Allogeneic cell compositions and methods of use thereof

Abstract

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

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 of making same, and methods of use same.

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

[0004] There is a long felt and unmet need in the art for allogeneic cell compositions that overcome the challenges presented by eliminating genes involved in graft versus host and host versus graft responses. The present disclosure provides allogeneic cell compositions, methods of making and using these compositions, including 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, where the activating component is isolated 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 or derived from a second protein, where the first protein and the second protein are not identical.

[0006] The activating component can include a portion of one or more of a T cell receptor (TCR), a TCR complex, a TCR co-receptor, a TCR co-stimulatory protein, 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 may comprise 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 may comprise a CD3 protein or a portion thereof. The CD3 protein may comprise a CD3 zeta protein or a portion thereof.

[0008] The endodomain may further comprise a cytoplasmic domain. The cytoplasmic domain may be isolated from or derived from a third protein. The first protein and the third protein may be identical. The ectodomain may further comprise a signal peptide. The signal peptide may be derived from a fourth protein. The first protein and the fourth protein may be identical. The transmembrane domain may be isolated from or derived from a fifth protein. The first protein and the fifth protein may be identical.

[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. 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 activating component, the signal peptide comprising a CD2 signal peptide or a portion thereof, and the activating component comprising 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, the cytoplasmic domain comprising a CD2 cytoplasmic domain or a portion thereof, and the at least one signal transduction domain comprising a CD3 zeta protein or a portion thereof. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence that is 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 a non-naturally occurring chimeric stimulating receptor (CSR) in which the ectodomain comprises a modification. The modification can comprise a mutation or truncation of the amino acid sequence of the activating component or the first protein when compared to the wild-type sequence of the activating component or the first protein. The mutation or truncation of the amino acid sequence of the activating component can comprise 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 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 that is 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 activating component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, the activating 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, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and 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 that is 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 cell disclosed herein can be an allogeneic cell or an autologous cell.In some preferred embodiments, the modified cell is an allogeneic cell.In some preferred embodiments, the modified cell is an allogeneic T cell or modified allogeneic CAR T cell.

[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), where the modification 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 moiety, where the activating moiety 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, 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 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.

[0018] The modified T cell can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising 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 located between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E can further comprise a first linker located between the B2M polypeptide and the peptide, and a second linker located 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 a composition comprising any of the modified T cells disclosed herein. The present disclosure also provides a composition comprising a population of modified T lymphocytes (T cells), where the population of a plurality of modified T cells comprises a CSR as disclosed herein. The present disclosure also provides a composition comprising a population of T lymphocytes (T cells), where the population of a plurality of T cells comprises a modified T cell as disclosed herein.

[0023] The present disclosure provides a method for treating a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of any composition 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 for 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 a method of 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 a composition 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 disclosure also provides the use of the composition produced by the method for treating a disease or disorder. The 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 produced by the method. The treatment method can further comprise administering to the subject an activator composition to activate the in vivo modified population of T cells, to induce cell division of the in vivo modified population of T cells, or a combination thereof.

[0025] The present disclosure provides a method of producing a population of engineered T cells, 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 a composition 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 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 the use of the composition 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 the composition produced by the method. In some embodiments, the modified T cells in the population of modified T cells administered to the subject no longer express CSR.

[0026] The disclosure provides a method of expanding a population of modified T cells, comprising introducing a composition comprising a CSR of the disclosure or a sequence encoding same into a plurality of primary human T cells under conditions that stably 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 stably express 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 a use of the composition expanded by the present 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 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 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 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 the use of the composition increased 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 increased by the method. In some embodiments, the modified T cells in the population of modified 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 those skilled in the art to which the present invention belongs. In this specification, unless the context clearly dictates otherwise, the singular can also include the plural, and by way of 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" are understood to mean the inclusion of a described 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 invention claimed. In case of conflict, the present specification, including definitions, shall control. In addition, 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 claims. [Brief description 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 the coreceptors CD28 and CD2. [Diagram 2] 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 by costimulatory receptors that promote immune responses. Primary and secondary costimulation can be delivered to T cells via treatment with agonist mAbs (e.g., anti-CD3, anti-CD28, and anti-CD2) and engagement of surface receptors with them. [Diagram 3] Schematic diagram showing that in the absence of TCR, only secondary costimulation is delivered to T cells via binding of agonistic 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 is suboptimal and therefore reduced. [Figure 4] Schematic diagram showing that in the absence of TCR, stimulation is enhanced by expression of chimeric stimulating receptor (CSR). In the absence of TCR, but in the presence of surface-expressed CSR / s, primary and secondary costimulatory signals are delivered when T cells are treated with standard agonist mAb. More complete T cell activation is achieved through stimulatory signals via CSR, thus enhancing T cell activation and expansion. [Diagram 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]Schematic diagram of a strategy for mutation of CSR CD2z to eliminate binding of the natural ligand (CD58). A panel of CSR CD2z mutants was designed within the extracellular domain of CD2. The aim 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 and prevent optimal performance of CSR, and 2) WT CD2z CSR may act as a natural ligand CAR, such that T cells expressing CSR may mediate cytotoxic activity against cells expressing CD58, including activated T cells. Thus, mutant CD2z CSR that cannot interact with CD58 but retain the ability to bind activating anti-CD2 reagents for optimal cell expansion is 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]1 is 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 normal donors 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 a CSR (either CD28z or CD2z), an mRNA encoding the super piggyBac™ transposase enzyme, an 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 first culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). In samples expressing either CD2z or CD28z CSR, a greater expansion of DKO cells was observed as was a higher frequency of CAR-only DKO cells (Figures 9A and 9B). In DKO CAR-T cell samples expressing either CD2z or CD28z CSR, an at least two-fold expansion of cells was observed compared to DKO CAR-T cells alone. [Figure 9B] Same as explanation for Figure 9A. [Figure 10A]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, the expression of TCR and MHCI (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, were still highly pure to DKO cells (>98.8% DKO). DKO CAR-T cells expressing either CD2z or CD28z CSR conferred enhanced expansion when compared to cells expressing neither CSR. [Figure 10B] Same as explanation for FIG. 10A. [Figure 11]Graph showing that cytokine supplementation can further expand purified DKO CAR-T cells expressing CSR upon restimulation. After initial genetic modification and first round of stimulation and expansion, cells expressing CSR 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 the second expansion, all purified DKO cells, including cells expressing either CD2z CSR or CD28z CSR, were still highly pure for TCR-MHCI- cells (>98.8% double knockout (data not shown)). In addition, cells grew robustly in the presence of IL7 and IL15, which was greater than without supplementation. These data show that exogenous cytokines can be added to further expand WT CAR-T cells expressing CSR. [Figure 12] 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]Graph showing that expression of CSR does not significantly affect 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 line K562-PSMA-luciferase (eK562-Luc.PSMA) at E:T ratios of 10:1, 3:1, or 1:1 for 48 hours. Luciferase signal was measured to determine cytotoxicity. Killing of eK562-Luc.PSMA is shown as dotted line, while killing of eK562-Luc.BCMA is shown as solid line. All CAR+ T cells expressed an anti-BCMA-specific CAR. DKO CAR-T cells exhibit similar in vitro cytotoxicity as WT CAR-TCR cells. This activity is not significantly affected by co-expression of CD2z or CD28z CSR. [Figure 14] 1 is a graph showing that expression of CSR does not significantly affect DKO CAR-T cell secretion of IFNg in vitro. Supernatants from the 48-hour killing assay were assayed for secreted IFNg as a measure of antigen-specific function of BCMA CAR-T cells. All CAR-T cells, regardless of the presence or absence of CD2z or CD28z CSR expression, secrete IFNg in response to co-culture with target cells expressing BCMA (eK562-Luc.BCMA), whereas cells expressing an irrelevant target (eK562-Luc.PSMA) do not secrete IFNg. [Figure 15]A series of plots showing that expression of CSR does not significantly affect proliferation of DKO CAR-T cells 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 are labeled with Cell Trace Violet (CTV) and diluted as cells proliferate. Cells were co-cultured with eK562-Luc.PSMA or eK562-Luc.BCMA cells at an E:T ratio of 1:2 for 5 days. All CAR-T cells, with or without CD2z or CD28z, proliferate in response to target cells expressing BCMA (eK562-Luc.BCMA) but not cells expressing an irrelevant antigen (eK562-Luc.PSMA). [Figure 16] A pair of graphs showing that the memory phenotype of DKO CAR-T 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-), Teff (CD45RA+CD45RO-CD62L-).WT and DKO CAR-T cells, with or without CD2z, consist primarily of very high levels of favorable 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 negatively impact the in vivo function of these CAR T cells as they appear more differentiated. [Figure 17]A series of graphs showing that the expression of activation / exhaustion markers in DKO CAR-T 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 exhaustion molecules Lag3, PD1, and Tim3. WT and DKO CAR-T cells, with or without CD2z, have little to no expression of exhaustion molecules when compared to mock T cells. However, expression of CD28z CSR in DKO CAR-T during the expansion process leads to a significant upregulation of exhaustion markers Lag3, PD1, and Tim3. This phenotype may negatively impact the function of these CAR T cells in vivo, as they appear to be 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]1 is a graph showing that CSR delivery enhances the expansion of CAR-T cells. CSR was delivered to CAR-T cells either transiently by mRNA or stably by 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 the BCMA CAR, and a selection gene, along with additional mRNA encoding CSR (either CD28z or CD2z; resulting in transient expression), or CD19 mRNA control, or a transposon encoding 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 first culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). Bars represent total live CAR-T cells in the wells, and numbers indicate 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 the expression of CSR is not significantly affected by the cytotoxicity of CAR-T cells. CSR was delivered to CAR-T cells either transiently by mRNA or stably by 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 the BCMA CAR, and a selection gene, together with additional mRNA encoding CSR (either CD28z or CD2z; resulting in transient expression), or a transposon encoding the BCMA CAR, a selection gene, and a CSR (either CD28z or CD2z; resulting in stable expression). Cells were subsequently stimulated with agonist mAbs, anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification over a culture period of 19 days. At the end of the first culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). To evaluate the killing capacity of CAR-T cells, cells were co-cultured with engineered K562-BCMA-luciferase (eK562-Luc.BCMA) or negative control line K562-luciferase (eK562-Luc) at E:T ratios of 10:1, 3:1, or 1:1 for 48 h. 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 showed similar in vitro cytotoxicity against BCMA+ target cells. In summary, this activity was not significantly affected by CSR co-expression, either transiently or stably. [Figure 20]Schematic diagram showing that in the presence of TCR, stimulation is enhanced by the expression of chimeric stimulating receptor (CSR). In the presence of CSR / s expressed on the surface, either transiently or stably expressed, and when T cells are treated with a reagent that represents agonist mAb, enhanced primary and secondary costimulatory signals are delivered. In one embodiment, this schematic diagram represents autologous cells. More complete T cell activation is achieved through stimulatory signals via CSR, thus enhancing T cell activation and expansion. [Figure 21] 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 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]1 is a series of line graphs showing that CSR molecules can be delivered temporarily during manufacturing for the expansion of enhanced 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 CC gene editing for the production of autologous (Auto) CAR-T cells, and 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 Allo CAR-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 provided to the cells transiently as mRNA only once in the first EP reaction, with varying amounts of mRNA at 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 over a 19-day culture period using the selection gene. At the end of the first culture period, all T cells expressed the CAR, indicating successful selection of genetically modified cells (data not shown). Each data is displayed in a line graph for various production dates. A greater degree of CAR-T cell expansion was observed in samples provided with CD2z CSR stably (as transposon-encoded (stable)) or transiently (as mRNA-encoded (mRNA)) compared to CAR-T cells generated without CSR.These data indicate that CSR can be delivered transiently as mRNA during manufacturing to enhance the expansion of both autologous and allogeneic CAR-T products. [Figure 23A] 23C is a bar graph showing CSR CD2z mutant staining data. A panel of CSR CD2z mutants was designed, constructed, and tested for surface expression and binding to several anti-CD2 antibody reagents. To do so, each mutant was synthesized 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 the next day by flow cytometry, and the data is shown in a bar graph. 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 is summarized in FIG. 23C. [Figure 23B]A series of bar graphs showing CSR CD2z variants degranulation data. A panel of CSR CD2z variants were tested for their ability to mediate degranulation against CD58 positive cell targets. T cell degranulation is a surrogate for T cell death and can be measured by FACS staining of intracellular CD107a expression following co-culture with a target cell line 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 variants and cultured overnight. The next day, 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, and negative controls were either non-electroporated Rat2 cells or CSR CD2z mutant expressing T cells only. 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 for D111H, K67R / Y110D, K67R / Q70K / Y110D / D111H, deltaK106-120, CD3z deletion, and mock controls, and 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 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 binds strongly to all staining reagents on the cell surface and completely abolishes anti-CD58 degranulation activity. [Figure 23D]A series of flow cytometry plots showing 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, were tested. Degranulation of engineered T cells was assessed against cell lines K562 and Rat2 made to overexpress the target ligands, and expression was confirmed by FACS staining. Red histograms are unstained cells, and blue histograms are cells electroporated / lipofected with mRNA and stained for expression of the respective markers by FACS. [Figure 23E]Figure 1 is a bar graph showing 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, were tested. Degranulation of engineered T cells was assessed against cell lines K562 and Rat2 made 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, T cells transfected with GFP were also included as a control. T cell degranulation is a surrogate for T cell death and can be measured by FACS staining of intracellular CD107a expression after co-culture 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, cells were co-cultured for 4–6 h in the presence of various target cell lines electroporated / lipofected with mRNA encoding human CD48, CD58, or CD59, while negative controls were either K562 or Rat2 cells that were not electroporated / lipofected, or electroporated T cells alone, respectively. 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 T cells expressing GFP. [Figure 24A]1 is a bar graph showing that delivery of 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 combined with the Cas-CLOVER™ gene editing system (CC) for the production of allogeneic (Allo) CAR-T cells, or without CC gene editing as a control for the production of autologous (Auto) CAR-T cells without CSR (No CSR), and 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 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 transposons encoding either CAR, selection gene, and CSR CD2z, as well as either WT or mutant (D111H) CSR CD2z, or a safety switch that did not encode CAR, selection gene, and CSR. For the latter, Allo CAR-T cells that did not receive transposon-encoded CSR for stable integration, WT or mutant (D111H) CSR CD2z was provided to cells transiently as mRNA only once in the first EP reaction. After EP, all cells were subsequently stimulated with a cocktail of agonistic 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 first 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 either stably (transposon-encoded) or transiently (mRNA-encoded) compared to Allo CAR-T cells generated without CSR. [Figure 24B]1 is a series of bar graphs showing that delivery of CSR CD2z-D111H mutant does not inhibit gene editing. Pan-T cells isolated from blood of healthy donors 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, a selection gene, and a safety switch that did not encode a CSR. For the latter, cells that did not receive the transposon-encoded CSR for stable integration, WT or mutant (D111H) CSR CD2z was provided transiently as mRNA only once in the first EP reaction. After EP, all cells were then stimulated with a cocktail of agonistic 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 first 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 in which WT or mutant (D111H) CD2z was provided stably (as transposon-encoded) or transiently (as mRNA-encoded) compared to Allo CAR-T cells produced without CSR. [Figure 24C]1 is a bar graph showing that the memory phenotype of Allo CAR-T is not significantly affected by delivery of CD2z CSR. Allo CAR-T cells without CSR and Allo CAR-T 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-), Teff (CD45RA+CD45RO-CD62L-). All samples were run in duplicate and data are shown in bar graphs with error bars representing standard deviation. Delivery of CSR did not dramatically affect the levels of favored Tscm and Tcm cells within the product. [Diagram 25] FIG. 1 is a schematic diagram showing 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] A pair of graphs showing that expression of single chain HLA-E reduces NK cell-mediated cytotoxicity against HLA-deficient T cells. B2M and TCRαβ were knocked out from T cells (Jurkat) using CRISPR. B2M / TCRαβ double knockout (DKO) T cells were electroporated with mRNA encoding the HLA-E molecule (HLA-bGBE) and expressed in single chain with B2M and the peptide VMAPRETLIL (SEQ ID NO: 17127) (B2M / peptide / HLA-E). DKOT cells electroporated with various amounts of mRNA encoding single chain HLA-E were used as targets for artificial antigen presenting cells (aAPC)-expanded NK cells in a 3-hour co-culture. 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 in 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] A series of flow cytometry plots showing targeted knockout of endogenous HLA-ABC, but not HLA-E. Having shown 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 strand HLA-E gene. To do so, multiple guide RNAs (gRNAs) were designed to disrupt expression of the primary targets of host versus graft (HvG), HLA-A, HLA-B, and HLA-C, while minimizing disruption of endogenous HLA-E. Specifically, guides were designed to target 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 displayed below. These data indicate 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. [Diagram 30] FIG. 1 is a schematic diagram of the loss-of-self hypothesis of natural killer cell-mediated toxicity towards MHCI-KO cells. [Diagram 31] Schematic representation of the Csy4-T2A-Clo051-G4Slinker-dCas9 construct map (embodiment 2). [Diagram 32] Schematic representation of the pRT1-Clo051-dCas9 double NLS construct map (embodiment 1). [Diagram 33] FIG. 1 is a schematic diagram showing an exemplary method for producing an allogeneic CAR-T of the present disclosure. [Figure 34A]Graph showing highly efficient gene integration 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™ (RNA-guided fusion protein containing dCas9-Clo051). The Cas-CLOVER system interrupted the expression of TCRα in rapidly proliferating Jurkat T cells and non-dividing resting T cells at relatively high levels. [Figure 34B] 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 that mediate alloreactivity, were efficiently edited by Cas-CLOVER in resting human T cells. [Diagram 35] A series of flow cytometry plots showing that Cas-CLOVER can 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. [Diagram 36]A series of graphs showing the reduction in alloreactivity after TCR and MHCI KO. 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 mixed in a 1:1 ratio with celltrace violet (CTV)-labeled and irradiated peripheral blood mononuclear cells (PBMCs) and incubated for 12 days or 20 hours, respectively, before analysis of proliferation or activation-induced secretion of IFNγ by ELISpot assay. WT or DKO CAR-T cells were incubated in 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, and these results showed significant proliferation of WT CAR-T cells when incubated with allogeneic PBMCs, with proliferation rates of 40% and 39% 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. On the other hand, DKO CAR-T cells did not proliferate when incubated with allogeneic PBMCs, indicating that KO of TCR and MHCI led to the elimination of graft-versus-host alloreactions. This was also true for short-term IFNγ by ELISpot assay (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 in 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, and the results showed 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 KO of TCR and MHCI resulted in the elimination of host vs. 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 PBMC when incubated with allogeneic CAR-T. [Figure 37] 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 / β2MDKO and WT T cells expressing 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 allo CAR-T did not significantly reduce the composition of memory CAR-T cells, which remained very high and predominantly retained the Tscm phenotype. [Figure 38] A series of graphs showing that DKO CAR-Ts are highly functional. Gene editing does not impact the functionality of CAR-T cells. TCRβ / β2M DKO and WT T cells expressing BCMA CARs were analyzed for function. Proliferation against H929 (BCMA+) tumor lines 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 H929 (BCMA+) tumor lines was 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 is normalized to tumor cell only samples. These data show that gene editing to produce DKO CAR-T cells does not significantly impact their functional capacity. [Figure 39A]FIG. 1 is a schematic showing 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 luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into 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 (2.5×10^6 or 4×10^6) 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 in the axilla and treated when tumors were established (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] FIG. 34A is a series of graphs showing tumor volume assessment of mice treated as described. Tumor volume assessment by caliper measurement for control mice (black), donor #1 FLP mice (red), donor #1 NT mice (blue), donor #2 FLP mice (orange), and donor #2 NT mice (green) as displayed as group averages with error bars (top) and individual mice (bottom). The y-axis shows 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 constructed SC LNCaP.luc solid tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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, 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 co-stimulatory 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 co-stimulatory 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 the CD2 extracellular domain or a portion thereof to which the agonist binds, or the ectodomain can comprise, consist essentially of, or consist of the CD28 extracellular domain or a portion thereof to which the agonist binds. The activating component can comprise, consist essentially of, or consist of the CD2 extracellular domain or a portion thereof to which the agonist binds, or the activating component can comprise, consist essentially of, or consist of the CD28 extracellular domain or a portion thereof to which the agonist binds. The CD2 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: 17111. The CD2 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: 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 co-receptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The second protein can comprise, consist 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 that is 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 that is at least 99% identical to the amino acid sequence of SEQ ID NO:17102. The CD3 zeta 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, essentially consist 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, essentially consist of, or consist of a CD2 cytoplasmic domain or a portion thereof, or the cytoplasmic domain can comprise, essentially consist 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 that is 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 that is 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 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 an 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 may be isolated 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 may comprise, essentially consist of, or consist of the CD2 transmembrane domain or a portion thereof, or the transmembrane domain may comprise, essentially consist of, or consist of the CD28 transmembrane domain or a portion thereof. The CD2 transmembrane domain comprises, essentially consists of, or consists of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, essentially consists of, or consists of an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO: 17112. The CD28 transmembrane domain comprises, essentially consists of, or consists of an amino acid sequence that is 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 cannot bind to a naturally occurring molecule. In some embodiments, the activating component of the CSR of the present disclosure binds or can bind 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 can bind to a non-naturally occurring molecule. The activating component of the CSR of the present disclosure selectively transduces a signal upon binding of the 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 that can bind 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 the 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 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 the 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 modifications. The modifications can include mutations or truncations of the amino acid sequence of the activating component or the first protein when compared to the wild-type sequence of the activating component or the first protein. The mutations or truncations of the amino acid sequence of the activating component or the first protein can include mutations or truncations of the CD2 extracellular domain or a portion thereof to which the agonist binds. The mutations or truncations of the CD2 extracellular domain reduce or eliminate binding to naturally occurring CD58.

[0043] A reduction in binding is a reduction in the binding capacity 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. An elimination in binding is a reduction in the binding capacity 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 of SEQ ID NO: 17119. The CSR comprising 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 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: 17118. The CSR comprising 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: 17118. The CSR comprising the mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 17118.

[0045] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) that comprises, consists essentially of, or consists of: (a) an ectodomain that includes 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 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, 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) that comprises, consists essentially of, or consists of: (a) an ectodomain that includes an activating moiety, where the activating moiety is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain that includes at least one signal transduction domain, where the at least one signal transduction domain is isolated 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) that comprises, consists essentially of, or consists of: (a) an ectodomain that includes an activating component, where the activating component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain that includes at least one signal transduction domain, where the at least one signal transduction domain is isolated 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 moiety, wherein the signal peptide comprises the CD2 signal peptide or a portion thereof, and the activating moiety comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises 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 CD3 zeta protein or a portion thereof.

[0049] 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 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 stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is at least 80% identical to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulating receptor (CSR) can comprise, consist essentially of, or consist of an amino acid sequence that is 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 activating component, the signal peptide comprising a CD2 signal peptide or a portion thereof, and the activating component comprising 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, the cytoplasmic domain comprising a CD2 cytoplasmic domain or a portion thereof, and the at least one signal transduction domain comprising a CD3 zeta protein or a portion thereof. In one embodiment, the 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 stimulating 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 stimulating 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 stimulating 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 that encodes any of the chimeric stimulating receptor (CSR) amino acid sequences disclosed herein. The present disclosure also provides a transposon, vector, donor sequence, or donor plasmid that comprises, consists essentially of, or consists of the nucleic acid sequence that encodes any of the chimeric stimulating receptor (CSR) amino acid sequences 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 a cell comprising, essentially consisting of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides a cell comprising, essentially consisting 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 a cell comprising, essentially consisting of, or consisting of a transposon, vector, donor sequence, or donor plasmid comprising, essentially consisting 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 cell of the present disclosure comprising, essentially consisting of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein can be an allogeneic cell or an autologous cell. In some preferred embodiments, the cell is an allogeneic cell.

[0054] The present disclosure also provides a composition comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides a composition 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 a composition 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 a composition 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 moiety, where the activating moiety 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, where the first protein and the second protein are not identical.

[0056] The 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 moiety, where the activating moiety 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, where the first protein and the second protein are not identical; and (b) an inducible pro-apoptotic polypeptide.

[0057] The 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 moiety, where the activating moiety is isolated 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 or derived from a second protein, where the first protein and the second protein 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 expression or activity of the TCR.

[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 moiety, where the activating moiety 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, where the first protein and the second protein are not identical.

[0060] The present disclosure provides modified T lymphocytes (T cells) that comprise, consist essentially of, or consist 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; (b) a chimeric stimulating receptor (CSR) that comprises: (i) an ectodomain that includes an activating moiety, where the activating moiety 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; 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) comprising: (i) an ectodomain comprising an activating component, the activating component being isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, the at least one signal transduction domain being isolated from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0062] The present disclosure relates to an engineered T lymphocyte (T cell), comprising: (a) an engineered T cell receptor (TCR) in an endogenous sequence that encodes the T cell receptor, the engineered TCR reducing or eliminating the level of expression or activity; (b) an engineered beta-2-microglobulin (B2M) in an endogenous sequence that encodes the major histocompatibility complex (MHC) class I (MHC-I) reducing or eliminating the level of expression or activity; and (c) a chimeric stimulating receptor (CSR), the engineered T cell comprising: (i) an ectodomain that comprises an activation component, the activation component being isolated from a first protein; The present invention provides 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 protein and the second protein are not identical; and (d) a non-naturally occurring chimeric antigen receptor.

[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 that includes an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulating receptor (CSR) that includes (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, 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 an engineered T lymphocyte (T cell) comprising: (a) an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of TCR expression or activity; (b) 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 that includes an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulating receptor (CSR) having an ectodomain that includes (i) an activation 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 or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein, and the first protein and the second protein are not identical; and (e) a non-naturally occurring chimeric antigen receptor.

[0065] The 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 of an HLA class I histocompatibility antigen, alpha chain A (HLA-A), an HLA class I histocompatibility antigen, alpha chain B (HLA-B), an HLA class I histocompatibility antigen, alpha chain C (HLA-C), or a combination thereof; and (c) a chimeric stimulating receptor (CSR), comprising: (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 is a modified T lymphocyte (T cell) comprising, consisting essentially of, or consisting of a chimeric stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating moiety, where the activating moiety 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 present disclosure (preferably modified T cells of the present 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 disclosure (preferably modified T cells of the 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 expression or activity of major histocompatibility complex (MHC) class I (MHC-I). 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 reduction in 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 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 naturally occurring wild-type T cells of the cell. An elimination in 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 the naturally occurring wild-type counterpart of the cell. An elimination in 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 naturally occurring wild-type T cells.

[0069] The modified cells of the present disclosure (preferably 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, essentially consist of, or consist of a linker molecule (herein referred to as linker). The non-naturally occurring polypeptide comprising HLA-E can further comprise, essentially consist of, or consist of a linker, the linker being located between the B2M polypeptide and the HLA-E polypeptide. The linker comprises, essentially consists 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, essentially consists 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, essentially consists 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 that is 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 that is at least 99% 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 of SEQ ID NO: 17128. The second 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 second 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 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 that is 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 that is 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 an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:17064. The 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 that is 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 that is at least 99% 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 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, the 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 in front of the HLA-E polypeptide (e.g., 5' in the context of a nucleic acid sequence, or amino-terminal in the context of an amino acid sequence). The 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. The 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. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:17068. The non-naturally occurring polypeptide comprising HLA-E can be encoded by a nucleic acid having a 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, essentially consist 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, essentially consists of, or consists of a chimeric antigen receptor (CAR). The CAR comprises, essentially consists 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, essentially consist of, or consist of a signal peptide. The ectodomain of the CAR can further comprise, essentially consist of, or consist of a hinge between the antigen recognition region and the transmembrane domain. The endodomain of the CAR can further comprise, essentially consist of, or consist of a human CD3 zeta 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 human CD28 and / or 4-1BB costimulatory domains.

[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 express the CSR of the present disclosure transiently or stably. 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 the present disclosure (preferably modified T cells of the present disclosure) can transiently or stably express a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure. In one embodiment, a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably modified T cells of the present disclosure). In one embodiment, a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure is stably expressed in a modified cell of the present disclosure (preferably modified T cells of the present 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 modified cells of the present disclosure (preferably modified 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, a sequence encoding a non-naturally occurring antigen receptor or a therapeutic protein of the present disclosure is transiently expressed in the modified cells of the present disclosure (preferably modified T cells of the present disclosure). In a preferred embodiment, a sequence encoding a non-naturally occurring antigen receptor or a therapeutic protein of the present disclosure is stably expressed in the modified cells of the present disclosure (preferably modified 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 the disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of the 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 the disclosure (preferably, an engineered T cell of the 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), which reduces or eliminates the level of expression or activity of the TCR; and (b) a sequence encoding a chimeric stimulating receptor (CSR), which comprises: (i) an ectodomain comprising an activating moiety, which is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, which 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, essentially consist of, or consist of a sequence encoding an inducible pro-apoptotic polypeptide. The modified cells can further comprise, essentially consist 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, essentially consist 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 selection marker. The transposon can be a piggyBac® transposon, a piggy-Bac®-like transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon, or a TcBuster transposon. The CSR-encoding sequence can be transiently expressed in the cell. The CSR-encoding sequence can be stably expressed in the cell. The inducible pro-apoptotic polypeptide-encoding sequence can be stably expressed in the cell. The sequence encoding the non-naturally occurring antigen receptor or the sequence encoding the therapeutic protein is stably expressed in the cell. In some embodiments, the sequence encoding the CSR can be transiently expressed in the cell, and the sequence encoding the inducible pro-apoptotic polypeptide can be stably expressed in the cell. In some embodiments, the sequence encoding the CSR can be stably expressed in the cell, and the sequence encoding the inducible pro-apoptotic polypeptide can be stably expressed in the cell. In some embodiments, the sequence encoding the CSR can be transiently expressed in the cell, and the sequence encoding the inducible pro-apoptotic polypeptide can be stably expressed in the cell, and the sequence encoding the non-naturally occurring antigen receptor or the sequence encoding the therapeutic protein is stably expressed in the cell.In some embodiments, the sequence encoding CSR can be stably expressed in cells, the sequence encoding inducible pro-apoptotic polypeptide can be stably expressed in cells, and the sequence encoding non-naturally occurring antigen receptor or the sequence encoding 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 and not active 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 cell of the present disclosure (preferably the modified T cell of the present disclosure) can further comprise, essentially consist of, or consist of a gene editing composition. The gene editing composition can comprise, essentially consist 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 expressed transiently by the modified cell. The gene editing composition can be expressed stably by the modified cell.

[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 the nuclease domain thereof 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 the nuclease domain thereof 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, consist essentially of, or consist 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, consist essentially of, or consist of the sequence of SEQ ID NO: 17059.

[0099] The gene editing composition can further comprise, essentially consist of, or consist of a guide sequence. The guide sequence can comprise, essentially consist of, or consist of an RNA sequence. In an embodiment when the modified cell is a T cell, the guide RNA can comprise, essentially consist of, or consist of a sequence that is complementary to a target sequence that encodes an endogenous TCR. The guide RNA can comprise, essentially consist of, or consist of a sequence that is complementary to a target sequence that encodes a B2M polypeptide. The guide RNA can comprise, essentially consist of, or consist of a sequence that is 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 gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding 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 gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding 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 an 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 an inactivated Cas9 (dCas9) and a sequence encoding Clo051 nuclease or a nuclease domain thereof.

[0104] The Clo051 nuclease or its nuclease domain can induce single- or double-stranded breaks in the target sequence. The donor sequence or donor plasmid can be integrated at the location of the single- or double-stranded 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 provides 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, the population of a plurality of modified cells comprising any of the non-naturally occurring chimeric stimulatory receptors (CSRs) disclosed herein, and provides a composition comprising a population of modified cells, the population of a plurality of modified cells comprising any of the modified cells disclosed herein. The population of modified cells can comprise, consist essentially of, or consist of immune cells or immune cell precursors. The population 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). The composition can include a pharmaceutically acceptable carrier.

[0108] The present disclosure provides a composition comprising a population of modified T lymphocytes (T cells), where 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), where the population of a plurality of T cells comprises any of the modified T cells disclosed herein. The composition can include a pharma- ceutically acceptable carrier.

[0109] Preferably, the disclosure provides a composition comprising a population of T lymphocytes (T cells), the population of a plurality of T cells comprising a non-naturally occurring chimeric stimulating receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an ectodomain comprising an activating moiety, the activating moiety being isolated or derived from a first protein, (b) a transmembrane domain, and (c) an endodomain comprising at least one signal transduction domain, the at least one signal transduction domain being isolated or derived from a second protein, the first protein and the second protein being non-identical. The composition may comprise a pharma- ceutically 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 CSR.

[0110] The population of 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 T cells can further comprise a modification of an endogenous sequence encoding a T cell receptor (TCR), which reduces or eliminates the level of expression or activity of the TCR. 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 modification of an endogenous sequence encoding a TCR, which reduces or eliminates the level of expression or activity of the TCR.

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

[0113] The population of multiple T cells can further comprise a modification in an 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 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.

[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 levels of TCR expression or activity, and a modification in the endogenous sequence encoding B2M that reduces or eliminates levels of MHC-I expression or activity.

[0115] The population of 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 classified as early memory T cells, stem cell-like T cells, and stem memory T cells (T SCM ), central memory T cells (T CM In some embodiments, the present invention can include stem cell-like T cells, stem cell memory T cells (T SCM ), and central memory T cells (T CM ) comprises 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 one or more cell surface markers of CD45RA and CD62L are expressed by the CD45-like cells.

[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 one or more cell surface markers of the CD45-like population include CD45RO 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 the treatment of a disease or disorder disclosed herein, or the use of the composition for the treatment of 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 of producing modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding the same into a primary human T cell under conditions that stably express the CSR in the modified T cell and maintain desirable stem-like properties of the modified T cell to produce the modified T cell. The primary human T cell can be a resting primary human T cell. The present disclosure provides modified T cells produced by the disclosed method. The present disclosure provides a method of administering modified T cells comprising a stably expressed CSR produced by the disclosed method. The present disclosure provides a method of administering modified T cells comprising a stably expressed CSR produced by the disclosed method to treat a disease or disorder.

[0123] The present disclosure provides a method of producing a population of modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding the 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 of administering a population of modified T cells comprising a stably expressed CSR produced by the disclosed method. The present disclosure provides a method of 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 of producing modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding the same into a primary human T cell under conditions that transiently express the CSR in the modified T cell and maintain the desired stem-like properties of the modified T cell to produce the modified T cell. The primary human T cell can be a resting primary human T cell. The present disclosure provides modified T cells produced by the disclosed method. The present disclosure provides a method of administering modified T cells comprising a transiently expressed CSR produced by the disclosed method. In one embodiment, the present disclosure provides a method of administering modified T cells comprising a transiently expressed CSR produced by the disclosed method after the modified T cell no longer expresses CSR. The present disclosure provides a method of administering modified T cells comprising a transiently expressed CSR produced by the disclosed method to treat a disease or disorder. In one embodiment, the present disclosure provides a method of administering modified T cells comprising a transiently expressed CSR produced by the disclosed method to treat a disease or disorder.

[0125] The present disclosure provides a method of producing a population of modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a chimeric stimulating receptor (CSR) of the present disclosure or a sequence encoding the 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 of administering a population of modified T cells comprising a transiently expressed CSR produced by the disclosed method. In one embodiment, the present disclosure provides a method of 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 of 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 of an endogenous sequence encoding a T cell receptor (TCR), where the modification reduces or eliminates the level of expression or activity of the TCR. The method of producing modified T cells or producing a population of modified T cells can further include introducing a modification of an endogenous sequence encoding beta-2-microglobulin (B2M), where the modification reduces or eliminates the level of expression or activity of major histocompatibility complex (MHC) class I (MHC-1). 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 of an endogenous sequence encoding a TCR, where the modification reduces or eliminates the level of expression or activity of the TCR, and introducing a modification of an endogenous sequence encoding B2M, where the modification reduces or eliminates the level of expression or activity of MHC-1.

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

[0128] The method of producing modified T cells or producing a population of modified T cells can further include contacting the modified T cells or 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 agonists / activators are antibodies or antibody fragments. The agonists / activators 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 agonists / activators 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 any 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 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 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 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 expanding method can comprise, consist essentially of, or consist of one or more agonists or activators that can bind to the CSR activation component of the engineered T cell or a 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 any combination thereof.

[0132] The conditions can include culturing the modified T cell or the plurality of modified T cells in a medium that includes 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 may include one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including the endpoint, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg including 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 a composition for use in the treatment of a disease or disorder disclosed herein, or the use of the composition for the treatment of any disease or disorder disclosed herein. The present disclosure also provides a method of 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 modified cells or populations of modified cells disclosed herein. Preferably, any of the modified T cells or CAR T cells disclosed herein. Any non-naturally occurring molecule that can bind to the activating component of CSR of the present disclosure 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 may 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 may 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. Thus, the present disclosure provides a method 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 population of modified cells contained in the disclosed compositions can be cultured in vitro or ex vivo before being administered to a subject in need of administration. 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 the composition comprising the modified cell population may be administered to the patient by any means known in the art. In some embodiments, the composition is administered by systemic administration. In some embodiments, the composition is administered by intravenous administration. Intravenous administration may be administered by intravenous injection or infusion. In some embodiments, the composition is administered by local administration. In some embodiments, the composition is administered by intrathecal, intraventricular, intraocular, or intraosseous injection or infusion.

[0142] The therapeutically effective amount can be a single dose or multiple doses of the modified cell composition or the composition comprising a population of modified cells.In some embodiments, the 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 during which they are 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 following 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 cell is stem cell.In some embodiments, the allogeneic cell is derived from stem cell.Exemplary stem cells include, but are not limited to, embryonic stem cell, adult stem cell, induced pluripotent stem cell (iPSC), multipotent stem cell, pluripotent stem cell, and hematopoietic stem cell (HSC).

[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 cell is an immune cell. In some embodiments, the allogeneic cell is a T lymphocyte (T cell). In some embodiments, the allogeneic cell is a T cell that does not express one or more components of a naturally occurring T cell receptor (TCR). In some embodiments, the allogeneic cell is a T cell that expresses a non-naturally occurring antigen receptor. Alternatively, or in addition, in some embodiments, the allogeneic cell is a T cell that expresses 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 a class of T cell receptor (TCR) and / or major histocompatibility complex (MHC). TCR mediates graft-versus-host (GvH) reactions, while MHC mediates host-versus-graft (HvG) reactions. In a preferred embodiment, any expression and / or function of TCR is reduced in the allogeneic cells of the present disclosure to prevent T cell-mediated GvH, which may cause death to the subject. Thus, in a particularly preferred embodiment, the present disclosure provides a pure TCR-negative allogeneic T cell composition (e.g., each cell of the composition expresses at such a low level that it is either undetectable or absent).

[0150] In a preferred embodiment, the expression and / or function of MHC class I (MHC-1, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated in the allogeneic cells of the present disclosure to prevent HvG and, as a result, to improve the engraftment of the allogeneic cells of the present disclosure in the subject. The improved engraftment of the allogeneic cells of the present disclosure results in a longer persistence of the cells and thus 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-1, is reduced or eliminated in the allogeneic cells of the present disclosure.

[0151] The above-mentioned strategy for generating allogeneic cells of the present disclosure induces additional challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), which is part of the TCR complex. Loss of CD3ζ in TCR-KO T cells dramatically reduces the ability to optimally activate and expand these cells using standard stimulatory / activator reagents, including but not limited to agonistic anti-CD3 mAbs. When expression or function of any one component of the TCR complex is disrupted, 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), are lost. Both CD3ε and CD3ζ are required for T cell activation and expansion. Agonistic anti-CD3 mAbs typically recognize CD3ε and possibly another protein in 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 by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL, etc.) that promote the immune response. However, in the absence of TCR, T cell expansion is greatly reduced when stimulating using standard activation / stimulation reagents that include agonistic anti-CD3 mAbs. In fact, T cell expansion is reduced to only 20-40% of normal expansion levels when stimulating using standard activation / stimulation reagents that include 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) upon stimulation using standard activation / stimulation reagents, including agonistic anti-CD3 mAbs.

[0153] In the absence of endogenous TCR, the chimeric stimulating receptor (CSR) of the present disclosure provides CD3ζ stimulation to enhance activation and expansion of allogeneic T cells. In other words, in the absence of endogenous TCR, the chimeric stimulating receptor (CSR) of the present disclosure rescues allogeneic cells from activation-based disadvantages when compared with non-allogeneic T cells expressing endogenous TCR. In some embodiments, the CSR of the present disclosure comprises an agonist mAb epitope extracellularly and a CD3ζ stimulating domain intracellularly, functionally converting anti-CD28 or anti-CD2 binding events on the surface into CD3z signaling events in allogeneic T cells modified to express CSR. In some embodiments, the CSR comprises wild-type CD28 or CD2 protein and a CD3z intracellular stimulating domain, generating CD28z CSR and CD2z CSR, respectively. In a preferred embodiment, the CD28z CSR and / or CD2z CSR 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 (sequence number 17096)

[0156] The nucleotide sequence encoding the wild type / native CD28 protein (NCBI: CCDS2361.1) comprises or consists of the following nucleotide sequence: (Sequence number 17097)

[0157] An exemplary CSR CD28z protein of the disclosure comprises or consists of the amino acid sequence of: (CD28 signal peptide, CD28 extracellular domain, CD28 transmembrane domain, CD28 cytoplasmic domain, CD3z intracellular domain). [ka] CD28 signal peptide: MLRLLLALNLFPSIQVTG (sequence number 17098) CD28 extracellular domain: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 17099) CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV (sequence number 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 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 (sequence number 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 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 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 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] Exemplary nucleotide sequences encoding the 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: 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 in 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 (sequence number 17112) CD2 cytoplasmic domain: KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 17113) CD3z intracellular domain: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 17102)

[0165] The 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 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 in 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 of a gene (such as a promoter) 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, a genomic target sequence, and a 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 MHCI, MHCII, or MHC activators of 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 MHCI, MHCII, or MHC activators of 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 makes them 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% are specific for foreign MHC and may mediate rejection of foreign cells and tissues. Targeted KO of MHCI, 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 due to the "missing self hypothesis." NK-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 to engineer 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 that reduces or prevents NK killing (such as single chain HLA-E) is introduced or delivered to the allogeneic cells. Alternatively, or in addition, the gene editing method of the disclosure preserves certain endogenous HLA molecules (such as endogenous HLA-E). For example, the first approach involves piggyBac® (PB) delivery of single chain (SC) HLA-E molecules to B2M KO T cells.

[0180] The second approach uses gene editing compositions that include guide RNAs selective for HLA-A, HLA-B, 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 referred to 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 amino acid sequences of (signal peptide, extracellular, TM, cytoplasmic). [ka]

[0184] Exemplary INFAR2 proteins of the disclosure comprise or consist of the amino acid sequences of (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 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 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 disclosure comprises or consists of the following amino acid sequence: [ka]

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

[0206] Exemplary gBE dimer (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 dimer (G and S) protein of the present disclosure comprises or consists of the following amino acid sequence: [ka]

[0209] An exemplary gBE dimer (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: MVDGTLLLLLSEALALTQTWAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETR SARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (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] An exemplary WT HLA-E monomer (R and S) protein of the present disclosure comprises or consists of the following nucleic acid sequence: [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: ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAA 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: GGGSGGGGSGGGGS (SEQ ID NO: 17128) B2M domain: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17129) Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130) HLA-E peptide: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETR SARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL (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: GGAGGAGGAGGCAGCGGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT (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: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETR SARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL (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: GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETR SARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYYKAEWSDSAQGSESHSL (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 precursor cells include any cell that can differentiate into one or more types of immune cells. In certain embodiments, immune precursor cells include pluripotent stem cells that can self-renew and develop into immune cells. In certain embodiments, immune precursor cells include hematopoietic stem cells (HSCs) or their progeny. In certain embodiments, immune precursor cells include progenitor cells that can develop into immune cells. In certain embodiments, immune precursor 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 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 HSC of the present disclosure can be isolated or derived from primary stem cells or cultured stem cells.The HSC 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 HSC 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 lymphoid cells (T cells), B lymphoid cells (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

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

[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 that contain stem cell-like T cells. The stem cell-like engineered T cells of the present disclosure have the greatest 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 then in turn produces T CM A linear pathway of differentiation that directly gives 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, SCM Cells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0234] In some embodiments of the methods of the present disclosure, 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 methods of the present disclosure, the immune cells are selected from 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 It 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 It is.

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

[0240] In some embodiments of the methods of the disclosure, the methods modify a plurality of modified T cells and / or the methods produce a plurality of modified T cells, where at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, of the plurality of modified T cells 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 comprises at least one modified T 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 methods of the disclosure, the methods modify a plurality of modified T cells, and / or the methods produce a plurality of modified T cells, where at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, of the plurality of modified T cells 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 in between, of the plurality of modified T cells are stem memory T cells (T SCM ). 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 in between, 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 in between, 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 in between, 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 in between, 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 comprises 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 prior to 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 comprises KCl, MgCl 2 , ClNa, glucose, and Ca(NO 3 ) 2 in any absolute or relative abundance or concentration, and optionally the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer contains 5 mM KCl, 15 mM MgCl 2 , 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO 3 ) 2 In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl 2 , 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO 3 ) 2 In certain embodiments, the buffer contains 5 mM KCl, 15 mM MgCl, and supplements including 20 mM HEPES and 75 mM Tris / HCl. 2 , 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO 3 ) 2 , and 40 mM Na 2 HPO 4 / NaH 2 PO 4In certain embodiments, the composition comprising primary human T cells comprises 100 μl of buffer and 5×10 6 ~25×10 6 In certain embodiments, the composition comprises 250×10 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 the transposon and / or transposase of the present disclosure into the immune cells of the present disclosure may further include contacting the immune cells with the T cell expansion composition. In some embodiments, including those in which the introduction step of the method includes an electroporation or nucleofection step, the electroporation or nucleofection step may involve contacting the immune cells 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 method of producing modified T cells of the present disclosure, the expansion supplement comprises 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.

[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, sterol, and 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 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 sterols 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 engineered T cells, wherein at least 2% of the plurality of engineered 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). In certain embodiments, the T cell expansion composition comprises or further comprises one or more of octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-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 sterols 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 sterols 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 about 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 2.12 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 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 sterol at a concentration of 0.25 μmol / kg to 25 μmol / kg including the endpoint. 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 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 includes a 10-fold higher amount of phosphorus than can be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as catalog number 12440053).

[0254] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may include the following components: 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 (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 the supplemented T cell expansion composition that is supplemented with the following components: 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) ester (bis(2-methylpropyl) ester) (CAS No. 126-86-3), ... The medium may be used interchangeably with media containing one or more of the following: tert-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 any of the following components: 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) 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 term "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 term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a medium comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate, and phosphate.

[0257] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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" may be used interchangeably with a medium comprising one or more of the following free amino acids at 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 supplementation of T cell expansion composition with the following free amino acids at 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 1.00%), arginine (about 1.00%), glycine (about 1.00%), aspartic acid (about 1.00%), glutamic acid (about 1.00%), threonine (about 1.00%), arginine (about 1.00%), glycine (about 1.00%), 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 includes a 10-fold higher amount of phosphorus than can be found in, for example, Iscove's Modified Dulbecco's Medium ((IMDM); available at ThermoFisher Scientific as catalog number 12440053).

[0259] In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg including the endpoint, palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg including the endpoint, and sterol at a concentration of about 0.1 mg / kg to 10 mg / kg including the endpoint (mg / kg = parts per million). In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a medium comprising one or more of octanoic acid at a concentration of about 9.19 mg / kg, palmitic acid at a concentration of about 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 term "supplemented T cell expansion composition" or "T cell expansion composition" may 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 to refer to 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 sterols at a concentration of about 2.5 μmol / kg.

[0260] In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with 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 sterol at a concentration of about 2.61 μmol / kg. In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with 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 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 a modified stem cell-like T cell of the present disclosure, the method comprises contacting the modified T cell 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 14 N 4 O 2Exemplary inhibitors of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO™). Further exemplary inhibitor moieties 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 targeting 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), aminofurazans (GSK690693), heterocycles (7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, ipatasertib (GDC-0068, RG7440), A-6 74563, and A-443654), phenylpyrazole derivatives (AT7867 and AT13148), thiophenecarboxamide derivatives (afuresertib (GSK2110183), 2-pyrimidyl-5-amidothiophene derivatives (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-OCH 3) Irmofosine (BM41.440), Miltefosine (hexadecylphosphocholine, HePC), Perifosine (D-21266), Erucylphosphocholine (ErPC), Erfosine (ErPC3, erucylphosphohomocholine), Indole-3-carbinol analogues (indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydr loindolo[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 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, medermicin, 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 method of 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, hyanotriazolodiazepines) 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 includes 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 of producing a IL-1 agonist, 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 a CD4+ CD5+ CD6+ CD7+ CD8+ CD9+ CD10+, the activation 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 activation complement comprises or consists of an activator complex that includes a human, humanized, or recombinant or chimeric antibody. In some embodiments, the activation complement comprises or consists of an activator complex that binds to CD3 and CD28. In some embodiments, the activation 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 precursor cells of the present disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes that differentiate from lymphoid precursor cells.

[0268] The modified NK cells of the present disclosure may 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 (500V, 700usec pulse length, 0.2mm electrode gap, 1 pulse). All Lonza 4D Nucleofector programs are contemplated as being within the scope of the disclosed methods.

[0271] In one particular 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 additional cell lines. In certain embodiments, the additional cell lines include artificial antigen presenting cells (aAPCs). In certain embodiments, the stimulation occurs on the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th day after electroporation. In certain embodiments, the stimulation occurs on the 2nd day after electroporation.

[0273] In certain embodiments, 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. The B cell receptors bind to specific antigens.

[0275] The modified B cells of the present disclosure may 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 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 thus 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 polypeptide of the present disclosure may be encoded by an amino acid or nucleic acid sequence having one or more modifications compared to the wild-type amino acid or nucleic acid sequence. The nucleic acid sequence encoding the caspase polypeptide of the present disclosure may be codon-optimized. The one or more modifications to the amino acid and / or nucleic acid sequence of the caspase polypeptide of the present disclosure may increase the interaction, cross-linking, cross-activation, or activation of the caspase polypeptide of the present disclosure compared to the wild-type amino acid or nucleic acid sequence. Alternatively, or in addition, the one or more modifications to the amino acid and / or nucleic acid sequence of the caspase polypeptide of the present disclosure may decrease the immunogenicity of the caspase polypeptide of the present disclosure compared to the wild-type amino acid or nucleic acid sequence.

[0283] The caspase polypeptide of the present disclosure may be truncated compared to the wild-type caspase polypeptide. For example, the caspase polypeptide 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 polypeptide of the present disclosure. The nucleic acid sequence encoding the caspase polypeptide of the present disclosure may be spliced ​​to form a variant amino acid sequence of the caspase polypeptide of the present disclosure compared to the wild-type caspase polypeptide. The caspase polypeptide of the present disclosure may be encoded by a recombinant and / or chimeric sequence. The recombinant and / or chimeric caspase polypeptide of the present disclosure may include sequences from one or more different caspase polypeptides. Alternatively, or in addition, the recombinant and / or chimeric caspase polypeptide of the present disclosure may include sequences from one or more species (e.g., human and non-human sequences). The caspase polypeptide 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 may comprise any polypeptide or functional domain thereof that allows induction using endogenous or non-naturally occurring ligands (i.e., inducers), e.g., non-naturally occurring synthetic ligands. The ligand-binding region may be internal or external to 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 may comprise one or more sequences from a receptor. Of particular interest are ligand-binding regions for which the ligand (e.g., small organic ligands) are known or can be readily generated. These ligand-binding regions or receptors may include, but are not limited to, FKBP and cyclophilin receptors, steroid receptors, tetracycline receptors, etc., as well as "non-naturally occurring" receptors (which may 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, including 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 amino acids. 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 suitable ligands (i.e., inducers). 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, specifically fragments, more specifically all or part of the variable region thereof, or fusions of heavy and light chains that produce high affinity binding, can be used as the ligand binding region of the present disclosure. Contemplated antibodies include those that are 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). Such examples include, but are not limited to, low affinity nerve growth factor receptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonic antigen). Still further, antibodies can be prepared against physiologically acceptable hapten molecules, and the individual antibody subunits thereof can be screened for binding affinity. The cDNAs encoding the subunits can be isolated and modified by deletion of constant regions, portions of variable regions, mutagenesis of variable regions, etc., to obtain binding protein domains with appropriate affinity for the ligand. In this manner, almost any physiologically acceptable hapten compound can be used as a ligand or to provide an epitope for the ligand. Instead of an antibody unit, an endogenous receptor 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 multimerizing receptors, the ligand for the ligand-binding region / receptor domain of an inducible pro-apoptotic polypeptide may be multimeric in the sense that the ligand may have at least two binding sites, each of which is capable of binding to a ligand receptor domain (i.e., a first binding site capable of binding a ligand-binding region of a first inducible pro-apoptotic polypeptide, and a second binding site capable of binding a ligand-binding region of a second inducible pro-apoptotic polypeptide, where 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. The multimeric ligands of the present disclosure include dimeric ligands. The dimeric ligands of the present disclosure may 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 more than about tetramers, with each molecule typically being at least about 150 Da, 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, etc. Alternatively, higher order ligands, such as trimers, can be used.For embodiments that include 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 that comprise the multimeric ligands of the present disclosure is that each binding site can bind a receptor with high affinity, and preferably, they can be chemically dimerized. Also, methods are available to balance the hydrophobicity / hydrophilicity of the ligands so that they can dissolve at a functional level in serum for most applications and can also 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. The pro-apoptotic polypeptides of the present disclosure are not only 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: C78H98N4O20; Molecular Weight: 1411.65). In certain embodiments, where 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: C82H107N5O20). 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 AP1903 injectable formulation is manufactured by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of non-ionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, the formulation is a clear, pale yellow solution. Upon refrigeration, the formulation undergoes a reversible phase transition resulting in a milky 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 AP1903 total for injection per vial). Once the need to administer AP1903 has been determined, the patient may receive a single fixed dose of AP1903 for injection (0.4 mg / kg) over 2 hours by IV infusion using a non-DEHP, non-ethylene oxide sterilized infusion set. The dose of AP1903 is calculated individually for every patient and is not recalculated unless the 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 IV over 2 hours. Plasma levels of AP1903 were directly proportional to dose, with mean Cmax values ​​ranging from approximately 10 to 1275 ng / mL for the dose range of 0.01 to 1.0 mg / kg. After the first infusion hour, 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, after dosing. 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, the fixed dose of AP1903 for injection used may be 0.4 mg / kg infused intravenously over 2 hours. The amount of AP1903 required in vitro for effective signaling of cells is 10-100 nM (1600 Da MW). This equates 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 mentioned above. Thus, 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 sequence encoding the ligand binding region of the present disclosure may contain one or more modified sequences compared to the wild-type amino acid or nucleic acid sequence. For example, the amino acid and / or nucleic acid sequence encoding the ligand binding region of the present disclosure may be a codon-optimized sequence. The one or more modifications may increase the binding affinity of a ligand (e.g., an inducer) to the ligand binding region 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 region of the present disclosure compared to the wild-type polypeptide. The ligand binding region of the present disclosure and / or the inducer 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, and 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) to 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 a truncated caspase-9 polypeptide 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 of the sequence. Alternatively, or in addition, 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 is encoded by an amino acid sequence that does not include an alanine (A) at position 282 of the sequence. 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

[0300] (The translated text seems to be incomplete in terms of grammar and context. It might need further refinement for better readability and comprehensibility.)

[0301] The inducible pro-apoptotic polypeptide of the present disclosure may be expressed in a cell under the transcriptional control of any promoter capable of initiating and / or controlling the expression of the inducible pro-apoptotic polypeptide of the present disclosure in the cell. As used herein, the term "promoter" refers to a promoter that serves as the first binding site for RNA polymerase to transcribe a gene. For example, the inducible pro-apoptotic polypeptide of the present disclosure may be expressed in a mammalian cell under the transcriptional control of any promoter capable of initiating and / or controlling the expression of the inducible pro-apoptotic polypeptide 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 polypeptide of the present disclosure may be expressed in a human cell under the transcriptional control of any promoter capable of initiating and / or controlling the expression of the inducible pro-apoptotic polypeptide 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 may 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 well known in the art to achieve expression of the inducible pro-apoptotic polypeptides of the present disclosure is similarly 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 that is regulated in response to a particular physiological or synthetic signal can allow for 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 allow for controlled expression of a gene of interest in mammalian cells. It consists of a tightly controlled expression mechanism that allows for virtually no basal level expression of the transgene but more than 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when an analog such as ecdysone or muristerone A binds to the receptor, the receptor activates the promoter to turn on expression of the downstream transgene and high levels of mRNA transcripts are obtained. In this system, the ecdysone-responsive promoter that drives expression of the gene of interest is on a separate plasmid, but both monomers of the heterodimeric receptor are constitutively expressed from one vector. Thus, engineering this type of system into a vector of interest can be useful. Another inducible system that may be useful 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, whereas 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 where tetracycline responsive elements are present in the plasmid. The second plasmid contains a regulatory element called tetracycline-controlled transactivator, which in the Tet-Off™ system is composed of the VP16 domain from herpes simplex virus and 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 making gene therapy vectors, the Tet-Off™ system can be used, whereby producer cells can grow in the presence of tetracycline or doxycycline to prevent expression of potentially toxic transgenes, but when the vector is introduced into a patient, gene expression can be constitutively turned on.

[0303] In some situations, it is desirable to regulate the expression of transgenes in gene therapy vectors. For example, different viral promoters with various activity strengths are utilized depending on the expression level desired. 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 expression level of the transgene is desired, a less strong modified version of the CMV promoter is also used. When expression of the transgene in hematopoietic cells is desired, retroviral promoters such as LTR from MLV or MMTV are often used. Other viral promoters used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviral promoters such as from E1A, E2A, or MLP regions, AAV LTR, HSV-TK, and avian sarcoma virus.

[0304] In other instances, 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 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 may result in reduced expression compared to stronger promoters such as CMV promoters, but may also result in 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), as well as Flt-1 (endothelial cells), GFAP (astrocytic cells).

[0307] In certain indications, it is desirable to activate transcription at specific times following administration of the gene therapy vector, This 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., (1989) Proc. Nat'l Acad. Sci. USA, 86, 8202-8206), and inflammatory protein responsive promoters. 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 al., (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, depending on 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 promoters and methods 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, translocation 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 a nucleic acid or amino acid sequence that is non-naturally occurring because the sequence is isolated 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] The 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 the 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 the 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 CAR 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 CAR of the present disclosure, at least one costimulatory domain may comprise CD28 and / or 4-1BB costimulatory domain. In certain embodiments of the CAR of the present disclosure, the hinge may comprise a sequence derived from human CD8α, IgG4, and / or CD4 sequence. In certain embodiments of the CAR of the present disclosure, the hinge may comprise a sequence derived from human CD8α sequence.

[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 can 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 may 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 may comprise a sequence derived from a human CD8α sequence. The hinge may 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 CD8 alpha hinge amino acid sequence can be encoded by a nucleic acid sequence comprising actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (sequence number 14482).

[0322] ScFv The present disclosure provides single chain variable fragment (scFv) compositions and methods for using these compositions to recognize and bind specific target proteins. ScFv compositions include heavy and light chain variable regions of an antibody. 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 immunoglobulins, where the VH and VL domains are 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 includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 14483).

[0323] Sentinel The centrin of the present disclosure specifically binds to an antigen. The chimeric antigen receptor of the present disclosure, which comprises one or more centrins that specifically bind to an antigen, can be used to target the specificity of cells (e.g., cytotoxic immune cells) to a specific antigen.

[0324] The centilin of the present disclosure may include a protein scaffold, which can specifically bind to an antigen. The centilin of the present disclosure may include a protein scaffold including a consensus sequence of at least one fibronectin type III (FN3) domain, which can specifically bind 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 include LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence may 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 that includes or consists of amino acid residues TEDS (SEQ ID NO: 14491) at positions 13 to 16 of the consensus sequence, (b) a BC loop that includes or consists of amino acid residues TAPDAAF (SEQ ID NO: 14492) at positions 22 to 28 of the consensus sequence, (c) a CD loop that includes or consists of amino acid residues SEKVGE (SEQ ID NO: 14493) at positions 38 to 43 of the consensus sequence, and (d) a BC loop that includes or consists of amino acid residues 51 to 54 of the consensus sequence. (e) a DE loop comprising or consisting of amino acid residues GSER (SEQ ID NO: 14494) at positions 60-64 of the consensus sequence; (f) an EF loop comprising or consisting of amino acid residues KGGHRSN (SEQ ID NO: 14495) at positions 75-81 of the consensus sequence; or (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) may be modified at one or more positions within the consensus sequence. 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 fifteen fibronectin type III (FN3) domains. The scaffold may comprise at least ten. -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 or less, and 10 -15 K below M D The antigen can bind to at least one of the antigens with an affinity selected from the group consisting of 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 a naturally occurring antibody. 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 manufacturing 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 molecule of the present disclosure comprises a protein scaffold that comprises or consists of one or more alpha helices that are free of any disulfide bridges.Preferably, the affibody molecule of the present disclosure comprises or consists of three alpha helices.For example, the antibody molecule of the present disclosure may comprise an immunoglobulin binding domain.The affibody molecule of the present disclosure may comprise the Z domain of protein A.

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

[0328] The affimer molecules of the present disclosure include protein scaffolds that include highly stable proteins engineered to present 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 on top of an antiparallel beta sheet.

[0329] The affitin molecules of the present disclosure include artificial protein scaffolds, the structure of which may be derived from, for example, DNA-binding proteins (e.g., DNA-binding protein Sac7d). The affitins of the present disclosure selectively bind to target sequences that may be the whole 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, in genomes or on the surface of peptides, proteins, viruses, or bacteria. In certain embodiments of the present disclosure, the affitin molecules may be used as specific inhibitors of enzymes. The affitin molecules of the present disclosure may include heat-resistant proteins or derivatives thereof.

[0330] The alpha body molecules of the present disclosure may be referred to as cell-penetrating alpha bodies (CPABs). The alpha body molecules of the present disclosure comprise small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). The alpha body molecules are capable of reaching and binding to intracellular target sequences. Structurally, the alpha body molecules of the present disclosure comprise artificial sequences that form single-stranded alpha helices (similar to naturally occurring coiled-coil structures). The alpha body molecules of the present disclosure may comprise a protein scaffold that comprises 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 alpha body 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 better tissue penetration and thermal stability than monoclonal antibodies or fragments thereof. An exemplary anticalin molecule of the present disclosure may include about 180 amino acids with a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure include a barrel structure that includes pairwise antiparallel beta strands connected by loops and linked by alpha helices. In a preferred embodiment, the anticalin molecules of the present disclosure include a barrel structure that includes 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 within different targets. When the avimers of the present disclosure recognize more than one target, the avimers mimic the function of bispecific antibodies. The artificial protein avimers may include two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. The amino acid sequence of one or more of the peptides of the avimer may be derived from the A domain of a membrane receptor. The 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 equal affinity and specificity as antibodies.

[0335] The Kunitz domain peptides of the present disclosure include protein scaffolds that contain Kunitz domains. The Kunitz domains contain active sites for inhibiting protease activity. Structurally, the Kunitz domains of the present disclosure include 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 can 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 about 94 amino acids and having a mass of about 10 kDa). These engineered proteins specifically bind to target sequences, including antigens. The monobodies of the present disclosure may 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 mimics 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 the variable domain of an antibody, the monobody lacks a binding site for metal ions as well as a central disulfide bond. Multispecific monobodies can be optimized by modifying loops BC and FG. The monobodies of the present disclosure may comprise 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) that comprises at least one VHH (VCAR). The chimeric antigen receptor of the present disclosure can comprise more than one VHH. For example, a bispecific VCAR can comprise two VHHs that specifically bind to two distinct antigens.

[0339] The VHH proteins of the present disclosure specifically bind to an antigen. The 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 known in the art.

[0342] Optionally, VHH proteins can be engineered while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, VHH proteins can be prepared by a process of analysis of parental sequences and various conceptual engineered products, using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display 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 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 its antigen. In this way, residues can be selected and combined from parental and reference sequences such that desired characteristics, such as affinity for the target antigen, are achieved. Alternatively, or in addition to the above procedures, other suitable engineering methods can be used.

[0343] Screening of 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 a large collection of peptides for individual members with the desired function or structure. The displayed nucleotide or peptide sequences can be 3-5000 or more nucleotides or amino acids long, often 5-100 amino acids long, and often about 8-25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence that codes for a particular displayed peptide sequence. The VHH proteins of the present disclosure can bind 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., between 0.1 and 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 equal to or less than M, or any range or value therebetween.

[0344] The affinity or avidity of a VHH or VCAR for an antigen can be determined experimentally 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 if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using standardized solutions of VHH or VCAR and antigen, and standardized buffers such as those described herein.

[0345] Competitive assays can be performed with the VHH or VCAR of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the VHH or VCAR of the present disclosure for binding to a target protein and / or share epitope regions. These assays, which are readily known to those skilled in the art, evaluate the competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and / or antibody are immobilized or insolubilized before or after the competition, and the sample bound to the target protein is separated from the unbound sample, for example, by decantation (if the protein / antibody was insolubilized beforehand) or centrifugation (the protein / antibody precipitated after the competitive reaction). Competitive binding can also be determined by whether the binding or lack of binding of the VHH or VCAR to the target protein changes function, for example, whether the VCAR molecule inhibits or enhances, for example, the enzyme activity of the 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 a chimeric antigen receptor (CAR) comprising a single domain antibody (VCAR). In certain embodiments, the single domain antibody comprises 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, the VHs are fully engineered using the UniRat™ (TeneoBio) system and “NGS-based discovery” to generate the 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 directly from a single clone (hybridoma) of a cell or cell line. These VHs were not subsequently cloned from said 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, and thus are human / rat chimeras without light chains, which differ from standard mAb formats. The native rat genes have been knocked out, and the only antibodies expressed in rats are from transgenes with VH domains linked to rat Fc (UniAbs). These are the exclusive Abs expressed in UniRat. 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 human VH domains with human Fc in vitro (to generate non-naturally occurring recombinant VH antibodies). In certain embodiments, VHs are fully humanized, but they are expressed in vivo as human / rat chimeras without light chains (human VH, rat Fc). Fully humanized VHs are expressed in vivo because the human / rat chimeras without light chains (human VH, rat Fc) are approximately 80 kDa (vs. 150 kDa).

[0349] The VCAR of the present disclosure may comprise at least one VH of the present disclosure.In certain embodiments, the VH of the present disclosure may be modified to remove Fc domain or a part thereof.In certain embodiments, the framework sequence of the VH of the present disclosure may be modified, for example, to improve expression, to reduce immunogenicity, or to 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 dosages and equivalents thereof known to those of skill in the art, and so forth.

[0351] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending 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 within 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. When a particular value is described in the present application and claims, the term "about" meaning within an acceptable error range of the particular value should be assumed unless otherwise indicated.

[0352] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular materials or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide does not include sequences (optimally protein-encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, an isolated polynucleotide may include less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 k...

Claims

1. A non-naturally occurring chimeric stimulatory receptor (CSR), (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide, the activation component comprises a CD2 extracellular domain to which an agonist binds, and the CD2 extracellular domain comprises the amino acid sequence of SEQ ID NO: 17119; (b) a transmembrane domain, the transmembrane domain comprising a CD2 transmembrane domain; (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, and the at least one signal transduction domain comprises a CD3ζ protein.

2. The CSR of claim 1, wherein the CSR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:17118.

3. The CSR of claim 1, wherein the CSR comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:17118.

4. The CSR of claim 1, wherein the CSR comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:17118.

5. The CSR of claim 1 , wherein the CSR comprises the amino acid sequence of SEQ ID NO: 17118.

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

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

8. A transposon comprising the nucleic acid of claim 6.

9. A cell comprising a CSR according to any one of claims 1 to 5, or a nucleic acid according to claim 6.

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

11. The cell of claim 9 , wherein the cell is an autologous cell.

12. A composition comprising a plurality of cells according to any one of claims 9 to 11.

13. 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) according to any one of claims 1 to 5, and a modified T lymphocyte (T cell).

14. 14. The modified T cell of claim 13, further comprising an inducible pro-apoptotic polypeptide, or further comprising a modification of an 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.

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

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

17. the non-naturally occurring polypeptide comprising HLA-E, a first linker located between the B2M signal peptide and the peptide; 17. The modified T cell of claim 15 or 16, further comprising a second linker positioned between the B2M polypeptide and the peptide encoding the HLA-E.

18. 14. The modified T cell of claim 13, further comprising a sequence encoding a non-naturally occurring antigen receptor, a therapeutic polypeptide, or a combination thereof.

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

20. 14. The modified T cell of claim 13, wherein the CSR is transiently expressed in the modified T cell, or alternatively, the CSR is stably expressed in the modified T cell.

21. The modified T cell of any one of claims 15 to 17, wherein the polypeptide comprising the HLA-E polypeptide is transiently expressed in the modified T cell, or the polypeptide comprising the HLA-E polypeptide is stably expressed in the modified T cell.

22. 15. The modified T cell of claim 14, wherein the inducible pro-apoptotic polypeptide is stably expressed in the modified T cell.

23. 20. The modified T cell of claim 18 or 19, wherein the sequence encoding the non-naturally occurring antigen receptor or therapeutic protein is stably expressed in the modified T cell.

24. The modified T cells may be allogeneic cells, autologous cells, 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.

25. 25. A pharmaceutical composition comprising a population of modified T cells and a pharma- ceutical acceptable carrier, wherein a plurality of said population of modified T cells comprises the CSR of any one of claims 1-5, or the modified T cells of any one of claims 13-24.

26. 26. The pharmaceutical composition of claim 25, wherein the composition further comprises at least one molecule that binds to the CSR.

27. 27. A pharmaceutical composition according to claim 25 or 26 for use in a method for treating a disease or disorder, the method comprising administering to a subject in need of treatment a therapeutically effective amount of the composition.

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

29. 11. A method of expanding a population of engineered T cells, comprising: introducing a CSR of claims 1-5, or a sequence encoding same, into a plurality of primary human T cells under conditions that stably or 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 express the CSR under the same conditions.

30. 30. A population of modified T cells expanded by the method of claim 29.

31. 25. The modified T cell of any one of claims 13-24 for use in a method of treating a disease or disorder, said method comprising administering to a subject in need of treatment a therapeutically effective amount of said modified T cell.

32. A pharmaceutical composition according to any one of claims 25 to 27 for use in a method for treating a disease or disorder, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject in need of treatment.

33. The population of modified T cells of claim 30 for use in a method for treating a disease or disorder, the method comprising administering to a subject in need of treatment a therapeutically effective amount of the population of modified T cells.

34. 32. The modified T cell of claim 31 , wherein the modified T cells within the population of modified T cells administered to the subject no longer express the CSR.

35. The pharmaceutical composition of claim 32, wherein the modified T cells within the population of modified T cells administered to the subject no longer express the CSR.

36. The population of modified T cells of claim 33, wherein the modified T cells within the population of modified T cells administered to the subject no longer express the CSR.

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