosure.

[0155] The wild-type / native human CD28 protein (NCBI: CD28_HUMAN; UniProt / Swiss-Prot: P10747.1) comprises or consists of the following amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 17096)

[0156] The nucleotide sequence encoding the wild-type / native CD28 protein (NCBI: CCDS2361.1) comprises or consists of the following nucleotide sequence: (SEQ ID NO: 17097)

[0157] An exemplary CSR CD28z protein of the present 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). [ka] CD28 signal peptide: MLRLLLALNLFPSIQVTG (SEQ ID NO: 17098) CD28 extracellular domain: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 17099) CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 17100) CD28 cytoplasmic domain: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 17101) CD3z intracellular domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17102)

[0158] An exemplary nucleotide sequence encoding a CSR CD28z protein of the present disclosure comprises or consists of the nucleotide sequence of (CD28 signal peptide, CD28 extracellular domain, CD28 transmembrane domain, CD28 cytoplasmic domain, CD3z intracellular domain). [ka] CD28 signal peptide: ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGACCGGC (SEQ ID NO: 17103) CD28 extracellular domain: AACAAGATCCTGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACAACGCCGTGAACCTGAGCTGCAAGTACAGCTACAACCTGTTCAGCAGAGAGTTCCGGGCCAGCCTGCACAAAGGACTGGATTCTGCTGTGGAAGTGTGCGTGGTGTACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACCGGCTTCAACTGCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACCTGTACGTGAACCAGACCGACATCTATTTCTGCAAGATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCT(SEQ ID NO: 17104) CD28 transmembrane domain: TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACAGTGGCCTTCATCATCTTTTGGGTC(SEQ ID NO: 17105) CD28 cytoplasmic domain: CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCC (SEQ ID NO: 17106) CD3z intracellular domain: AGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCT GTACAATGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 17107)

[0159] The wild-type / native human CD2 protein (NCBI: CD2_HUMAN; UniProt / Swiss-Prot: P06729.2) comprises or consists of the following amino acid sequence: MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLDIYLIIGICGGGSLLMVFVALLVFYITKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17108)

[0160] The nucleotide sequence encoding the wild-type / native CD2 protein (NCBI: CCDS889.1) comprises or consists of the following nucleotide sequence:

[0161] Exemplary CSR CD2z proteins of the present disclosure comprise or consist of the amino acid sequence of (CD2 signal peptide, CD2 extracellular domain, CD2 transmembrane domain, CD2 cytoplasmic domain, CD3z intracellular domain). [ka] CD2 signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 17110) CD2 extracellular domain: KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (SEQ ID NO: 17111) CD2 transmembrane domain: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 17112) CD2 cytoplasmic domain: KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17113) CD3z intracellular domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17102)

[0162] The present disclosure provides a non-naturally occurring CSR CD2 protein comprising, consisting essentially of, or consisting of an amino acid sequence that is 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 essentially of, or consisting of an amino acid sequence that is 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 essentially of, or consisting of an amino acid sequence that is 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 transmembrane domain comprising, consisting essentially of, or consisting of an amino acid sequence that is 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 essentially of, or consisting of an amino acid sequence that is 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: 17113.The present disclosure provides a CD3z intracellular domain comprising, consisting essentially of, or consisting of an amino acid sequence that is 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.

[0163] 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). [ka] CD2 signal peptide: ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGGGCGCCGTGTCC (SEQ ID NO: 17114) CD2 extracellular domain: AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACATCTATAAGGTGTCCATCTACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGAC (SEQ ID NO: 17115) CD2 transmembrane domain: ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACATCACC (SEQ ID NO: 17116) CD2 cytoplasmic domain: AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCACCTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCA CAGACCTCCACCACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAAC (SEQ ID NO: 17117) CD3z intracellular domain: AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCT GTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 17107)

[0164] An exemplary mutant CSR CD2z-D111H protein of the present disclosure comprises or consists of the amino acid sequence of (CD2 signal peptide, CD2 extracellular domain with a D111H mutation within the CD2 extracellular domain, CD2 transmembrane domain, CD2 cytoplasmic domain, CD3z intracellular domain). [ka] CD2 signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 17110) CD2 extracellular domain with D111H mutation within the CD2 extracellular domain: KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGL (SEQ ID NO: 17119) CD2 transmembrane domain: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 17112) CD2 cytoplasmic domain: KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17113) CD3z intracellular domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17102)

[0165] The present disclosure provides a non-naturally occurring CSR CD2 protein comprising, consisting essentially of, or consisting of an amino acid sequence that is 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: 17118. The present disclosure provides a CD2 extracellular domain comprising, consisting essentially of, or consisting of an amino acid sequence that is 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: 17119.

[0166] An exemplary nucleotide sequence encoding a mutant CSR CD2z-D111H protein of the present disclosure comprises or consists of the amino acid sequence of (CD2 signal peptide, CD2 extracellular domain with a D111H mutation within the CD2 extracellular domain, CD2 transmembrane domain, CD2 cytoplasmic domain, CD3z intracellular domain). [ka] CD2 extracellular domain with D111H mutation within the CD2 extracellular domain: [ka] CD2 transmembrane domain: ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACATCACC (SEQ ID NO: 17116) CD2 cytoplasmic domain: AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCACCTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCA CAGACCTCCACCACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAAC (SEQ ID NO: 17117) CD3z intracellular domain: AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGA GGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 17107)

[0167] Endogenous TCR knockout Gene editing compositions of the present disclosure, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target and reduce or delete the expression of endogenous T cell receptors in allogeneic cells of the present disclosure. In a preferred embodiment, the gene editing compositions of the present disclosure target and delete a gene, a portion of a gene, or a regulatory element (such as a promoter) of a gene that encodes an endogenous T cell receptor in an allogeneic cell of the present disclosure.

[0168] Non-limiting examples of primers (comprising a T7 promoter, genomic target sequence, and gRNA scaffold) for generating guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α) are provided in Table 10.

[0169] [Table 1-1] [Table 1-2]

[0170] Non-limiting examples of primers for generating guide RNA (gRNA) templates for targeting and deleting TCR-beta (TCR-β) are shown in Table 11.

[0171] [Table 2-1] [Table 2-2]

[0172] Non-limiting examples of primers for generating guide RNA (gRNA) templates for targeting and deleting beta-2-microglobulin (β2M) are provided in Table 12.

[0173] [Table 3]

[0174] Endogenous MHC knockout Gene editing compositions of the present disclosure, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target and reduce or delete expression of endogenous MHC1, MHCII, or MHC activators in allogeneic cells of the present disclosure. In a preferred embodiment, the gene editing compositions of the present disclosure target and delete genes, portions of genes, or regulatory elements of genes (such as promoters) that encode one or more components of endogenous MHC1, MHCII, or MHC activators in allogeneic cells of the present disclosure.

[0175] Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are provided in Tables 13 and 14.

[0176] [Table 4]

[0177] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0178] Genetically engineered HLA-E compositions MHCI knockout (KO) not only renders cells resistant to killing by T cells but also susceptible to natural killer (NK) cell-mediated cytotoxicity (the "missing self hypothesis" (see Figure 30). It is hypothesized that NK rejection may reduce the in vivo efficacy and / or persistence of these KO cells in therapeutic settings such as allogeneic (allo)CAR-T therapy. Retention of MHCI on the surface of allogeneic CAR-T cells may render them susceptible to killing by host T cells, as observed in classical mixed lymphocyte reaction (MLR) experiments. It is estimated that up to 10% of HLAs are specific for foreign MHC and can mediate the rejection of foreign cells and tissues. Targeted KO of HLA-C1s, particularly HLA-A, B, and C, can be achieved by targeted KO of B2M, resulting in the loss of additional HLA molecules, including HLA-E. For example, loss of HLA-E renders KO cells susceptible to NK cell-mediated cytotoxicity, according to the "missing self hypothesis." NK cell-mediated cytotoxicity against missing-self cells is a defense mechanism against pathogens that downregulate MHC on the surface of infected cells to avoid detection and killing by cells of the adaptive immune system.

[0179] Two strategies are contemplated by the disclosure for genetically engineering allogeneic (MHCI-negative) T cells (including CAR-T cells) that are 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 cell killing is introduced or delivered into the allogeneic cells. Alternatively, or in addition, the gene editing methods of the disclosure preserve certain endogenous HLA molecules (such as endogenous HLA-E). For example, the first approach involves the delivery of piggyBac® (PB) of single-chain (SC) HLA-E molecules to B2M KO T cells.

[0180] The second approach uses gene editing compositions containing guide RNAs selective for HLA-A, HLA-B, and HLA-C, but not, for example, HLA-E or other molecules that protect against natural killer cell-mediated cytotoxicity in MHCI KOs.

[0181] Alternative or additional molecules to HLA-E that protect against NK cell-mediated cytotoxicity include, but are not limited to, CD47, interferon alpha / beta receptor 1 (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 carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), viral hemagglutinin, CD48, LLT1 (also known as C-type lectin domain family 2 member (CLC2D)), ULBP2, ULBP3, and sMICA or variants thereof.

[0182] Exemplary CD47 proteins of the present disclosure comprise or consist of the following amino acid sequences: (signal peptide, extracellular, TM, cytoplasmic). [ka]

[0183] Exemplary INFAR1 proteins of the present disclosure comprise or consist of the following amino acid sequences: (signal peptide, extracellular, TM, cytoplasmic). [ka]

[0184] Exemplary INFAR2 proteins of the present disclosure comprise or consist of the following amino acid sequences: (signal peptide, extracellular, TM, cytoplasmic). [ka]

[0185] An exemplary HLA-G1 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3). [ka]

[0186] An exemplary HLA-G2 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3). [ka]

[0187] An exemplary HLA-G3 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3). [ka]

[0188] An exemplary HLA-G4 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3). [ka]

[0189] An exemplary HLA-G5 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3, intron 4). [ka]

[0190] An exemplary HLA-G5 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3, intron 4). [ka]

[0191] An exemplary HLA-G5 protein of the present disclosure comprises or consists of the amino acid sequence of (alpha chain 1, alpha chain 2, alpha chain 3, intron 2). [ka]

[0192] Exemplary CEACAM1 proteins of the present disclosure comprise or consist of the amino acid sequence of (extracellular, TM, cytoplasmic). [ka]

[0193] An exemplary viral hemagglutinin protein of the present disclosure comprises or consists of the amino acid sequence of the HA of influenza A virus (A / New Caledonia / 20 / 1999(H1N1); TM). [ka]

[0194] Exemplary CD48 proteins of the present disclosure comprise or consist of the amino acid sequence of (signal peptide, chain, propeptide removed in the mature form). [ka]

[0195] Exemplary LLT1 proteins of the present disclosure comprise or consist of the amino acid sequence of (cytoplasmic, TM, extracellular). [ka]

[0196] An exemplary ULBP2 protein of the present disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank Accession No. AAQ89028). [ka]

[0197] An exemplary ULBP3 protein of the present disclosure comprises or consists of the amino acid sequence of (also known as NKG2D ligand; Genbank Accession No. NP_078794). [ka]

[0198] An exemplary sMICA protein of the present disclosure comprises or consists of the amino acid sequence of (signal peptide, portion of the extracellular domain, TM and cytoplasmic domains) (Genbank Accession No. Q29983). [ka]

[0199] Exemplary sMICA proteins of the present disclosure comprise or consist of the amino acid sequence of (alpha-1, alpha-2, alpha-3). [ka]

[0200] Exemplary sMICA proteins of the present disclosure comprise or consist of the amino acid sequence of (signal peptide; alpha-1, alpha-2, alpha-3). [ka]

[0201] Exemplary sMICA proteins of the present disclosure comprise or consist of the amino acid sequence of (signal peptide). [ka]

[0202] An exemplary bGBE trimer (270G and 484S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0203] An exemplary bGBE trimer (270G and 484S) protein of the present disclosure comprises or consists of the following nucleic acid sequence: [ka]

[0204] An exemplary bGBE trimer (270R and 484S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0205] An exemplary bGBE trimer (270R and 484S) protein of the present disclosure comprises or consists of the following nucleic acid sequence: [ka]

[0206] Exemplary gBE dimeric (R and S) proteins of the present disclosure comprise or consist of the following amino acid sequences: [ka]

[0207] Exemplary gBE dimeric (R and S) proteins of the present disclosure comprise or consist of the following nucleic acid sequences: [ka]

[0208] An exemplary gBE dimeric (G and S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0209] An exemplary gBE dimeric (G and S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0210] The wild-type / native human HLA-E protein (NCBI: HLAE_HUMAN; UniProt / Swiss-Prot: P13747.4) comprises or consists of the following amino acid sequence: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 17122)

[0211] The nucleotide sequence encoding the wild-type / native HLA-E protein (NCBI: CCDS34379.1) comprises or consists of the following nucleotide sequence:

[0212] An exemplary WT HLA-E monomer (R and S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0213] Exemplary WT HLA-E monomer (R and S) proteins of the present disclosure comprise or consist of the following nucleic acid sequences: [ka]

[0214] An exemplary WT HLA-E monomer (G and S) protein of the present disclosure comprises or consists of the following nucleic acid sequence: [ka]

[0215] An exemplary WT HLA-E monomer (G and S) protein of the present disclosure comprises or consists of the following nucleic acid sequence: [ka]

[0216] The wild-type / native human B2M protein (NCBI: B2MG_HUMAN; UniProt / Swiss-Prot: P61769.1) comprises or consists of the following amino acid sequence: MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17124)

[0217] The nucleotide sequence encoding the wild-type / native B2M protein (NCBI: CCDS10113.1) comprises or consists of the following nucleotide sequence: ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAA TGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA (SEQ ID NO: 17125)

[0218] An exemplary HLA-bGBE (single chain trimer) protein of the present disclosure comprises or consists of the amino acid sequence of (B2M signal peptide, peptide, linker, B2M domain, linker, HLA-E peptide). [ka] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126) Peptide: VMAPRTLIL (SEQ ID NO: 17127) Linker: GGGGSGGGGSGGGGS (SEQ ID NO: 17128) B2M domain: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17129) Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (SEQ ID NO: 17131)

[0219] An exemplary nucleotide sequence encoding an HLA-bGBE (single-chain trimer) protein of the present disclosure comprises or consists of the nucleotide sequence of (B2M signal peptide, peptide, linker, B2M domain, linker, HLA-E peptide). [ka] B2M signal peptide: ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGGCCTGGAGGCC (SEQ ID NO: 17132) Peptide: GTGATGGCCCCCCGGACCCTGATCCTG (SEQ ID NO: 17133) Linker: GGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT (SEQ ID NO: 17134) B2M domain: ATCCAGCGCACACCTAAGATCCAGGTGTATTCTCGGCACCCAGCCGAGAACGGCAAGAGCAACTTCCTGAATTGCTACGTGAGCGGCTTTCACCCTTCCGACATCGAGGTGGATCTGCTGAAGAATGGCGAGAGAATCGAGAAGGTGGAGCACTCCGACCTGAGCTTCTCCAAGGATTGGTCTTTTTATCTGCTGTACTATACCGAGTTTACCCCTACAGAGAAGGACGAGTACGCCTGTCGCGTGAACCACGTGACACTGTCCCAGCCAAAGATCGTGAAGTGGGACCGGGATATG (SEQ ID NO: 17135) Linker: GGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCT (SEQ ID NO: 17136)

[0220] An exemplary HLA-gBE (single chain dimer) protein of the present disclosure comprises or consists of the amino acid sequence of (B2M signal peptide, B2M domain, linker, HLA-E peptide). [ka] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126) B2M domain: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17129) Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL (SEQ ID NO: 17131)

[0221] An exemplary nucleotide sequence encoding an HLA-gBE (single-chain dimer) protein of the present disclosure comprises or consists of the nucleotide sequence of (B2M signal peptide, B2M domain, linker, HLA-E peptide). [ka] B2M signal peptide: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 17132) B2M domain: ATCCAGCGGACCCCTAAGATCCAGGTGTACAGCAGACCCCCGCCGAGAACGGCAAGAGCAACTTCCTGAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGGCGAGCGGATCGAGAAGGTGGAA CACAGCGATCTGAGCTTCAGCAAGGACTGGTCCTTCTACCTGCTGTACTACACCGAGTTCACCCCTACCGAGAAGGACGAGTACGCCTGCAGAGTGAACCACGTGACACTGAGCCAGCCTAAGATCGTGAAGTGGGACAGAGATATG (SEQ ID NO: 17135) Linker: GGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCT (SEQ ID NO: 17136)

[0222] An exemplary HLA-bE (monomeric) protein of the present disclosure comprises or consists of the amino acid sequence of (B2M signal peptide, HLA-E peptide). [ka] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL (SEQ ID NO: 17131)

[0223] An exemplary nucleotide sequence encoding an HLA-bE (monomer) protein of the present disclosure comprises or consists of the nucleotide sequence of (B2M signal peptide, HLA-E peptide). [ka] B2M signal peptide: ATGTCTCGCAGCGTGGCCCTGGCCGTGCTGGCCCTGCTGTCCCTGTCTGGCCTGGAGGCC (SEQ ID NO: 17132)

[0224] Immune and Immune Progenitor Cells In certain embodiments, the immune cells of the present disclosure include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem and memory T cells (T SCM cells), central memory T cells (T CM ), 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.

[0225] In certain embodiments, immune progenitor cells include any cells that can differentiate into one or more types of immune cells. In certain embodiments, immune progenitor cells include pluripotent stem cells that can self-renew and develop into immune cells. In certain embodiments, immune progenitor cells include hematopoietic stem cells (HSCs) or their progeny. In certain embodiments, immune progenitor cells include progenitor cells that can develop into immune cells. In certain embodiments, immune progenitor cells include hematopoietic progenitor cells (HPCs).

[0226] hematopoietic stem cells (HSC) 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.

[0227] The HSCs of the present disclosure can be isolated or derived from primary stem cells or cultured stem cells.The HSCs of the present disclosure can be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0228] The immune progenitor cells of the present disclosure may include HSCs or HSC progeny cells. Exemplary HSC progeny cells of the present disclosure include, but are not limited to, pluripotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells (T cells), B lymphocyte cells (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0229] HSCs produced by the disclosed methods can be isolated from or derived from adult stem cells, while retaining the characteristics of "primitive" stem cells that share embryonic stem cell characteristics while committed to a single lineage. For example, "primitive" HSCs generated by the disclosed methods retain their "stemness" after division and do not differentiate. As a result, as adoptive cell therapy, "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs generated by the disclosed methods can be therapeutically effective when administered as a single dose. In some embodiments, the disclosed primitive HSCs are CD34+. In some embodiments, the disclosed primitive HSCs are CD34+ and CD38-. In some embodiments, the disclosed primitive HSCs are CD34+, CD38-, and CD90+. In some embodiments, the primitive HSCs of the present disclosure are CD34+, CD38-, CD90+, and CD45RA-. In some embodiments, the primitive HSCs of the present disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+. In some embodiments, the most primitive HSCs of the present disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0230] In some embodiments of the present disclosure, primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the methods of the present disclosure to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). In some embodiments of the present disclosure, the modified primitive HSCs, modified HSCs, and / or modified HSC progeny cells can be forward differentiated to produce modified immune cells, including, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells of the present disclosure.

[0231] T cells The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0232] Unlike traditional biologics and chemotherapy, the modified T cells of the present disclosure have the ability to rapidly regenerate upon antigen recognition, potentially eliminating the need for repeated treatment. To accomplish this, in some embodiments, the modified T cells of the present disclosure are maintained in the patient as a stable population of viable memory T cells not only to drive the initial response but also to prevent potential relapse. Alternatively, in some embodiments, the modified T cells of the present disclosure are not maintained in the patient when this is undesirable.

[0233] Antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, and early memory T cells, especially stem cell memory (T SCM Intensive efforts have focused on the development of engineered T cell products containing stem cell-like T cells. The stem cell-like engineered T cells of the present disclosure possess maximum capacity for self-renewal as well as central memory (T CM ) T cells or T CM -like cells, effector memory (T EM ) and effector T cells (T E ), resulting in better tumor eradication and long-term engraftment of modified T cells. Nis the parent progenitor cell, and T SCM directly, which in turn produces T CM A linear pathway of differentiation, such as directly giving rise to naive T cells (T N )>T SCM >T CM >T EM >T E >T TE may be involved in the generation of these cells. The composition of T cells of the present disclosure may include one or more of each parental T cell subset, and may include T SCM Cells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0234] In some embodiments of the disclosed methods, the immune cell precursors include early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE In some embodiments, the immune cell precursor is a primitive HSC, HSC, or HSC progeny cell of the present disclosure.

[0235] In some embodiments of the disclosed methods, the immune cells include early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE is.

[0236] In some embodiments of the methods of the present disclosure, the immune cells are early memory T cells.

[0237] In some embodiments of the methods of the present disclosure, the immune cells are stem cell-like T cells.

[0238] In some embodiments of the disclosed methods, the immune cells are TSCM is.

[0239] In some embodiments of the disclosed methods, the immune cells are T CM is.

[0240] In some embodiments of the methods of the present disclosure, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween, of the plurality of modified T cells express one or more cell surface markers of early memory T cells. 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 expresses at least one modified T cell surface marker. SCM In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T CM Includes:

[0241] In some embodiments of the disclosed methods, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween, of the plurality of modified T cells express one or more cell surface markers of stem cell-like T cells. In certain embodiments, the plurality of modified stem cell-like T cells expresses at least one modified T SCM In certain embodiments, the plurality of engineered stem cell-like T cells comprises at least one engineered T CM Includes:

[0242] In some embodiments of the disclosed methods, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween, of the plurality of modified T cells are stem memory T cells (T SCM ) express one or more cell surface markers. In certain embodiments, the cell surface markers include CD62L and CD45RA. In certain embodiments, the cell surface markers include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. In certain embodiments, the cell surface markers include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0243] In some embodiments of the disclosed methods, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween, of the plurality of modified T cells are central memory T cells (T CM ) In certain embodiments, the cell surface markers include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0244] In some embodiments of the disclosed methods, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween, of the plurality of modified T cells are naive T cells (TN ) In certain embodiments, the cell surface markers include one or more of CD45RA, CCR7, and CD62L.

[0245] In some embodiments of the disclosed methods, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween of the plurality of modified T cells are effector T cells (modified T EFF ) In certain embodiments, the cell surface markers include one or more of CD45RA, CD95, and IL-2Rβ.

[0246] In some embodiments of the methods of the disclosure, the method modifies a plurality of modified T cells, and / or the method produces 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 therebetween of the plurality of modified T cells are stem cell-like T cells, stem memory T cells (T SCM ), or central memory T cells (T CM ) express one or more cell surface markers.

[0247] In some embodiments of the disclosed methods, the buffer contains immune cells or their precursors. The buffer maintains or enhances the level of cell survival and / or stem-like phenotype of immune cells or their precursors, including T cells. In certain embodiments, the buffer maintains or enhances the level of cell survival and / or stem-like phenotype of primary human T cells before nucleofection. In certain embodiments, the buffer maintains or enhances the level of cell survival and / or stem-like phenotype of primary human T cells during nucleofection. In certain embodiments, the buffer maintains or enhances the level of cell survival and / or stem-like phenotype of primary human T cells after nucleofection. In certain embodiments, the buffer contains one or more of KCl, MgCl, ClNa, glucose, and Ca(NO) at 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 phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO). In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 40 mM NaHPO / NaHPO, pH 7.2. In certain embodiments, a composition comprising primary human T cells is prepared by mixing 100 μl of buffer and 5×10 6 ~25×10 6 In certain embodiments, the composition comprises 250 x 10 cells per milliliter of buffer or other medium during the introduction step. 6 Contains a measurable proportion of primary human T cells.

[0248] In some embodiments of the methods of the present disclosure, the methods include contacting immune cells of the present disclosure, including T cells of the present disclosure, with a T cell expansion composition. In some embodiments of the methods of the present disclosure, the step of introducing a transposon and / or transposase of the present disclosure into an immune cell of the present disclosure may further include contacting the immune cell and the T cell expansion composition. In some embodiments, including those in which the introducing step of the method includes an electroporation or nucleofection step, the electroporation or nucleofection step may involve contacting the immune cell with the T cell expansion composition of the present disclosure.

[0249] In some embodiments of the disclosed methods, the T cell expansion composition comprises, consists essentially of, or consists of phosphorus; one or more of octanoic acid, palmitic acid, linoleic acid, and oleic acid; a sterol; and an alkane.

[0250] In certain embodiments of the methods of producing modified T cells of the present disclosure, the expansion supplement comprises one or more cytokines. The one or more cytokines can include any cytokine, including, but not limited to, lymphokines. Exemplary lymphokines 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 (INFγ). The one or more cytokines can include IL-2.

[0251] In some embodiments of the disclosed methods, the T cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement. In certain embodiments of the methods, the T cell expansion composition further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, a sterol, and an alkane. In certain embodiments of the methods, 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 the method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg inclusive, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg inclusive. In certain embodiments of the 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 the method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg inclusive of the endpoint, palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg inclusive of the endpoint, linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive of the endpoint, oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive of the endpoint, and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg inclusive of the endpoint.In certain embodiments of the method, the T cell expansion composition further comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg.

[0252] 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 are early memory T cells, stem cell-like T cells, stem memory T cells (T SCM ) and / or central memory T cells (T CM), expressing one or more cell surface markers of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, 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 0.9 mg / kg to 90 mg / kg inclusive, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg inclusive (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 (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 9.19 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 sterols at a concentration of about 1.01 mg / kg (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 about 2.13 mg / kg, oleic acid at a concentration of about 2.13 mg / kg, and sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg inclusive, palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg inclusive, linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive, oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive, and a sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg inclusive. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the T cell expansion composition comprises octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg.

[0253] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium at 37°C that includes one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium that includes phosphorus, octanoic acid fatty acid, palmitic acid fatty acid, linoleic acid fatty acid, and oleic acid. In certain embodiments, the medium contains a 10-fold higher amount of phosphorus than can be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available from ThermoFisher Scientific as catalog number 12440053).

[0254] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium at 37°C that includes one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium that includes 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" can be used interchangeably with a medium that includes one or more of the following elements present at corresponding average concentrations: 3.7 mg / L boron, 3000 mg / L sodium, 18 mg / L magnesium, 29 mg / L phosphorus, 15 mg / L potassium, and 4 mg / L calcium.

[0255] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium at 37°C that includes one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can refer to a medium containing one or more of the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-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 The medium may be used interchangeably with media containing one or more of the following: hydroxybenzoates (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), sterols (e.g., cholesterol) (CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5).In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" refers to a composition that is supplemented with the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-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) esters. It can be used interchangeably with media containing one or more of the following: butyl ether (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), sterols (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 term "supplemented T cell expansion composition" or "T cell expansion composition" refers to a composition that contains the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-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-dimethyl-2,4-benzotriazole (DMSO) (CAS No. 126-86-3 ... 2,4,7,9-tetramethyl-5-decyne-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-dimethyl-2,4-benzotriazole (DMSO) (CAS No. 126-86-3), 1,2-dimethyl-2,4-benzotriazole (DMSO) (CAS No. 126-86- -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. 143-74-8), and lanolin alcohol may be used interchangeably with media containing one or more of the following:

[0256] In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements at 37° C. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate, and phosphate.

[0257] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and expansion supplements at 37° C. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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" can be used interchangeably with a medium comprising one or more of the following free amino acids in the corresponding average molar 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 term "supplemented T cell expansion composition" or "T cell expansion composition" refers to the composition containing the following free amino acids in the corresponding average molar percentages: histidine (about 0.78%), 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 1.92%), arginine (about 1.92%), glycine (about 1.92%), aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine (about 1.92%), arginine (about 1.92%), glycine (about 1.92%), aspartic acid (about 1.92%), glutamic acid (about 1.92%), threonine (about 1.92%), argin ... (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%).

[0258] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium at 37°C that includes one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's MDM, and expansion supplements. Alternatively, or in addition, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium that includes phosphorus, octanoic acid fatty acid, palmitic acid fatty acid, linoleic acid fatty acid, and oleic acid. In certain embodiments, the medium contains a 10-fold higher amount of phosphorus than can be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available from ThermoFisher Scientific as catalog number 12440053).

[0259] In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium 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" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg inclusive, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg inclusive, and a sterol at a concentration of about 0.1 mg / kg to 10 mg / kg inclusive (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium 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 sterols at a concentration of about 1 mg / kg (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium 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 sterols at a concentration of about 1.01 mg / kg (mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium 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 sterols at a concentration of 1.01 mg / kg (mg / kg = parts per million).In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg inclusive of the endpoint, palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg inclusive of the endpoint, linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive of the endpoint, oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg inclusive of the endpoint, and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg inclusive of the endpoint. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 64 μmol / kg, palmitic acid at a concentration of about 7 μmol / kg, linoleic acid at a concentration of about 7.5 μmol / kg, oleic acid at a concentration of about 7.5 μmol / kg, and a sterol at a concentration of about 2.5 μmol / kg.

[0260] In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of about 7.56 μmol / kg, and a sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably to refer to a medium comprising one or more of octanoic acid at a concentration of about 63.75 μmol / kg, palmitic acid at a concentration of about 7.27 μmol / kg, linoleic acid at a concentration of about 7.57 μmol / kg, oleic acid at a concentration of 7.56 μmol / kg, and a sterol at a concentration of 2.61 μmol / kg.

[0261] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM , and / or T CMIn certain embodiments of the method for producing the modified stem cell-like T cells of the present disclosure, the method comprises contacting the modified T cells with an inhibitor of the P13K-Akt-mTOR pathway. SCM , and / or T CM The modified T cells of the present disclosure, comprising: 18 H 14Exemplary inhibitors of the PI3K pathway include, but are not limited to, inhibitors of GSK3β, such as BB007 (BLUEBIRDBIO™), which has CAS number 601514-19-6 and has N4O2. Additional exemplary inhibitor components of the PI3K pathway include allosteric Akt inhibitor VIII (also referred to as Akti-1 / 2 having compound number 10196499), ATP-competitive inhibitors (orthosteric inhibitors that target the ATP-binding pocket of protein kinase B (Akt)), isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), azepane derivatives (a series of structures derived from (-)-balanol), aminofurazan (GSK690693), heterocycles (7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidines, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, ipatasertib (GDC-0068, RG7440), A-674563, and and A-443654), phenylpyrazole derivatives (AT7867 and AT13148), thiophenecarboxamide derivatives (afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivative (DC120), aprosertib (GSK2141795)), allosteric inhibitors (superior to orthosteric inhibitors, offering higher specificity, reduced side effects, and less toxicity), 2,3-diphenylquinoxaline analogues (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivatives (MK-2206)), alkylphospholipids (edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3), irmofosine (BM41).440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), elfosine (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 (SR13668), OSU-A9), sulfonamide derivatives (PH-316 and PHT-427), thiourea derivatives (PIT-1, PIT-2, DM-PIT-1, N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), purine derivatives (triciribine (TCN, NSC 154020), Triciribine Monophosphate Active Analog (TCN-P), 4-Amino-pyrido[2,3-d]pyrimidine Derivative API - 1,3-Phenyl-3H-imidazo[4,5-b]pyridine Derivative, ARQ 092), BAY These include, but are not limited to, 1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucaric acid, 3α- and 3β-acetoxy-tirucaric acid, acetoxy-tirucaric acid, and irreversible inhibitors (antibiotics, lactoquinomycin, frenolicin B, calafungin, medelmicin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-1,2-pyridine derivatives).

[0262] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM , and / or T CMIn certain embodiments of the methods for producing a T cell, the method comprises contacting the modified T cell and T cell effector differentiation. Exemplary inhibitors of T cell effector differentiation include, but are not limited to, BET inhibitors (e.g., JQ1, hyenotriazolodiazepines) and / or inhibitors of the BET family of proteins (e.g., BRD2, BRD3, BRD4, and BRDT).

[0263] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM , and / or T CM In certain embodiments of the method for producing Acss1, the method comprises contacting the modified T cell with an agent that reduces nuclear-cytoplasmic acetyl-CoA. Exemplary agents that reduce nuclear-cytoplasmic acetyl-CoA include, but are not limited to, 2-hydroxycitric acid (2-HC) and agents that increase expression of Acss1.

[0264] In certain embodiments of the methods of producing modified T cells (e.g., stem cell-like T cells, TSCMs, and / or TCMs) of the present disclosure, the method comprises contacting the modified T cells and a composition comprising a histone deacetylase (HDAC) inhibitor. In some embodiments, the composition comprising the HDAC inhibitor comprises or consists of valproic acid, sodium phenylbutyrate (NaPB), or a combination thereof. In some embodiments, the composition comprising the HDAC inhibitor comprises or consists of valproic acid. In some embodiments, the composition comprising the HDAC inhibitor comprises or consists of sodium phenylbutyrate (NaPB).

[0265] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CMIn certain embodiments of the method for producing IL-1, the activation supplement may include one or more cytokines. The one or more cytokines may include any cytokine, including, but not limited to, lymphokines. Exemplary lymphokines 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 (INFγ). The one or more cytokines may include IL-2.

[0266] The modified T cells of the present disclosure (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the method for producing CD3, the activator complement may comprise one or more activator complexes. Exemplary, non-limiting activator complexes may include monomeric, dimeric, trimeric, or tetrameric antibody complexes that bind to one or more of CD3, CD28, and CD2. In some embodiments, the activator complement comprises or consists of an activator complex comprising a human, humanized, or recombinant or chimeric antibody. In some embodiments, the activator complement comprises or consists of an activator complex that binds to CD3 and CD28. In some embodiments, the activator complement comprises or consists of an activator complex that binds to CD3, CD28, and CD2.

[0267] Natural killer (NK) cells In certain embodiments, the modified immune cells or immune progenitor cells of the present disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes that differentiate from lymphoid progenitor cells.

[0268] The modified NK cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0269] In certain embodiments, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0270] In certain embodiments, NK cells are electroporated using a Lonza 4D Nucleofector or BTX ECM 830 (500 V, 700 usec pulse length, 0.2 mm electrode gap, 1 pulse). All Lonza 4D Nucleofector programs are contemplated as being within the scope of the disclosed methods.

[0271] In one specific embodiment, 5x10E6 cells were electroporated per electroporation in 100 μL of P3 buffer in a cuvette, however, this ratio of cells per volume is scalable for commercial production methods.

[0272] In certain embodiments, NK cells are stimulated by co-culture with an additional cell line. In certain embodiments, the additional cell line comprises an artificial antigen-presenting cell (aAPC). In certain embodiments, stimulation occurs on day 1, 2, 3, 4, 5, 6, or 7 after electroporation. In certain embodiments, stimulation occurs on day 2 after electroporation.

[0273] In certain embodiments, the NK cells express CD56.

[0274] B cells In certain embodiments, the modified immune cells or immune precursor cells of the present 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.

[0275] The modified B cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0276] In certain embodiments, HSPCs are modified using the methods of the present disclosure and then primed for B cell differentiation in the 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 present disclosure and then primed for B cell differentiation in the presence of human IL-3, Flt3L, TPO, SCF, and G-CSF for 5 days.

[0277] In certain embodiments, after priming, the modified HSPC cells are transferred to a layer of feeder cells and fed every other week with a new layer of feeder cells once a week. In certain embodiments, the feeder cells are MS-5 feeder cells.

[0278] In certain embodiments, the 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, the modified HSPC cells are cultured with MS-5 feeder cells for 33 days.

[0279] Inducible Pro-Apoptotic Polypeptides The inducible pro-apoptotic polypeptides of the present disclosure are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure are much less immunogenic. The inducible pro-apoptotic polypeptides of the present disclosure are recombinant polypeptides, and therefore the non-naturally occurring sequences that are recombined to produce the inducible pro-apoptotic polypeptides of the present disclosure do not contain non-human sequences that the host human immune system may recognize as "non-self" and thereby induce an immune response in a subject receiving the inducible pro-apoptotic polypeptides of the present disclosure, cells comprising the inducible pro-apoptotic polypeptides, or compositions comprising the inducible pro-apoptotic polypeptides, or cells comprising the inducible pro-apoptotic polypeptides.

[0280] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the pro-apoptotic 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. The inducible pro-apoptotic polypeptide of the present disclosure may be non-naturally occurring.

[0281] Caspase polypeptides of the present 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 present disclosure include, but are not limited to, 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 present disclosure include, but are not limited to, caspase polypeptides that initiate apoptosis, including caspase 2, caspase 8, caspase 9, and caspase 10. Caspase polypeptides of the present disclosure include, but are not limited to, caspase polypeptides that execute apoptosis, including caspase 3, caspase 6, and caspase 7.

[0282] The caspase polypeptides of the present disclosure can be encoded by amino acid or nucleic acid sequences having one or more modifications compared to the wild-type amino acid or nucleic acid sequence. The nucleic acid sequences encoding the caspase polypeptides of the present disclosure can be codon-optimized. One or more modifications to the amino acid and / or nucleic acid sequences of the caspase polypeptides of the present disclosure can increase the interaction, cross-linking, cross-activation, or activation of the caspase polypeptides of the present disclosure compared to the wild-type amino acid or nucleic acid sequence. Alternatively, or in addition, one or more modifications to the amino acid and / or nucleic acid sequences of the caspase polypeptides of the present disclosure can decrease the immunogenicity of the caspase polypeptides of the present disclosure compared to the wild-type amino acid or nucleic acid sequence.

[0283] The caspase polypeptides of the present disclosure may be truncated compared to wild-type caspase polypeptides. For example, the caspase polypeptides may be truncated to eliminate the sequence encoding the caspase activation and recruitment domain (CARD) to eliminate or minimize the possibility of activating a local inflammatory response in addition to initiating apoptosis in cells containing the inducible caspase polypeptides of the present disclosure. The nucleic acid sequence encoding the caspase polypeptides of the present disclosure may be spliced ​​to form a variant amino acid sequence of the caspase polypeptides of the present disclosure compared to the wild-type caspase polypeptides. The caspase polypeptides of the present disclosure may be encoded by recombinant and / or chimeric sequences. The recombinant and / or chimeric caspase polypeptides of the present disclosure may contain sequences from one or more different caspase polypeptides. Alternatively, or in addition, the recombinant and / or chimeric caspase polypeptides of the present disclosure may contain sequences from one or more species (e.g., human and non-human sequences). The caspase polypeptides of the present disclosure may be non-naturally occurring.

[0284] The ligand-binding region of an inducible pro-apoptotic polypeptide of the present disclosure may comprise any polypeptide sequence that promotes or facilitates dimerization of a first inducible pro-apoptotic polypeptide of the present disclosure with a second inducible pro-apoptotic polypeptide of the present disclosure, which dimer activates or induces cross-linking of the pro-apoptotic polypeptides and the initiation of apoptosis in a cell.

[0285] The ligand-binding ("dimerization") region can include any polypeptide or functional domain thereof that allows induction using an endogenous or non-naturally occurring ligand (i.e., inducer), for example, a non-naturally occurring synthetic ligand. The ligand-binding region can be inside or outside the cell membrane, depending on the nature of the inducible pro-apoptotic polypeptide and the choice of ligand (i.e., inducer). A wide variety of ligand-binding polypeptides and their functional domains, including receptors, are known. The ligand-binding region of the present disclosure can include one or more sequences from receptors. Of particular interest are ligand-binding regions for which the ligands (e.g., small organic ligands) are known or can be readily generated. These ligand-binding regions or receptors can include, but are not limited to, FKBP and cyclophilin receptors, steroid receptors, tetracycline receptors, etc., as well as "non-naturally occurring" receptors (which can be obtained from antibodies, particularly heavy or light chain subunits, their mutant sequences, random amino acid sequences obtained by stochastic procedures, combinatorial synthesis, etc.). In certain embodiments, the ligand binding region is selected from the group consisting of an FKBP ligand binding region, a cyclophilin receptor ligand binding region, a steroid receptor ligand binding region, a cyclophilin receptor ligand binding region, and a tetracycline receptor ligand binding region.

[0286] The ligand-binding region, comprising one or more receptor domains, either as an endogenous domain or a truncated active portion thereof, can be at least about 50 amino acids and less than about 350 amino acids, usually less than 200. The binding region can be, for example, a small (less than 25 kDa to allow efficient transfection in a viral vector), monomeric, non-immunogenic, synthetically accessible, cell-permeable, non-toxic ligand that can be configured for dimerization.

[0287] The ligand-binding region, which includes one or more receptor domains, can be intracellular or extracellular, depending on the design of the inducible pro-apoptotic polypeptide and the availability of an appropriate ligand (i.e., an inducer). For hydrophobic ligands, the binding region can be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region is usually on the outside of the cell membrane unless a transport system exists to internalize the ligand in a form available for binding. For intracellular receptors, the inducible pro-apoptotic polypeptide, or a transposon or vector containing the inducible pro-apoptotic polypeptide, can encode a signal peptide and a transmembrane domain 5' or 3' to the receptor domain sequence, or can have a lipid attachment signal sequence 5' to the receptor domain sequence. If the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain is extracellular.

[0288] Antibodies and antibody subunits, such as heavy or light chains, particularly fragments, and more particularly all or part of their variable regions, or fusions of heavy and light chains that produce high-affinity binding, can be used as ligand-binding regions in the present disclosure. Contemplated antibodies include ectopically expressed human products, such as extracellular domains, that do not provoke an immune response and are not generally expressed in the periphery (i.e., outside the CNS / brain region). Examples include, but are not limited to, the low-affinity nerve growth factor receptor (LNGFR) and embryonic surface proteins (i.e., carcinoembryonic antigen). Furthermore, antibodies can be prepared against physiologically acceptable hapten molecules, and their individual antibody subunits can be screened for binding affinity. The cDNAs encoding the subunits can be isolated and modified, such as by deletion of portions of the constant region or variable region, or by mutagenesis of the variable region, to obtain binding protein domains with appropriate affinity for the ligand. In this manner, almost any physiologically acceptable haptenic compound can be used as a ligand or to provide an epitope for the ligand. Instead of antibody units, endogenous receptors can be used for which the binding region or domain is known and for which there are useful or known ligands for binding.

[0289] With respect to receptor multimerization, a ligand for a ligand-binding region / receptor domain of an inducible pro-apoptotic polypeptide can be multimeric in the sense that the ligand can have at least two binding sites, each of which is capable of binding to a ligand receptor domain (i.e., a ligand having a first binding site capable of binding the ligand-binding region of a first inducible pro-apoptotic polypeptide and a second binding site capable of binding the ligand-binding region of a second inducible pro-apoptotic polypeptide, wherein the ligand-binding region of the first inducible pro-apoptotic polypeptide and the ligand-binding region of the second inducible pro-apoptotic polypeptide are either the same or different). Thus, as used herein, the term "multimeric ligand-binding region" refers to the ligand-binding region of an inducible pro-apoptotic polypeptide of the present disclosure that binds to a multimeric ligand. Multimeric ligands of the present disclosure include dimeric ligands. Dimeric ligands of the present disclosure can have two binding sites capable of binding to a ligand receptor domain. In certain embodiments, the multimeric ligands of the present disclosure are dimers or higher-order oligomers of synthetic organic small molecules, usually no greater than about tetramers, with individual molecules typically being at least about 150 Da and less than about 5 kDa, usually less than about 3 kDa. Various pairs of synthetic ligands and receptors can be used. For example, in embodiments involving endogenous receptors, dimeric FK506 can be used with the FKBP12 receptor, dimerized cyclosporin A can be used with the cyclophilin receptor, dimerized estrogen with the estrogen receptor, dimerized glucocorticoid with the glucocorticoid receptor, dimerized tetracycline with the tetracycline receptor, dimerized vitamin D with the vitamin D receptor, and so on. Alternatively, higher-order ligands, such as trimers, can be used.For embodiments involving non-naturally occurring receptors, such as antibody subunits, modified antibody subunits, single-chain antibodies consisting of heavy and light chain variable regions separated by a flexible linker, or modified receptors, as well as mutant sequences thereof, any of a wide variety of compounds can be used. An important characteristic of the units comprising the multimeric ligands of the present disclosure is that each binding site is capable of binding the receptor with high affinity, and preferably, that they can be chemically dimerized. Also, methods are available for balancing the hydrophobicity / hydrophilicity of the ligands so that they are functionally soluble in serum for most applications and can diffuse across the plasma membrane.

[0290] Activation of the inducible pro-apoptotic polypeptides of the present disclosure can be achieved, for example, through chemically induced dimerization (CID) mediated by an inducing agent to produce a conditionally regulated protein or polypeptide. Not only are the pro-apoptotic polypeptides of the present disclosure inducible, but induction of these polypeptides is also reversible due to degradation of labile dimerizing agents or administration of monomeric competitive inhibitors.

[0291] In certain embodiments, the ligand-binding region comprises an FK506-binding protein 12 (FKBP12) polypeptide. In certain embodiments, the ligand-binding region comprises an FKBP12 polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V). In certain embodiments, in which the ligand-binding region comprises an FKBP12 polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V), the inducer may comprise the synthetic drug AP1903 (CAS index name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1(R*),2R*[S*[S*[1(R*),2R*]]]]]-(9Cl) CAS registry number: 195514-63-7; molecular formula: CHNO; molecular weight: 1411.65). In certain embodiments, in which the ligand-binding region comprises an FKBP12 polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V), the inducer may comprise AP20187 (CAS Registry Number: 195514-80-8 and molecular formula: CHO). In certain embodiments, the inducer is an AP20187 analog, such as AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 may be used interchangeably.

[0292] AP1903 API is manufactured by Alphora Research Inc., and the AP1903 injectable formulation is manufactured 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, pale yellow solution. Upon refrigeration, this formulation undergoes a reversible phase transition, resulting in an opaque solution. This phase transition is reversed by warming back to room temperature. The fill is 2.33 mL in a 3 mL glass vial (approximately 10 mg total AP1903 for injection per vial). Once the need for AP1903 administration is determined, patients can receive a single fixed dose of AP1903 for injection (0.4 mg / kg) over 2 hours via IV infusion using, for example, a non-DEHP, non-ethylene oxide sterilized infusion set. The AP1903 dose is calculated individually for every patient and is not recalculated unless body weight changes by ≥10%. The calculated dose is diluted to 100 mL in 0.9% saline prior to infusion. In a previous Phase I study of AP1903, 24 healthy volunteers were treated with a single injectable dose of AP1903 at dose levels of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg infused intravenously over 2 hours. AP1903 plasma levels were directly proportional to dose, with mean Cmax values ​​ranging from approximately 10 to 1275 ng / mL for the 0.01 to 1.0 mg / kg dose range. After the initial infusion period, blood concentrations showed a rapid distribution phase, with plasma levels declining to approximately 18%, 7%, and 1% of the maximum concentration at 0.5, 2, and 10 hours, respectively. Injectable AP1903 has been shown to be safe and well tolerated at all dose levels and has demonstrated a favorable pharmacokinetic profile. Iuliucci JD, et al., J Clin Pharmacol. 41:870-9, 2001.

[0293] For example, a fixed dose of AP1903 for injection may be 0.4 mg / kg infused intravenously over a 2-hour period. The amount of AP1903 required in vitro for effective cell signaling is 10-100 nM (1600 Da MW). This is equivalent to 16-160 μg / L or approximately 0.016-1.6 μg / kg (1.6-160 μg / 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 therapeutic cells.

[0294] The amino acid and / or nucleic acid sequences encoding the ligand binding domains of the present disclosure may contain one or more modified sequences compared to the wild-type amino acid or nucleic acid sequences. For example, the amino acid and / or nucleic acid sequences encoding the ligand binding domains of the present disclosure may be codon-optimized sequences. The one or more modifications may increase the binding affinity of a ligand (e.g., an inducer) to the ligand binding domain of the present disclosure compared to the wild-type polypeptide. Alternatively, or in addition, the one or more modifications may decrease the immunogenicity of the ligand binding domain of the present disclosure compared to the wild-type polypeptide. The ligand binding domains of the present disclosure and / or the inducers of the present disclosure may be non-naturally occurring.

[0295] The modified cells, transposons, and / or vectors of the present disclosure may comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-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. The inducible pro-apoptotic polypeptide of the present disclosure may also be referred to as an "iC9 safety switch." In certain embodiments, the modified cells and / or transposons of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the modified cells and / or transposons of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the transposon of the present 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 pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or truncated caspase 9 polypeptide of the present disclosure, the ligand-binding region may comprise an FK506-binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand-binding region comprising the FK506-binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of phenylalanine (F) with valine (V) at position 36 (F36V).

[0296] In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 14635).

[0297] In certain embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 14636). In certain embodiments, inducers specific for the ligand-binding region, which may include an FK506 binding protein 12 (FKBP12) polypeptide having a phenylalanine (F) to valine (V) substitution at position 36 (F36V), include AP20187 and / or AP1903, both of which are synthetic drugs.

[0298] In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase-9 polypeptides of the present disclosure, the linker region is encoded by an amino acid sequence comprising GGGGS (SEQ ID NO: 14637) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 14638). In certain embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0299] In certain embodiments of the truncated caspase-9 polypeptides of the present disclosure, the truncated caspase-9 polypeptide is encoded by an amino acid sequence that does not include an arginine (R) at position 87. Alternatively, or in addition, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase-9 polypeptides of the present disclosure, the truncated caspase-9 polypeptide is encoded by an amino acid sequence that does not include an alanine (A) at position 282.

[0023] In certain embodiments of an inducible pro-apoptotic polypeptide, an inducible caspase polypeptide, or a truncated caspase 9 polypeptide of the present disclosure, the truncated caspase 9 polypeptide comprises the amino acid sequence GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14639):Or it is encoded by a nucleic acid sequence containing TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC(SEQ ID NO: 14640).

[0300]

[0301] The inducible pro-apoptotic polypeptides of the present disclosure may be expressed in a cell under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present disclosure in the cell. As used herein, the term "promoter" refers to a promoter that serves as the initial binding site for RNA polymerase, which transcribes a gene. For example, the inducible pro-apoptotic polypeptides of the present disclosure may be expressed in a mammalian cell under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present disclosure in a mammalian cell, including, but not limited to, a native promoter, an endogenous promoter, an exogenous promoter, and a heterologous promoter. Preferred mammalian cells include human cells. Thus, the inducible pro-apoptotic polypeptides of the present disclosure may be expressed in a human cell under the transcriptional control of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present disclosure in a human cell, including, but not limited to, a human promoter or a viral promoter. Exemplary promoters for expression in human cells include, but are not limited to, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, the β-actin promoter, the rat insulin promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter, each of which can be used to obtain high levels of expression of the inducible pro-apoptotic polypeptides of the present disclosure. The use of other viral or mammalian cell or bacterial phage promoters known in the art to achieve expression of the inducible pro-apoptotic polypeptides of the present disclosure is also contemplated, provided that the expression level is sufficient to initiate apoptosis in the cells. By using a promoter with well-known properties, the level and pattern of expression of the protein of interest after transfection or transformation can be optimized.

[0302] Selection of a promoter regulated in response to a specific physiological or synthetic signal can enable inducible expression of the inducible pro-apoptotic polypeptide of the present disclosure. The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system. This system is designed to enable controlled expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows for virtually no basal level expression of the transgene, but over 200-fold inducibility. The system is based on the Drosophila heterodimeric ecdysone receptor; when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates the promoter, turning on expression of the downstream transgene and achieving high levels of mRNA transcripts. In this system, the ecdysone-responsive promoter driving expression of the gene of interest is on a separate plasmid, but both monomers of the heterodimeric receptor are constitutively expressed from a single vector. Therefore, engineering this type of system into a vector of interest can be useful. Another potentially useful inducible system is the Tet-Off™ or Tet-On™ system (Clontech, Palo Alto, Calif.), originally developed by Gossen and Bujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; Gossen et al., Science, 268:1766-1769, 1995). This system also allows high levels of gene expression to be controlled in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On™ system, gene expression is turned on in the presence of doxycycline, while in the Tet-Off™ system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. coli: the tetracycline operator sequence (to which the tetracycline repressor binds) and the tetracycline repressor protein.The gene of interest is cloned into the plasmid behind a promoter in which a tetracycline-responsive element is present in the plasmid. The second plasmid contains a regulatory element called a tetracycline-controlled transactivator, which in the Tet-Off™ system is composed of the VP16 domain from herpes simplex virus and a wild-type tetracycline repressor. Thus, in the absence of doxycycline, transcription is constitutively turned on. In the Tet-On™ system, the tetracycline repressor activates transcription in the presence of doxycycline, but not in the wild-type. When creating gene therapy vectors, the Tet-Off™ system can be used, allowing producer cells to grow in the presence of tetracycline or doxycycline, preventing the expression of potentially toxic transgenes, but allowing gene expression to be constitutively turned on when the vector is introduced into a patient.

[0303] In some situations, it is desirable to regulate the expression of transgenes in gene therapy vectors. For example, different viral promoters with varying activity strengths are utilized depending on the desired expression level. In mammalian cells, the CMV immediate-early promoter is often used to provide strong transcriptional activation. The CMV promoter is reviewed in Donnelly, JJ, et al., 1997. Annu. Rev. Immunol. 15:617-48. When a low level of transgene expression is desired, a less powerful modified version of the CMV promoter is also used. When transgene expression in hematopoietic cells is desired, retroviral promoters such as the LTR from MLV or MMTV are often used. Other viral promoters that can be used depending on the desired effect include adenoviral promoters such as those from SV40, RSV LTR, HIV-1 and HIV-2 LTR, E1A, E2A, or MLP regions, AAV LTR, HSV-TK, and avian sarcoma virus.

[0304] In other examples, promoters can be selected that are developmentally regulated and active in particular differentiated cells. Thus, for example, a promoter can be inactive in pluripotent stem cells, but can then be activated, for example, when the pluripotent stem cells differentiate into more mature cells.

[0305] Similarly, tissue-specific promoters are used to induce transcription in specific tissues or cells, so as to reduce potential toxicity or undesirable effects on non-target tissues.These promoters can cause reduced expression compared with stronger promoters such as CMV promoters, but can also cause more restricted expression and immunogenicity (Bojak, A., et al., 2002.Vaccine.20:1975-79; Cazeaux, N., et al., 2002.Vaccine 20:3322-31).For example, tissue-specific promoters such as PSA-related promoters or prostate-specific glandular kallikrein or muscle creatine kinase genes can be used as needed.

[0306] Examples of tissue- or differentiation-specific promoters include, but are not limited to, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells, healing tissues), and Flt-1 (endothelial cells), GFAP (astrocytes).

[0307] In certain indications, it is desirable to activate transcription at specific times after administration of the gene therapy vector, which can be accomplished using promoters such as hormone- or cytokine-regulatable promoters. Cytokine and inflammatory protein responsive promoters that can be used include K and T kininogen (Kageyama et al., (1987) J. Biol. Chem., 262, 2345-2351), c-fos, TNF-alpha, C-reactive protein (Arcone, et al., (1988) Nucl. Acids Res., 16(8), 3195-3207), haptoglobin (Oliviero et al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C / EBP alpha, IL-1, IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86, 8202-8206), complement C3 (Wilson et al. al., (1990) Mol. Cell. Biol., 6181-6191), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, (1988) Mol. Cell. Biol., 8, 42-51), alpha-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et (1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (inducible by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), stromelysin (inducible by phorbol esters, interleukin-1, and EGF), alpha-2 macroglobulin, and alpha-1 antichymotrypsin. Other promoters include, for example, SV40, MMTV, human immunodeficiency virus (MV), Moloney virus, ALV, Epstein-Barr virus, Rous sarcoma virus, human actin, myosin, hemoglobin, and creatine.

[0308] It is envisioned that any of the above promoters can be used alone or in combination with another promoter according to the desired effect.Promoter and other regulatory elements are selected so that they are functional in desired cells or tissues.In addition, this list of promoters should not be interpreted as exhaustive or limiting, and other promoters can be used with the promoter and method disclosed herein.

[0309] antigen receptor In some embodiments of the compositions and methods of the present disclosure, the modified autologous cells of the present disclosure comprise an antigen receptor.

[0310] In some embodiments of the compositions and methods of the present disclosure, the vector comprises a sequence encoding a chimeric antigen receptor or a portion thereof. Exemplary vectors of the present disclosure include, but are not limited to, viral vectors, non-viral vectors, plasmids, nanoplasmids, minicircles, transposition systems, liposomes, polymersomes, micelles, and nanoparticles.

[0311] In some embodiments of the compositions and methods of the present disclosure, the transposon comprises a sequence encoding a chimeric antigen receptor or a portion thereof, hi some embodiments, the transposon is integrated into the genomic sequence of the self-cell by a transposase.

[0312] In some embodiments of the compositions and methods of the present disclosure, the donor oligonucleotide or donor plasmid comprises a sequence encoding a chimeric antigen receptor or a portion thereof, hi some embodiments, the donor oligonucleotide or donor plasmid is fully or partially integrated into the chromosomal sequence of the autologous cell after single- or double-strand break and, optionally, cell-mediated repair.

[0313] Exemplary antigen receptors include non-naturally occurring transmembrane proteins that bind antigens at the site of their extracellular domains and transmit or induce an intracellular signal via their intracellular domains.

[0314] In some embodiments, non-naturally occurring antigen receptors include, but are not limited to, recombinant, mutant, chimeric, or synthetic T cell receptors (TCRs). In some embodiments, mutant TCRs contain one or more sequence mutations in either the nucleotide or amino acid sequence encoding the TCR when compared to a wild-type TCR. In some embodiments, synthetic TCRs contain at least one synthetic or modified nucleic acid or amino acid encoding the TCR. In some embodiments, recombinant and / or chimeric TCRs are encoded, over their entire length or a portion thereof, by nucleic acid or amino acid sequences that are non-naturally occurring because the sequences are isolated from or derived from one or more source sequences.

[0315] In some embodiments, non-naturally occurring antigen receptors include, but are not limited to, chimeric antigen receptors.

[0316] Chimeric Antigen Receptor In some embodiments of the compositions and methods of the present disclosure, the modified autologous cells of the present disclosure comprise a chimeric antigen receptor.

[0317] In some embodiments of the compositions and methods of the present disclosure, the transposon comprises a sequence encoding a chimeric antigen receptor or a portion thereof.

[0318] A chimeric antigen receptor (CAR) of the present disclosure may comprise (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments of a CAR of the present disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In certain embodiments of a CAR of the present disclosure, the signal peptide may comprise a sequence encoding a human CD8α signal peptide. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. In certain embodiments of the CARs of the present disclosure, the transmembrane domain may comprise a sequence encoding a human CD8α transmembrane domain. In certain embodiments of the CARs of the present disclosure, the endodomain may comprise a human CD3ζ endodomain.

[0319] In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain may comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In certain embodiments of the CARs of the present disclosure, at least one costimulatory domain may comprise a CD28 and / or 4-1BB costimulatory domain. In certain embodiments of the CARs of the present disclosure, the hinge may comprise a sequence derived from human CD8α, IgG4, and / or CD4 sequences. In certain embodiments of the CARs of the present disclosure, the hinge may comprise a sequence derived from human CD8α sequences.

[0320] The CD28 costimulatory domain may comprise an amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14477), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14477). The CD28 costimulatory domain can be encoded by a nucleic acid sequence comprising: cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 14478). The 4-1BB costimulatory domain may comprise an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14479), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14479).The 4-1BB costimulatory domain can be encoded by a nucleic acid sequence comprising: aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 14480). The 4-1BB costimulatory domain can be located between the transmembrane domain and the CD28 costimulatory domain.

[0321] In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α, IgG4, and / or CD4 sequence. In certain embodiments of the CARs of the present disclosure, the hinge can comprise a sequence derived from a human CD8α sequence. The hinge can comprise a human CD8α amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 14481), or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to an amino acid sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 14481). The human CD8α hinge amino acid sequence can be encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 14482).

[0322] ScFv The present disclosure provides single-chain variable fragment (scFv) compositions and methods for using these compositions to recognize and bind to specific target proteins. ScFv compositions comprise the heavy and light chain variable regions of an antibody. The ScFv compositions can be incorporated into the antigen recognition region of a chimeric antigen receptor of the present disclosure. ScFvs are fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, with the VH and VL domains connected by a short peptide linker. ScFvs retain the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. An exemplary linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 14483).

[0323] Sentinel The centilins of the present disclosure specifically bind to antigens. Chimeric antigen receptors of the present disclosure, comprising one or more centilins that specifically bind to antigens, can be used to target the specificity of cells (e.g., cytotoxic immune cells) to specific antigens.

[0324] The centilin of the present disclosure may comprise a protein scaffold, which is capable of specifically binding to an antigen. The centilin of the present disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, which is capable of specifically binding to an antigen. The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be tenascin-C. The consensus sequence may comprise LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence can comprise an amino acid sequence at least 74% identical to LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence can be encoded by a nucleic acid sequence comprising: atgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcgatagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctcttatgacctgacagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcaccacca (SEQ ID NO: 14490).The consensus sequence includes (a) an AB loop comprising or consisting of amino acid residues TEDS (SEQ ID NO: 14491) at positions 13 to 16 of the consensus sequence, (b) a BC loop comprising or consisting of amino acid residues TAPDAAF (SEQ ID NO: 14492) at positions 22 to 28 of the consensus sequence, (c) a CD loop comprising or consisting of amino acid residues SEKVGE (SEQ ID NO: 14493) at positions 38 to 43 of the consensus sequence, and (d) a 51 (e) a DE loop comprising or consisting of amino acid residues GSER (SEQ ID NO: 14494) at positions 10-54 of the consensus sequence; (f) an EF loop comprising or consisting of amino acid residues GLKPG (SEQ ID NO: 14495) at positions 60-64 of the consensus sequence; (g) an FG loop comprising or consisting of amino acid residues KGGHRSN (SEQ ID NO: 14496) at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). The centrin of the present disclosure may comprise at least five fibronectin type III (FN3) domains, at least ten fibronectin type III (FN3) domains, or a consensus sequence of at least 15 fibronectin type III (FN3) domains. The scaffold may comprise at least 10 fibronectin type III (FN3) domains. -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M and below, and 10 -15 K below M D It can bind to an antigen with at least one affinity selected from K D can be determined by surface plasmon resonance.

[0325] The term "antibody mimic" is intended to describe an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimics may include proteins, nucleic acids, or small molecules. The target sequence to which the antibody mimics of the present disclosure specifically bind may be an antigen. Antibody mimics may offer superior properties to antibodies, including, but not limited to, superior solubility, tissue penetration, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimics include, but are not limited to, affibodies, affrylins, affimers, affitins, alphabodies, anticalins, and avimers (also known as avidity multimers), DARPins (designed ankyrin repeat proteins), Fynomers, Kunitz domain peptides, and monobodies.

[0326] The affibody molecules of the present disclosure comprise a protein scaffold that comprises or consists of one or more alpha helices that are completely free of disulfide bridges. Preferably, the affibody molecules of the present disclosure comprise or consist of three alpha helices. For example, the antibody molecules of the present disclosure may comprise an immunoglobulin-binding domain. The affibody molecules of the present disclosure may comprise the Z domain of Protein A.

[0327] The affilin molecules of the present disclosure include protein scaffolds produced by modifying the exposed amino acids of, for example, gamma B crystallin or ubiquitin. Affilin molecules functionally mimic the affinity of antibodies for antigens, but do not structurally mimic antibodies. In any protein scaffold used to create affilins, amino acids that are accessible to solvent or potential binding partners in a properly folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids can be modified to specifically bind to a target sequence or antigen.

[0328] The Affimer molecules of the present disclosure comprise protein scaffolds that include highly stable proteins genetically engineered to display peptide loops that provide high-affinity binding sites for specific target sequences. Exemplary Affimer molecules of the present disclosure include protein scaffolds based on cystatin proteins or their tertiary structures. Exemplary Affimer molecules of the present disclosure may share a common tertiary structure that includes an alpha helix positioned above an antiparallel beta sheet.

[0329] The affitin molecules of the present disclosure include artificial protein scaffolds, the structure of which may be derived, for example, from DNA-binding proteins (e.g., the DNA-binding protein Sac7d). The affitins of the present disclosure selectively bind to target sequences, which may be all or part of an antigen. Exemplary affitins of the present disclosure are produced by randomizing one or more amino acid sequences on the binding surface of a DNA-binding protein and subjecting the resulting protein to ribosome display and selection. The target sequences of the affitins of the present disclosure may be found, for example, within genomes or on the surfaces of peptides, proteins, viruses, or bacteria. In certain embodiments of the present disclosure, the affitin molecules can be used as specific inhibitors of enzymes. The affitin molecules of the present disclosure may include heat-stable proteins or derivatives thereof.

[0330] The alphabody molecules of the present disclosure may be referred to as cell-penetrating alphabodies (CPABs). The alphabody molecules of the present disclosure comprise small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). The alphabody molecules can reach and bind to intracellular target sequences. Structurally, the alphabody molecules of the present disclosure comprise an artificial sequence that forms a single-stranded alpha helix (similar to a naturally occurring coiled-coil structure). The alphabody molecules of the present disclosure may comprise a protein scaffold containing one or more amino acids that have been modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the alphabody molecules of the present disclosure maintain correct folding and thermal stability.

[0331] The anticalin molecules of the present disclosure include artificial proteins that bind to target sequences or sites in either proteins or small molecules. The anticalin molecules of the present disclosure may include artificial proteins derived from human lipocalins. The anticalin molecules of the present disclosure may be used, for example, in place of monoclonal antibodies or fragments thereof. The anticalin molecules may exhibit superior tissue penetration and thermal stability compared to monoclonal antibodies or fragments thereof. An exemplary anticalin molecule of the present disclosure may contain approximately 180 amino acids with a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure comprise a barrel structure comprising pairwise antiparallel beta strands connected by loops and linked by alpha helices. In a preferred embodiment, the anticalin molecules of the present disclosure comprise a barrel structure comprising eight pairwise antiparallel beta strands connected by loops and linked by alpha helices.

[0332] The avimer molecules of the present disclosure include artificial proteins that specifically bind to a target sequence (which may also be an antigen). The avimers of the present disclosure may recognize multiple binding sites within the same target or different targets. When the avimers of the present disclosure recognize more than one target, they mimic the function of a bispecific antibody. The artificial protein avimers may contain two or more peptide sequences, each approximately 30-35 amino acids long. These peptides may be connected via one or more linker peptides. One or more of the amino acid sequences of the avimer peptides may be derived from the A domain of a membrane receptor. Avimers have a rigid structure that may optionally include disulfide bonds and / or calcium. The avimers of the present disclosure may exhibit higher thermal stability compared to antibodies.

[0333] The DARPins (designed ankyrin repeat proteins) of the present disclosure comprise engineered, recombinant, or chimeric proteins with high specificity and high affinity for target sequences. In certain embodiments, the DARPins of the present disclosure are derived from ankyrin proteins and optionally comprise at least three repeat motifs (also referred to as repeat structural units) of ankyrin proteins. Ankyrin proteins mediate high-affinity protein-protein interactions. The DARPins of the present disclosure comprise large target interaction surfaces.

[0334] Fynomers of the present disclosure comprise small binding proteins (approximately 7 kDa) derived from the human Fyn SH3 domain and engineered to bind target sequences and molecules with affinity and specificity equivalent to antibodies.

[0335] The Kunitz domain peptides of the present disclosure include protein scaffolds containing Kunitz domains. The Kunitz domains contain active sites for inhibiting protease activity. Structurally, the Kunitz domains of the present disclosure contain a disulfide-rich alpha + beta fold. This structure is exemplified by bovine pancreatic trypsin inhibitor. The Kunitz domain peptides recognize specific protein structures and act as competitive protease inhibitors. The Kunitz domains of the present disclosure may include ecallantide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

[0336] The monobodies of the present disclosure are small proteins comparable in size to single-chain antibodies (containing approximately 94 amino acids and having a mass of approximately 10 kDa). These engineered proteins specifically bind to target sequences, including antigens. The monobodies of the present disclosure can specifically target one or more distinct proteins or target sequences. In a preferred embodiment, the monobodies of the present disclosure comprise a protein scaffold that mimics the structure of human fibronectin, more preferably the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, and its monobody mimics, contain seven beta sheets and three exposed loops that form a barrel on each side, corresponding to the three complementarity-determining regions (CDRs) of an antibody. In contrast to the structure of an antibody variable domain, monobodies lack a binding site for metal ions and a central disulfide bond. Multispecific monobodies can be optimized by modifying the BC and FG loops. The monobodies of the present disclosure may also include adnectins.

[0337] VHH In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VHH.

[0338] The present disclosure provides a chimeric antigen receptor (CAR) comprising at least one VHH (VCAR). The chimeric antigen receptor of the present disclosure may comprise more than one VHH. For example, a bispecific VCAR may comprise two VHHs that specifically bind to two distinct antigens.

[0339] The VHH proteins of the present disclosure specifically bind to antigens. Chimeric antigen receptors of the present disclosure, comprising one or more VHHs that specifically bind to an antigen, can be used to target the specificity of cells (e.g., cytotoxic immune cells) to a specific antigen.

[0340] At least one VHH protein or VCAR of the present disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, for example, Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0341] Amino acids from a VHH protein may be changed, added, and / or deleted to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility, or any other suitable property, as is known in the art.

[0342] Optionally, VHH proteins can be engineered while retaining high affinity for antigens and other favorable biological properties. To achieve this goal, VHH proteins can be prepared by a process of analysis of parent sequences and various conceptual engineered products using three-dimensional models of the parent and engineered sequences. Three-dimensional models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformations of selected candidate sequences, allowing potential immunogenicity to be measured (e.g., the Immunofilter program from Xencor, Inc. (Monrovia, Calif.)). Inspection of these displays allows for analysis of the possible role of residues in the function of the candidate sequences, i.e., analysis of residues that affect the ability of the candidate VHH protein to bind to its antigen. In this way, residues can be selected and combined from parent and reference sequences to achieve desired characteristics, such as affinity for the target antigen. Alternatively, or in addition to the above procedures, other suitable engineering methods can be used.

[0343] Screening VHHs for specific binding to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. This method involves screening large collections of peptides for individual members with the desired function or structure. The displayed nucleotide or peptide sequences can be 3 to 5,000 or more nucleotides or amino acids in length, often 5 to 100 amino acids in length, and often about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves displaying peptide sequences on the surface of bacteriophage or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. The VHH proteins of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one VHH of the present disclosure has high affinity, e.g., 0.1 to 9.9 (or any range or value therebetween) x 10, as determined by surface plasmon resonance or Kinexa methods, as practiced by one of skill in the art. -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 Such as, but not limited to, about 10 -7 It can optionally bind to the target protein with a KD of less than or equal to M, or any range or value therebetween.

[0344] The affinity or avidity of a VHH or VCAR for an antigen can be experimentally determined using any suitable method (see, for example, Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992), and the methods described herein). The measured affinity of a particular VHH-antigen or VCAR-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using standardized solutions of the VHH or VCAR and antigen, and standardized buffers such as those described herein.

[0345] Competition assays can be performed on the VHHs or VCARs of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the VHHs or VCARs of the present disclosure for binding to a target protein and / or share epitope regions. These assays, readily known to those skilled in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The proteins and / or antibodies are immobilized or insolubilized before or after competition, and the target protein-bound sample is separated from the unbound sample by, for example, decantation (if the protein / antibody was insolubilized beforehand) or centrifugation (if the protein / antibody precipitated after the competition reaction). Competitive binding can also be determined by whether binding or lack of binding of the VHHs or VCARs to the target protein alters function, for example, whether the VCAR molecule inhibits or enhances the enzymatic activity of a label. ELISA and other functional assays can be used, as are well known in the art.

[0346] VH In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VH.

[0347] The present disclosure provides chimeric antigen receptors (CARs), including single-domain antibodies (VCARs). In certain embodiments, the single-domain antibodies comprise a VH. In certain embodiments, the VH is isolated or derived from a human sequence. In certain embodiments, the VH comprises human CDR sequences and / or human framework sequences, and a non-human or humanized sequence (e.g., a rat Fc domain). In certain embodiments, the VH is a fully humanized VH. In certain embodiments, the VH is not a naturally occurring antibody or a fragment of a naturally occurring antibody. In certain embodiments, the VH is not a fragment of a monoclonal antibody. In certain embodiments, the VH is a UniDab™ antibody (TeneoBio).

[0348] In certain embodiments, VHs are fully engineered using the UniRat™ (TeneoBio) system and "NGS-based discovery" to generate VHs. Using this method, specific VHs are generated using a non-naturally occurring, fully engineered system. The VHs are not derived from naturally occurring monoclonal antibodies (mAbs) isolated directly from a host (e.g., mouse, rat, or human) or from a single clone of a cell or cell line (hybridoma). These VHs were not subsequently cloned from the cell line. Instead, the VH sequences are fully engineered using the UniRat™ system as transgenes containing human variable regions (VH domains) with a rat Fc domain, making them human / rat chimeras lacking light chains and distinct from standard mAb formats. The native rat gene has been knocked out, and the only antibodies expressed in rats are from transgenes with VH domains linked to rat Fc (UniAbs). These are exclusively UniRat-expressed Abs. Next-generation sequencing (NGS) and bioinformatics are used to identify the complete antigen-specific repertoire of heavy chain antibodies generated by UniRat™ after immunization. A proprietary gene assembly method is then used to convert the antibody repertoire sequence information into a large collection of fully human heavy chain antibodies that can be screened in vitro for various functions. In certain embodiments, fully humanized VHs are generated by fusing a human VH domain with a human Fc in vitro (to generate non-naturally occurring recombinant VH antibodies). In certain embodiments, although the VHs are fully humanized, they are expressed in vivo as human / rat chimeras (human VH, rat Fc) without light chains. The fully humanized VHs are expressed in vivo because the human / rat chimeras (human VH, rat Fc) without light chains are approximately 80 kDa (vs. 150 kDa).

[0349] A VCAR of the present disclosure may comprise at least one VH of the present disclosure. In certain embodiments, a VH of the present disclosure may be modified to remove the Fc domain or a portion thereof. In certain embodiments, the framework sequence of a VH of the present disclosure may be modified, for example, to improve expression, reduce immunogenicity, or improve function.

[0350] As used throughout this disclosure, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes a plurality of such methods, and reference to a "dosage" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0351] The terms "about" or "approximately" mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Where particular values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable range of error for the particular value should be assumed.

[0352] The present disclosure provides isolated or substantially purified polynucleotide or protein composi...

Claims

1. A non-naturally occurring chimeric stimulatory receptor (CSR), (a) an ectodomain comprising an activation component, said activation component being isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, said at least one signal transduction domain being isolated or derived from a second protein; A non-naturally occurring chimeric stimulatory receptor (CSR), wherein the first protein and the second protein comprise non-identical endodomains.

2. 2. The CSR of claim 1, wherein the activating component comprises 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, and a chemokine receptor to which an agonist of the activating component binds.

3. The CSR of claim 1 , wherein the activating component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds.

4. The CSR of claim 1 , wherein the signal transduction domain comprises one or more of a human signal transduction domain, 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, and a chemokine receptor.

5. The CSR of claim 1 , wherein the signal transduction domain comprises a CD3 protein or a portion thereof.

6. The CSR of claim 5 , wherein the CD3 protein comprises a CD3ζ protein or a portion thereof.

7. The CSR of claim 1 , wherein the endodomain further comprises a cytoplasmic domain.

8. The CSR of claim 7 , wherein the cytoplasmic domain is isolated or derived from a third protein.

9. The CSR of claim 8 , wherein the first protein and the third protein are identical.

10. The CSR of claim 1 , wherein the ectodomain further comprises a signal peptide.

11. The CSR of claim 10 , wherein the signal peptide is derived from a fourth protein.

12. The CSR of claim 11 , wherein the first protein and the fourth protein are identical.

13. The CSR of claim 1 , wherein the transmembrane domain is isolated or derived from a fifth protein.

14. The CSR of claim 13 , wherein the first protein and the fifth protein are identical.

15. The CSR of claim 1 , wherein the activating component does not bind to a naturally occurring molecule.

16. The CSR of claim 1 , wherein the CSR does not transduce a signal upon binding of the activating component to a naturally occurring molecule.

17. The CSR of claim 1 , wherein the activating moiety binds to a non-naturally occurring molecule.

18. The CSR of claim 1 , wherein the CSR selectively transduces a signal upon binding of the activating component to a non-naturally occurring molecule.

19. A non-naturally occurring chimeric stimulatory receptor (CSR), (a) an ectodomain comprising a signal peptide and an activation moiety, said signal peptide comprising a CD2 signal peptide or a portion thereof, and said activation moiety comprising a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, the transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; (c) a non-naturally occurring chimeric stimulatory receptor (CSR), comprising: a cytoplasmic domain and an endodomain comprising at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

20. 20. The CSR of claim 19, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 17062.

21. 20. The CSR of claim 19, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17062.

22. 20. The CSR of claim 19, comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 17062.

23. 20. The CSR of claim 19, comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 17062.

24. The CSR of claim 19 comprising the amino acid sequence of SEQ ID NO: 17062.

25. The CSR of claim 1 , wherein the ectodomain comprises a modification.

26. 26. The CSR of claim 25, wherein the modification comprises a mutation or 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.

27. 27. The CSR of claim 26, wherein the mutation or truncation of the amino acid sequence of the activating component comprises a mutation or truncation of the CD2 extracellular domain or a portion thereof to which an agonist binds.

28. 28. The CSR of claim 27, wherein the CSR comprises a mutation or truncation of the CD2 extracellular domain or a portion thereof to which an agonist binds, and does not bind to CD58.

29. The CSR of claim 27, wherein the CD2 extracellular domain containing the mutation or truncation comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 17119.

30. The CSR of claim 27, wherein the CD2 extracellular domain containing the mutation or truncation comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 17119.

31. The CSR of claim 27, wherein the CD2 extracellular domain containing the mutation or truncation comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17119.

32. The CSR of claim 27, wherein the CD2 extracellular domain containing the mutation or truncation comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 17119.

33. The CSR of claim 27, wherein the CD2 extracellular domain containing the mutation or truncation comprises the amino acid sequence of SEQ ID NO: 17119.

34. A non-naturally occurring chimeric stimulatory receptor (CSR), (a) an ectodomain comprising a signal peptide and an activation moiety, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and the activation moiety comprises an agonist-binding CD2 extracellular domain or a portion thereof, wherein the agonist-binding CD2 extracellular domain or portion thereof comprises a mutation or truncation; (b) a transmembrane domain, the transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; (c) a non-naturally occurring chimeric stimulatory receptor (CSR), comprising: a cytoplasmic domain and an endodomain comprising at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

35. The CSR of claim 34, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 17118.

36. The CSR of claim 34, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 17118.

37. The CSR of claim 34, comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 17118.

38. The CSR of claim 34, comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 17118.

39. The CSR of claim 34 comprising the amino acid sequence of SEQ ID NO: 17118.

40. A nucleic acid sequence encoding a CSR according to any one of claims 1 to 39.

41. A vector comprising the nucleic acid sequence of claim 40.

42. A transposon comprising the nucleic acid sequence of claim 40.

43. A cell comprising a CSR according to any one of claims 1 to 39.

44. A cell comprising the nucleic acid of claim 40.

45. A cell comprising the vector of claim 41.

46. A cell comprising the transposon of claim 42.

47. The cell of any one of claims 43 to 46, wherein the cell is an allogeneic cell.

48. The cell according to any one of claims 43 to 46, wherein the cell is an autologous cell.

49. A composition comprising a CSR according to any one of claims 1 to 39.

50. A composition comprising the nucleic acid sequence of claim 40.

51. A composition comprising the vector of claim 41.

52. A composition comprising the transposon of claim 42.

53. A composition comprising a cell according to any one of claims 43 to 46.

54. A composition comprising a plurality of cells according to any one of claims 43 to 46.

55. 1. A modified T lymphocyte (T cell), comprising: (a) modification of an endogenous sequence encoding a T cell receptor (TCR), the modification reducing or eliminating the level of expression or activity of said TCR; (b) a chimeric stimulating receptor (CSR), (i) an ectodomain comprising an activation component, said activation component being isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) a chimeric stimulating receptor (CSR) comprising an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein, and the first protein and the second protein are not identical.

56. 56. The modified T cell of claim 55, further comprising an inducible pro-apoptotic polypeptide.

57. 56. The modified T cell of claim 55, further comprising a modification of the endogenous sequence encoding beta-2-microglobulin (B2M), wherein said modification reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

58. 56. The modified T cell of claim 55, further comprising a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

59. 59. The modified T cell of claim 58, wherein the non-naturally occurring polypeptide comprising HLA-E further comprises a B2M signal peptide.

60. 60. The modified T cell of claim 59, wherein the non-naturally occurring polypeptide comprising HLA-E further comprises a B2M polypeptide.

61. The modified T cell of claim 60, wherein the non-naturally occurring polypeptide comprising HLA-E further comprises a linker, the linker being positioned between the B2M polypeptide and the HLA-E polypeptide.

62. 62. The modified T cell of claim 61, wherein the non-naturally occurring polypeptide comprising HLA-E further comprises a peptide and a B2M polypeptide.

63. the non-naturally occurring polypeptide comprising HLA-E, a first linker located between the B2M signal peptide and the peptide; The modified T cell of claim 62, further comprising a second linker positioned between the B2M polypeptide encoding the HLA-E and the peptide.

64. 56. The modified T cell of claim 55, further comprising a sequence encoding a non-naturally occurring antigen receptor, a therapeutic polypeptide, or a combination thereof.

65. 65. The modified T cell of claim 64, wherein the non-naturally occurring antigen receptor comprises a chimeric antigen receptor (CAR).

66. 56. The modified T cell of claim 55, wherein the CSR is transiently expressed in the modified T cell.

67. 56. The modified T cell of claim 55, wherein the CSR is stably expressed in the modified T cell.

68. The modified T cell of claim 58, wherein the polypeptide comprising the HLA-E polypeptide is transiently expressed in the modified T cell.

69. The modified T cell of claim 58, wherein the polypeptide comprising the HLA-E polypeptide is stably expressed in the modified T cell.

70. 57. The modified T cell of claim 56, wherein the inducible pro-apoptotic polypeptide is stably expressed in the modified T cell.

71. 65. The modified T cell of claim 64, wherein the sequence encoding the non-naturally occurring antigen receptor or therapeutic protein is stably expressed in the modified T cell.

72. 56. The modified T cell of claim 55, wherein the modified T cell is an allogeneic cell.

73. 56. The modified T cell of claim 55, wherein the modified T cell is an autologous cell.

74. The modified T cells may be early memory T cells, stem cell-like T cells, stem memory T cells (T SCM ), central memory T cells (T CM ), or a stem cell-like T cell.

75. A composition comprising the modified T cell of any one of claims 55 to 74.

76. 40. A composition comprising a population of modified T cells, wherein a plurality of said population of modified T cells comprises a CSR according to any one of claims 1 to 39.

77. 10. A composition comprising a population of modified T cells, wherein a plurality of said population of modified T cells comprises the modified T cell of any one of claims 55-74.

78. At least 25% of the population of modified T cells are stem memory T cells (T SCM ) or T SCM The composition of claim 76 or 77, wherein the composition expresses one or more cell surface markers of a CD45-like cell, the one or more cell surface markers including CD45RA and CD62L.

79. At least 50% of the population of modified T cells are central memory T cells (T CM ) or T CM The composition of claim 76 or 77, wherein the composition expresses one or more cell surface markers of a CD45-like cell, said one or more cell surface markers including CD45RO and CD62L.

80. At least 75% of the population of modified T cells are central memory T cells (T CM ) or T CM The composition of claim 76 or 77, wherein the composition expresses one or more cell surface markers of a CD45-like cell, said one or more cell surface markers including CD45RO and CD62L.

81. 78. A composition according to any one of claims 76 or 77 for use in the treatment of a disease or disorder.

82. 80. Use of a composition according to any one of claims 76 or 77 for the treatment of a disease or disorder.

83. 80. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition of any one of claims 76 or 77.

84. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a composition described in claim 76 or 77 and at least one non-naturally occurring molecule that binds to the CSR.

85. 40. A method of producing a population of engineered T cells, comprising introducing a composition comprising a CSR of claims 1-39 or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of engineered T cells and maintain desirable stem-like properties of the plurality of engineered T cells, to produce the plurality of engineered T cells.

86. At least 25% of the population of modified T cells are stem memory T cells (T SCM ) or T SCM 86. The method of claim 85, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RA and CD62L.

87. At least 50% of the population of modified T cells are central memory T cells (T CM ) or T CM 86. The method of claim 85, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

88. At least 75% of the population of modified T cells are central memory T cells (T CM ) or T CM 86. The method of claim 85, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

89. 86. A composition comprising a population of modified T cells produced by the method of claim 85.

90. 90. The composition of claim 89 for use in treating a disease or disorder.

91. 90. Use of the composition of claim 89 for the treatment of a disease or disorder.

92. 90. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of the composition of claim 89.

93. 93. The method of claim 92, further comprising administering to the subject an activator composition to activate the population of in vivo modified T cells, to induce cell division of the population of in vivo modified T cells, or a combination thereof.

94. 40. A method of producing a population of engineered T cells, comprising introducing a composition comprising a CSR of claims 1-39 or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express a CSR in the plurality of engineered T cells and maintain desirable stem-like properties of said plurality of engineered T cells, to produce said plurality of engineered T cells.

95. At least 25% of the population of modified T cells are stem memory T cells (T SCM ) or T SCM 95. The method of claim 94, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RA and CD62L.

96. At least 50% of the population of modified T cells are central memory T cells (T CM ) or T CM 95. The method of claim 94, wherein the cells express one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

97. At least 75% of the population of modified T cells are central memory T cells (T CM ) or T CM 95. The method of claim 94, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

98. 95. A composition comprising a population of modified T cells produced by the method of claim 94.

99. 99. The composition of claim 98 for use in treating a disease or disorder.

100. 100. Use of the composition of claim 98 for the treatment of a disease or disorder.

101. 100. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of the composition of claim 98.

102. 102. The method of claim 101, wherein the modified T cells within the population of modified T cells administered to the subject no longer express the CSR.

103. 40. A method of expanding a population of engineered T cells, comprising: introducing a composition comprising a CSR of claims 1-39 or a sequence encoding same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of engineered T cells and maintain desirable stem-like properties of the plurality of engineered T cells to produce the plurality of engineered T cells; and contacting the cells with an activator composition to produce a plurality of activated engineered T cells, wherein the expansion of the plurality of engineered T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the CSR under the same conditions.

104. At least 25% of the population of modified T cells are stem memory T cells (T SCM ) or T SCM The method of claim 103, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RA and CD62L.

105. At least 50% of the population of modified T cells are central memory T cells (T CM ) or T CM The method of claim 103, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

106. At least 75% of the population of modified T cells are central memory T cells (T CM ) or T CM The method of claim 103, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

107. 104. A composition comprising a population of modified T cells expanded by the method of claim 103.

108. 108. The composition of claim 107 for use in treating a disease or disorder.

109. 108. Use of the composition of claim 107 for the treatment of a disease or disorder.

110. 108. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of the composition of claim 107.

111. The method of claim 110, further comprising administering to the subject an activator composition to activate the population of in vivo modified T cells, to induce cell division of the population of in vivo modified T cells, or a combination thereof.

112. 40. A method of expanding a population of engineered T cells, comprising: introducing a composition comprising a CSR of claims 1-39 or a sequence encoding same into a plurality of primary human T cells under conditions that transiently express the CSR in a plurality of engineered T cells and maintain desirable stem-like properties of the plurality of engineered T cells to produce the plurality of engineered T cells; and contacting the cells with an activator composition to produce a plurality of activated engineered T cells, wherein the expansion of the plurality of engineered T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not transiently express the CSR under the same conditions.

113. At least 25% of the population of modified T cells are stem memory T cells (T SCM ) or T SCM The method of claim 112, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RA and CD62L.

114. At least 50% of the population of modified T cells are central memory T cells (T CM ) or T CM The method of claim 112, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

115. At least 75% of the population of modified T cells are central memory T cells (T CM ) or T CM The method of claim 112, wherein the cell expresses one or more cell surface markers of a CD45-like cell, and the one or more cell surface markers include CD45RO and CD62L.

116. 113. A composition comprising a population of modified T cells expanded by the method of claim 112.

117. 117. The composition of claim 116 for use in treating a disease or disorder.

118. 117. Use of the composition of claim 116 for the treatment of a disease or disorder.

119. 117. A method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of the composition of claim 116.

120. 120. The method of claim 119, wherein the modified T cells within the population of modified T cells administered to the subject no longer express the CSR.