Allogeneic cell compositions and methods of use

Abstract

The present invention relates to allogeneic cell compositions and methods of use. Chimeric stimulating receptors (CSRs), cell compositions comprising CSRs, methods of making the same, and methods of using the same to treat a disease or condition in an individual are disclosed.

Description

[0001] This application is a divisional application. The application date of the original application is September 5, 2019, the application number is 201980072595.2 (PCT / US2019 / 049816), and the name of the invention is “Allogeneic cell compositions and methods of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 727,498, filed September 5, 2018, U.S. Provisional Application No. 62 / 744,073, filed October 10, 2018, U.S. Provisional Application No. 62 / 815,334, filed March 7, 2019, and U.S. Provisional Application No. 62 / 815,880, filed March 8, 2019. The contents of each of these applications are incorporated herein by reference in their entirety. Technical Field

[0004] The present disclosure relates to molecular biology and, more particularly, to chimeric receptors, allogeneic cell compositions, methods of making and using the same.

[0005] Sequence Listing is incorporated by reference

[0006] The contents of the file named "POTH-046_001WO_SequenceLisfing.txt," created on September 5, 2019, and 55.7 MB in size, are hereby incorporated by reference in their entirety. Background Art

[0007] There has been a long-standing but unmet need in the art for allogeneic cell compositions that overcome the challenges posed by eliminating genes involved in graft-versus-host and host-versus-graft reactions. The present disclosure provides allogeneic cell compositions, methods of making these compositions, and methods of using these compositions that contain non-naturally occurring structural modifications to restore allogeneic cell responsiveness to environmental stimuli and reduce or prevent natural killer cell-mediated cytotoxic rejection. Summary of the Invention

[0008] The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0009] The activating component may comprise a portion of one or more of a component of a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activating component binds. The activating component may comprise the extracellular domain of CD2, or a portion thereof, to which an agonist binds.

[0010] The signal transduction domain may comprise one or more of the following: a component of a human signal transduction domain, a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. 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.

[0011] The intracellular domain may further comprise a cytoplasmic domain. The cytoplasmic domain may be isolated or derived from a third protein. The first and third proteins may be identical. The extracellular domain may further comprise a signal peptide. The signal peptide may be derived from a fourth protein. The first and fourth proteins may be identical. The transmembrane domain may be isolated or derived from a fifth protein. The first and fifth proteins may be identical.

[0012] In some aspects, the activating component does not bind to a naturally occurring molecule. In some aspects, the activating component binds to a naturally occurring molecule, but the CSR does not transduce a signal after the activating component binds to the naturally occurring molecule. In some aspects, the activating component binds to a non-naturally occurring molecule. In some aspects, the activating component does not bind to a naturally occurring molecule, but does bind to a non-naturally occurring molecule. The CSR may selectively transduce a signal after the activating component binds to the non-naturally occurring molecule.

[0013] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises an extracellular domain of CD2 or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and wherein the at least one signaling domain comprises a CD3 zeta protein or a portion thereof. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 17062. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 17062.

[0014] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) in which the extracellular domain comprises a modification. When compared to the wild-type sequence of the activating component or the first protein, the modification may comprise a mutation or truncation of the amino acid sequence of the activating component or the first protein. The mutation or truncation of the amino acid sequence of the activating component may 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 may reduce or eliminate binding to naturally occurring CD58. In some aspects, 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 aspect, the CD2 extracellular domain comprising the mutation or truncation comprises the amino acid sequence of SEQ ID NO: 17119.

[0015] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and wherein the activation component comprises an extracellular domain of CD2 or a portion thereof to which an agonist binds, and wherein the extracellular domain of CD2 or a portion thereof to which an agonist binds comprises a mutation or truncation; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and wherein the at least one signaling domain comprises a CD3 zeta protein or a portion thereof. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 17118. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 17118.

[0016] 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.

[0017] 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.

[0018] The modified cells disclosed herein can be allogeneic cells or autologous cells. In some preferred aspects, the modified cells are allogeneic cells. In some preferred aspects, the modified cells are allogeneic T cells or modified allogeneic CART cells.

[0019] The present disclosure provides compositions comprising any of the CSRs disclosed herein. The present disclosure provides compositions comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a modified cell disclosed herein or a plurality of modified cells disclosed herein.

[0020] The present disclosure provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR; and (b) a chimeric stimulatory receptor (CSR), wherein the chimeric stimulatory receptor comprises: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0021] The modified T cells may further comprise an inducible pro-apoptotic polypeptide. The modified T cells may further comprise a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I).

[0022] The modified T cell may 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 the HLA-E polypeptide may further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding HLA-E.

[0023] The modified T cells may further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor may comprise a chimeric antigen receptor (CAR).

[0024] CSRs can be transiently expressed in modified T cells. CSRs can be stably expressed in modified T cells. Polypeptides comprising HLA-E polypeptides can be transiently expressed in modified T cells. Polypeptides comprising HLA-E polypeptides can be stably expressed in modified T cells. Inducible pro-apoptotic polypeptides can be transiently expressed in modified T cells. Inducible pro-apoptotic polypeptides can be stably expressed in modified T cells. Non-naturally occurring antigen receptors or sequences encoding therapeutic proteins can be transiently expressed in modified T cells. Non-naturally occurring antigen receptors or sequences encoding therapeutic proteins can be stably expressed in modified T cells.

[0025] 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-like T cells.

[0026] The present disclosure provides compositions comprising any modified T cells disclosed herein. The present disclosure also provides compositions comprising a modified T lymphocyte (T cell) population, wherein a plurality of modified T cells of the population comprise a CSR disclosed herein. The present disclosure also provides compositions comprising a T lymphocyte (T cell) population, wherein a plurality of T cells of the population comprise a modified T cell disclosed herein.

[0027] The present disclosure provides a method for treating a disease or condition comprising administering to an individual in need thereof a therapeutically effective amount of any composition disclosed herein; or a composition for treating a disease or condition. In one aspect, the composition is a modified T cell or a modified T cell population as disclosed herein. The present disclosure also provides a method for treating a disease or condition comprising administering to an individual in need thereof a therapeutically effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds to a CSR.

[0028] The present disclosure provides a method for producing a modified T cell colony, which comprises, is essentially composed of, or is composed of: a composition comprising a CSR of the present disclosure or a sequence encoding the same is introduced into a plurality of nascent human T cells to produce a plurality of modified T cells under conditions of stably expressing the CSR in the T cells of the plurality of modifications and retaining the desired stem-like properties of the T cells of the plurality of modifications. The present disclosure provides a composition comprising a modified T cell colony produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 colony comprising the CSR exhibits stem memory T cells (T SCM ) or T sCM In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population express central memory T cells (T cells). CM ) or T CM One or more cell surface markers of T-like cells; and wherein the one or more cell surface markers include CD45RO and CD62L. The composition can be used to treat a disease or condition. The present disclosure also provides the use of the composition produced by the method for treating a disease or condition. The present disclosure also provides a method for treating a disease or condition, which comprises administering a therapeutically effective amount of the composition produced by the method to an individual in need. The treatment method may further comprise administering an activator composition to the individual to activate the modified T cell population in vivo, to induce cell division of the modified T cell population in vivo, or a combination thereof.

[0029] The present disclosure provides a method for producing a modified T cell colony, which comprises, is essentially composed of, or is composed of: a composition comprising a CSR of the present disclosure or a sequence encoding the same is introduced into a plurality of nascent human T cells to produce a plurality of modified T cells under conditions of transiently expressing the CSR in the T cells of the plurality of modifications and retaining the desired stem-like properties of the T cells of the plurality of modifications. The present disclosure provides a composition comprising a modified T cell colony produced by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 colony comprising the CSR exhibits stem memory T cells (T SCM ) or T SCM In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population express central memory T cells (T cells). CM ) or T CM The invention also provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of a composition produced by the method to an individual in need thereof. In some aspects, the modified T cells within the modified T cell population administered to an individual no longer express CSR.

[0030] The present disclosure provides a method for expanding a modified T cell population, comprising introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of naive human T cells to produce a plurality of modified T cells under conditions in which the CSR is stably expressed within the plurality of modified T cells and the desired stem-like properties of the plurality of modified T cells are retained, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the CSR under the same conditions. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising CSRs express stem memory T cells (T cells). SCM ) or T SCM In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population express central memory T cells (T cells). CM ) or T CM One or more cell surface markers of T-cell-like cells; and wherein the one or more cell surface markers include CD45RO and CD62L. The present disclosure provides a composition comprising a modified T cell population expanded by the method. The composition can be used to treat a disease or condition. The present disclosure also provides the use of a composition expanded by the method for treating a disease or condition. The present disclosure also provides a method for treating a disease or condition, which comprises administering a therapeutically effective amount of a composition expanded by the method to an individual in need. The treatment method may further comprise administering an activator composition to the individual to activate the modified T cell population in vivo, to induce cell division of the modified T cell population in vivo, or a combination thereof.

[0031] The present disclosure provides a method for expanding a modified T cell population, comprising introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of naive human T cells to produce a plurality of modified T cells under conditions in which the CSR in the plurality of modified T cells is transiently expressed and the desired stem-like properties of the plurality of modified T cells are retained, 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 a modified T cell population expanded by the method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising CSRs express stem memory T cells (T cells). SCM ) or T SCM In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population express central memory T cells (T cells). CM ) or T CM The invention also provides a method for treating a disease or condition, comprising administering to an individual in need thereof a therapeutically effective amount of a composition amplified by the method. In some aspects, the modified T cells within the modified T cell population administered to an individual no longer express CSR.

[0032] Any of the above aspects may be combined with any other aspect.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Unless the context clearly dictates otherwise, in the specification, the singular also includes the plural; for example, the terms "a / an" and "the" are to be understood as singular or plural, and the term "or" is to be understood as inclusive. For example, "an element" means one or more elements. Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of the stated elements, integers, or steps, or groups of elements, integers, or steps, but not the exclusion of any other elements, integers, or steps, or groups of elements, integers, or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0034] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. It is not recognized that the references cited herein are prior art to the claimed invention. In the event of a conflict, this specification (including definitions) will prevail. In addition, materials, methods and examples are merely illustrative and are not intended to be restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] This patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0036] Figure 1 is a schematic depicting the T cell receptor (TCR) and co-receptors CD28 and CD2.

[0037] Figure 2 It is a schematic diagram depicting the delivery of primary and secondary co-stimulation to T cells by the combination of agonist mAbs (anti-CD3, anti-CD28 and anti-CD2). Complete T cell activation depends critically on the combination of TCR and the second signal of the co-stimulatory receptor by enhancing the immune response. Primary and secondary co-stimulation can be delivered to T cells by treating surface receptors with agonist mAbs (e.g., anti-CD3, anti-CD28 and anti-CD2) and binding them to surface receptors.

[0038] Figure 3is a schematic diagram demonstrating that secondary co-stimulation is delivered to T cells solely through binding of an agonist mAb in the absence of a TCR. Because full T cell activation is critically dependent on primary stimulation via CD3ζ and secondary signals through co-stimulatory receptors, T cell activation and expansion are suboptimal and therefore reduced.

[0039] Figure 4 This figure is a schematic diagram demonstrating enhanced stimulation in the absence of a TCR through expression of a chimeric stimulatory receptor (CSR). In the absence of a TCR, but in the presence of surface-expressed CSRs, when T cells are treated with a standard agonist mAb, both primary and secondary co-stimulatory signals are delivered. Because the CSR-mediated stimulatory signaling enables more complete T cell activation, T cell activation and expansion are enhanced.

[0040] Figure 5 is a schematic diagram depicting an exemplary CSR CD28z of the present disclosure.

[0041] Figure 6 is a schematic diagram depicting an exemplary CSR CD2z of the present disclosure.

[0042] Figure 7 Schematic diagram of a strategy for mutating CSR CD2z to eliminate binding of the natural ligand (CD58). A group of CSR CD2z mutants were designed within the extracellular domain of CD2. The goal of this group is to identify mutants that no longer bind CD58 but retain their receptivity for binding to anti-CD2 activator reagents. This may be desirable for two main reasons: 1) CD58 expression by activated T cells can interact with wild-type (WT) CD2z CSR and may interfere with the optimal performance of CSR; and 2) since WT CD2z CSR may act as a natural ligand CAR, it is possible that T cells expressing CSR can mediate cytotoxic activity against cells expressing CD58 (including activated T cells). Therefore, a mutant CD2z CSR is needed that cannot interact with CD58 but retains its ability to bind to activated anti-CD2 reagents to achieve optimal cell expansion.

[0043] Figure 8 is a schematic diagram depicting an exemplary CSR CD2z-D111H of the present disclosure. The D111H mutation is located within the CD2 extracellular domain of the CSR CD2z-D111H construct.

[0044] Figures 9A-9B It is to show CSR A series of images showing that delivery enhances the expansion of TCRb / b2M double knockout CAR-T cells. Pan T cells isolated from the blood of normal donors were used DNA modification system and Cas-CLOVERTM In a single reaction, cells were electroporated with: a transposon encoding a CAR, a selection gene, and a CSR (CD28z or CD2z), a transposon encoding a super piggyBac TM Transposase mRNA, encoding Cas-CLOVER TM The mRNA of CAR b and b2M is expressed, and multiple guide RNAs (gRNAs) of TCR b and b2M are knocked out TCR and MHCI (double knockout; DKO). Cells are then stimulated with agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later selected for genetic modification in the culture time course of 16 days. At the end of the initial culture period, all T cells express CAR, indicating that genetically modified cells (data not shown) have been successfully selected. In samples expressing CD2z or CD28z CSR, it was observed that DKO cells had a higher degree of amplification because the frequency of independent CAR DKO cells was higher ( Figure 9A and 9B In samples of DKO CAR-T cells expressing either CD2z or CD28z CSRs, at least two-fold cell expansion was observed compared to DKO CAR-T cells alone.

[0045] Figures 10A-10B It is a series of figures, showing the CSRCD2z or CD28z in the DKOCAR-T cells of purification so that amplification is enhanced after restimulation. After initial genetic modification and the first round of stimulation and amplification, magnetic beads are used to purify the cells from each group (simulation (WT CAR-T cells), DKO CAR-T cells, DKO CAR-T cells+CD2z CSR and DKO CAR-T cells+CD28z CSR) for TCR-MHCI- cells. The purified cells are then stimulated again using anti-CD2, anti-CD3 and anti-CD28 agonist mAbs. At the end of the 14-day culture period, TCR and MHCI expression (A) and the amplitude (B) of cell colony amplification were determined. After this secondary amplification, all purified DKO cells, including those expressing CD2z or CD28z CSR cells, are still extremely clean (>98.8% DKO) for DKO cells. When compared to those cells not expressing any CSR, the DKOCAR-T cells expressing CD2z or CD28z CSR enhance amplification.

[0046] Figure 11It is a diagram showing that cytokine supplementation can further amplify the purified DKO CAR-T cells expressing CSR after restimulation. After the initial genetic modification and the first round of stimulation and amplification, magnetic beads are used to purify cells expressing CSR for use in DKO cells. Then, in the presence of exogenous purified recombinant IL7 and IL15, anti-CD2, anti-CD3 and anti-CD28 agonist mAbs are used to stimulate the purified cells. At the end of the 14-day culture period, the amplitude of cell colony amplification is determined. After secondary amplification, all purified DKO cells, including those expressing CD2z or CD28z CSR, are still extremely pure for TCR-MHCI- cells (>98.8% double knockout (data not shown)). In addition, in the presence of IL7 and IL15, cell growth is strong, which is stronger than when there is no supplementation. These data show that exogenous cytokines can be added to further amplify WTCAR-T cells expressing CSR.

[0047] Figure 12 It is a diagram showing that the surface expression of CAR is not significantly affected by the co-expression of CSR in DKO cells. After secondary amplification, cells (simulation (WTT cells), WTCAR-T cells, DKO CAR-T cells, DKO CAR-T cells+CD2zCSR and DKO CAR-T cells+CD28z CSR) were stained for surface expression of CAR and compared with control WT CAR-T cells and simulated T cells. The expression of CD2z or CD28z CSR had no significant effect on the expression of CAR molecules on the T cell surface.

[0048] Figure 13 It is a graph showing that the expression of CSR does not significantly affect the cytotoxicity of DKO CAR-T cells in vitro. After secondary amplification, cells (mock (WTT cells), WTCAR-T cells, DKOCAR-T cells, DKOCAR-T cells+CD2z CSR and DKOCAR-T cells+CD28z CSR) were co-cultured with engineered K562-BCMA-luciferase (eK562-Luc.BCMA) or negative control K562-PSMA-luciferase (eK562-Luc.PSMA) at a 10:1, 3:1 or 1:1 E:T ratio for 48 hours. Luciferase signals were measured to determine cytotoxicity. The killing of eK562-Luc.PSMA is shown with a dotted line, while the killing of eK562-Luc.BCMA is shown with a solid line. All CAR + Both T cells expressed an anti-BCMA-specific CAR. DKO CAR-T cells exhibited in vitro cytotoxicity similar to that of WT CAR-TCR cells. This activity was not significantly affected by co-expression of CD2z or CD28z CSRs.

[0049] Figure 14 It is a graph showing that the expression of CSR does not significantly affect the secretion of IFNg by DKO CAR-T cells in vitro. The supernatant from the 48-hour killing assay was analyzed for secreted IFNg as a measure of the antigen-specific function of BCMACAR T cells. All CAR-T cells with or without CD2z or CD28z CSR expression secreted IFNg in response to co-culture with BCMA-expressing target cells (eK562-Luc.BCMA), but this was not the case for cells expressing irrelevant targets (eK562-Luc.PSMA).

[0050] Figure 15 It is a series of figures showing that the expression of CSR does not significantly affect the proliferation of DKO CAR-T cells in vitro. The mock (WT T cells), WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z CSR and DKO CAR-T cells + CD28z CSR cells were labeled with Cell Trace Violet (CTV), and CTV was diluted as the cells proliferated. The cells were co-cultured with eK562-Luc.PSMA or eK562-Luc.BCMA cells at a 1:2E:T ratio for 5 days. All CAR-T cells with or without CD2z or CD28z proliferated in response to target cells expressing BCMA (eK562-Luc.BCMA), but this was not the case for cells expressing irrelevant antigens (eK562-Luc.PSMA).

[0051] Figure 16 A pair of figures showing that the memory phenotype of DKO CAR-T is not significantly affected by co-expression of CD2z CSR. WT CAR-T cells, DKO CAR-T cells, DKO CAR-T cells + CD2z and DKO CAR-T cells + CD28z were stained for the expression of surface CD45RA, CD45RO and CD62L 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, were primarily composed of abnormally high levels of favorable Tscm and Tcm cells. However, when CD28z was expressed in DKO CAR-T cells, the phenotype became significantly more differentiated, favoring Tcm and TEM cells. This phenotype could negatively impact the in vivo function of these CAR T cells, as they appear more differentiated.

[0052] Figure 17 It is a series of figures showing that the expression of activation / exhaustion markers in DKO CAR-T is not significantly affected by CD2z CSR co-expression. About the expression of important exhaustion molecules Lag3, PD1 and Tim3, simulation (WTT 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. When compared to simulated T cells, WT and DKO CAR-T cells with or without CD2z have very little or no expression of exhaustion molecules. However, during the amplification process, the expression of CD28zCSR in DKOCAR-T significantly increases the exhaustion markers Lag3, PD1 and Tim3. This phenotype may have a negative impact on the in vivo function of these CAR T cells because it seems to be more exhausted. In contrast, CD2z expression has almost no effect on the exhaustion phenotype of DKO CAR-T cells, while significantly enhancing the amplification capacity of the cells.

[0053] Figure 18 Figure 2 shows that CSR delivery enhances CAR-T cell expansion. CSR is delivered transiently via mRNA or via Stable delivery to CAR-T cells. Pan T cells isolated from the blood of normal donors are used Genetic modification was performed using the DNA modification system and standard Poseida methods. In a single reaction, cells were co-electroporated with: TMThe mRNA of transposase (SPB), encoding BCMACAR and the transposon of selection gene and encoding CSR (CD28z or CD2z;Causing transient expression) other mRNA or CD19mRNA controls, or encoding BCMACAR, selection gene and CSR (CD28z or CD2z;Causing stable expression) transposon.Subsequently stimulate cells with agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later select for genetic modification in the culture time course of 19 days.At the end of the initial culture period, all T cells express CAR, indicating that genetically modified cells (data not shown) have been successfully selected.Bar represents the total live CAR-T cells in wells, and the amplification multiple on the CAR-T cells produced in the absence of CSR or CD19mRNA controls is enhanced.In the sample of transient or stably expressed CD2z or CD28z CSR, the amplification degree of CAR-T cells is higher.

[0054] Figure 19 is a series of bar graphs showing that the expression of CSR does not significantly affect the cytotoxicity of CAR-T cells. Stable delivery to CAR-T cells. Pan T cells isolated from the blood of normal donors are used Genetic modification was performed using the DNA modification system and standard Poseida methods. In a single reaction, cells were co-electroporated with: TM Transposase (SPB) mRNA, encoding BCMA CAR and the transposon of selection gene and encoding CSR (CD28z or CD2z;Causing transient expression) other mRNA, or encoding BCMACAR, selection gene and CSR (CD28z or CD2z;Causing stable expression) transposon.Subsequently stimulate cells with agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later select for genetic modification in the culture time course of 19 days.At the end of the initial culture period, all T cells express CAR, indicating that genetically modified cells (data not shown) were successfully selected.In order to evaluate the killing ability of CAR-T cells, cells and engineered K562-BCMA-luciferase (eK562-Luc.BCMA) or negative control system K562-luciferase (eK562-Luc) were co-cultured for 48 hours with 10: 1, 3: 1 or 1: 1E: T ratio.Measure luciferase signal to determine cytotoxicity. The left bar graph shows killing by eK562-Luc, while the right bar graph shows killing by eK562-Luc.BCMA. All CARs +All T cells expressed anti-BCMA-specific CARs and exhibited similar in vitro cytotoxicity against BCMA+ target cells. Overall, this activity was not significantly affected by transient or stable CSR co-expression.

[0055] Figure 20 Schematic diagram showing enhanced stimulation by expression of chimeric stimulatory receptors (CSRs) in the presence of TCRs. In the presence of surface-expressed CSR / s (transient or stably expressed), when T cells are treated with reagents displaying agonist mAbs, enhanced primary and secondary costimulatory signals are delivered. In one aspect, this schematic diagram represents autologous cells. Since more complete T cell activation is achieved by the stimulation signal mediated by CSRs, T cell activation and amplification are enhanced.

[0056] Figure 21 It is a series of figures, showing that CSR is expressed on T cell surface, and does not cause cell activation in the absence of exogenous stimulation.In standard T cell culture medium, pan-T cells from normal blood donors are stimulated with anti-CD3 / anti-CD28 beads, then rested.Then these cells are electroporated (BTX ECM 830 electroporators, at 500V, for 700 μs) together with the mRNA of 10 μg encoding CD28CSR, CD2CSR or wild-type CD19 controls. Two days later, the surface expression of each molecule of electroporated cells was examined by flow cytometry, and data were displayed as stacked histograms. In addition, cell size (FSC-A) and CD69 expression were assessed as possible instructions for cell activation above simulated electroporation control cells. The surface expression of the increase of CD28, CD2 and CD19 was detected in the T cells electroporated with CD28zCSR, CD2z CSR or CD19 respectively. In the absence of exogenous stimulation, the expression of these molecules on T cell surface was not activated in nature.

[0057] Figure 22 is a series of line graphs demonstrating that CSR molecules can be transiently delivered during manufacturing to enhance the expansion of CAR-T cells. Pan T cells isolated from healthy donor blood were used DNA modification system and Cas-CLOVER TM Genetic modification with a combination of gene editing systems (CC) to produce allogeneic (Allo) CAR-T cells, or without CC gene editing to produce autologous (Auto) CAR-T cells; Autologous CAR-T cells are modified by encoding super Transposase (SPB) mRNA and the nuclear transfection of the transposon encoding CAR, selection gene and safety switch are produced. In order to produce Allo CAR-T, cells are electroporated (EP) together with the following in a single reaction: mRNA encoding SPB enzyme, mRNA encoding CC, targeting TCRb and b2M to knock out multiple guide RNAs (gRNA) of TCR and MHICI, and transposon encoding CAR, selection gene and CSR CD2z, or transposon encoding CAR, selection gene and safety switch that do not encode CSR. For CAR-T cells that do not receive CSR encoded in transposon to stably integrate, CD2z CSR is only provided to cells once as mRNA with 5 μg, 10 μg and 20 μg mRNA in 100 μl EP reactants in the initial EP reaction. After EP, all cells are subsequently stimulated with a mixture of agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later selected using gene selection for genetic modification in a 19-day culture time course. At the end of the initial culture period, all T cells expressed CAR, indicating that genetically modified cells were successfully selected (data not shown). The data of each are shown in line graphs on different dates of production. Compared to the CAR-T cells produced in the absence of CSR, a greater degree of CAR-T cell expansion was observed in samples providing CD2zCSR stably (as encoded in transposons (stable)) or transiently (as encoded in mRNA (mRNA)). These data show that CSR can be delivered as mRNA transiently during manufacturing to enhance the amplification of autologous and allogeneic CAR-T products.

[0058] Figure 23A is a bar graph showing staining data for CSR CD2z mutants. A panel of CSR CD2z mutants were designed, constructed, and tested for surface expression and binding to several anti-CD2 antibody reagents. To this end, each mutant was synthesized, subcloned into an in-house mRNA production vector, and high-quality mRNA was then produced for each mutant. K562 cells were electroporated with 9 μg of mRNA, and the next day the surface expression of each molecule was analyzed by flow cytometry, and the data are displayed as 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 are summarized in Figure 23C middle.

[0059] Figure 23BIt is a series of bar graphs showing CSR CD2z mutant degranulation data. The ability of the CSR CD2z mutant group to mediate degranulation for CD58 positive cell targets was tested. T cell degranulation is a substitute for T cell killing, which can be measured by FACS staining for intracellular CD107a expression after 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 ECM 830 electroporator, at 500V, for 700μs) together with 9μg of mRNA expressing CSRCD2z mutants and cultured overnight. The next day, the cells were co-cultured for 4-6 hours in the presence of various target cell lines. Positive target cell lines included K562 cells or Rat2 cells electroporated or lipofected, respectively, with mRNA encoding human CD58, while negative controls were unelectroporated Rat2 cells or T cells expressing the CSR CD2z mutant alone. Only T cells expressing the CSR CD2z mutant that recognized surface-expressed human CD58 were able to degranulate above background levels. Very little reactivity was observed for D111H, K67R / Y110D, K67R / Q70K / Y110D / D111H, ΔK106-120, CD3z-deficient, and mock controls, and the data are summarized in Figure 23C middle.

[0060] Figure 23C The table summarizes the staining and degranulation data. Data from surface expression and binding studies, as well as degranulation experiments for each CSRCD2z mutant, are summarized in the table. Two candidates that expressed on the surface and / or retained binding to anti-CD2 activator reagents that did not mediate anti-CD58 degranulation activity were the D111H and K67R / Y110D CSRCD2z mutants. Only the D111H mutant robustly bound to all stains on the cell surface while completely abolishing anti-CD58 degranulation activity.

[0061] Figure 23D Is a series of flow cytometry graphs showing the expression of CD48, CD58 or CD59 on K562 and Rat2 cells. In order to confirm the possible ligands of CSRWTCD2z molecules, a group of known and suspected ligands, including human CD48, CD58 and CD59, were tested. For the cell lines K562 and Rat2 that overexpress the target ligand and confirm expression by FACS staining, the degranulation of engineered T cells was assessed. The red histogram is unstained cells, and the blue histogram is electroporated / lipofected together with mRNA, and then stained by FACS to express the cells of the corresponding marker.

[0062] Figure 23EIt is a bar graph showing that CSRCD2z recognizes human CD58 but not CD48 or CD59. In order to confirm the possible ligands of the CSRWT CD2z molecule, a group of known and suspected ligands were tested, including human CD48, CD58 and CD59. The degranulation of engineered T cells was assessed for the cell lines K562 and Rat2 that overexpress the target ligand and confirm expression by FACS staining. The cells were electroporated / lipofected together with mRNA and then stained by FACS to express the corresponding markers. As controls, BCMACAR and K562 cell lines that overexpress BCMA were included. In addition, T cells transfected with GFP were also included as controls. T cell degranulation is a substitute for T cell killing, which can be measured by FACS staining for 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. Then these T cells were electroporated together with the mRNA expressing CSRWT CD2z, BCMACAR or GFP, and cultured overnight. The next day, in the presence of various target cell lines, cells were co-cultured for 4-6 hours, and the target cell lines were electroporated / lipofected together with the mRNA encoding human CD48, CD58 or CD59, while the negative control was the K562 or Rat2 cells not electroporated / lipofected, or each of the electroporated T cells alone. When respectively co-cultured with the cell lines over-expressing human CD58 or BCMA and not for human CD48 or CD59, the T cells expressing CSRWT CD2z or BCMACAR can degranulate at a level higher than background. For the T cells expressing GFP, very little reactivity was observed.

[0063] Figure 24A This is a bar graph showing that delivery of the CSR CD2z-D111H mutant enhances the expansion of Allo CAR-T cells. Pan T cells isolated from healthy donor blood were used DNA modification system and Cas-CLOVER TM Genetic modification with a combination of gene editing systems (CC) to produce allogeneic (Allo) CAR-T cells, or without CC gene editing to produce CSR-free autologous (Auto) CAR-T cells (no CSR); autologous CAR-T cells are modified by encoding super Transposase (SPB) mRNA and the nuclear transfection of the transposon encoding CAR, selection gene and safety switch are produced. In order to produce AlloCAR-T, in a single reaction, cells are electroporated (EP) together with the following: mRNA encoding SPB enzyme, mRNA encoding CC, targeting TCRb and b2M to knock out multiple guide RNAs (gRNA) of TCR and MHCI, and encoding CAR, selection gene and WT or mutant (D111H) CSRCD2z transposon, or the transposon encoding CAR, selection gene and safety switch of CSR are not encoded. For the latter, the CSR encoded in the transposon is not accepted to stably integrate Allo CAR-T cells, and WT or mutant (D111H) CSR CD2z is only once transiently provided to cells as mRNA in the initial EP reaction. After EP, all cells are subsequently stimulated with the mixed solution of agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later selected using gene selection for genetic modification in a culture time course of up to 15 days. At the end of the initial culture period, all T cells expressed CAR, indicating that genetically modified cells had been successfully selected (data not shown), and then all unedited TCR positive cells were exhausted by negative selection to produce> 99% TCR negative allogeneic CAR-T cell colonies (data not shown). Except for the autologous (no CSR) control, all samples were performed in duplicate, and the peak amplification data of each sample were shown in the bar graph (display peak amplification day), where the error bars represent standard deviations. Compared to the Allo CAR-T cells produced in the absence of CSR, a greater degree of Allo CAR-T cell expansion was observed in samples in which WT or mutant (D111H) CD2z was provided stably (as encoded in transposons (stable)) or transiently (as encoded in mRNA (mRNA)).

[0064] Figure 24B is a series of bar graphs demonstrating that delivery of the CSRCD2z-D111H mutant does not inhibit gene editing. DNA modification system and Cas-CLOVER TMThe combination of gene editing system (CC) is genetically modified to the pan T cells separated from healthy donor blood to produce allogeneic (Allo) CAR-T cells. In a single reaction, cells are electroporated (EP) with the following: mRNA encoding SPB enzymes, mRNA encoding CC, targeting TCRb and b2M to knock out multiple guide RNAs (gRNA) of TCR and MHCI, and encoding CAR, selection gene and WT or mutant (D111H) CSRCD2z transposon, or the transposon encoding CAR, selection gene and safety switch of CSR are not encoded. For the latter, the CSR encoded in the transposon is not accepted to stably integrate cells, and only WT or mutant (D111H) CSRCD2z is transiently provided to cells as mRNA once in the initial EP reaction. After EP, all cells are subsequently stimulated with a mixed solution of agonist mAb anti-CD2, anti-CD3 and anti-CD28, and later selected using gene selection in a culture time course of up to 14 days for genetic modification. At the end of the initial culture period, all T cells expressed CAR, indicating that genetically modified cells (data not shown) were successfully selected. All samples were carried out in duplicate, and data are shown in bar graphs, where error bars represent standard deviations. Compared to the AlloCAR-T cells produced in the absence of CSR, similar or greater AlloCAR-T cell gene editing was observed in the sample providing WT or mutant (D111H) CD2z in a stable (such as encoding (stable) in transposon) or transient (such as encoding (mRNA) in mRNA).

[0065] Figure 24C Figure 2 is a bar graph showing that the memory phenotype of AlloCAR-T cells is not significantly affected by the delivery of CD2z CSR. Stable or transiently delivered AlloCAR-T cells without CSR and allogeneic CAR-T cells with CSR were stained for the expression of surface CD45RA, CD45RO and CD62L 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 the data are shown in bar graphs with error bars representing standard deviations. Delivery of CSR did not significantly affect the content of favorable Tscm and Tcm cells in the product.

[0066] Figure 25 is a schematic diagram depicting an exemplary HLA-bGBE composition of the present disclosure.

[0067] Figure 26 is a schematic diagram depicting an exemplary HLA-gBE composition of the present disclosure.

[0068] Figure 27 It is a pair of figures showing that the expression of single-chain HLA-E reduces the cytotoxicity of HLA-deficient T cells mediated by NK cells. B2M and TCRαβ are knocked out T cells (Jurkat) using CRISPR. B2M / TCRαβ double knockout (DKO) T cells are electroporated together with mRNA encoding HLA-E molecules (HLA-bGBE) and expressed on a single chain together with B2M and peptide VMAPRETLIL (SEQ ID NO: 17127) (B2M / peptide / HLA-E). In 3 hours of co-culture, DKOT cells electroporated with different amounts of mRNA encoding single-chain HLA-E are used as targets for NK cells amplified by artificial antigen presenting cells (aAPC). Cytotoxicity % is calculated based on the number of target cells remaining after 3 hours compared to individual target cells. These data show that the surface expression of HLA-E in DKO T cells reduces the total level of cells killed by NK cells in a dose-dependent manner.

[0069] Figure 28 is a list of gRNA sequences (from top to bottom) and primer sequences (from top to bottom).

[0070] Figure 29It is a series of flow cytometry diagrams, which show the targeted knockout of endogenous HLA-ABC instead of HLA-E. Since we have shown that the surface expression of HLA-E in MHCI KO T cells can increase its resistance to NK cell-mediated cytotoxicity, we have explored other strategies except introducing single-chain HLA-E genes. To this end, multiple guide RNAs (gRNAs) were designed to destroy the expression of the main targets of host-versus-graft (HvG), HLA-A, HLA-B and HLA-C, while minimizing the damage to endogenous HLA-E. Specifically, guides were designed to target the conserved regions that are present in all three MHCI protein targets but not in HLA-E. Pan-human T cells were electroporated with mRNA encoding CRISPR Cas9 and various gRNAs, and the efficiency of MHCI knockout was measured by surface HLA-A and HLA-E expression. After a single round of T cell expansion, FACS analysis of HLA-A and HLA-E expression was performed, and the data are shown below. These data suggest that gene editing technology can be used to target the disruption of MHCI while preserving endogenous HLA-E content on the surface of gene-edited T cells.

[0071] Figure 30 Schematic representation of the missing-self hypothesis for natural killer-mediated toxicity to MHC I-KO cells.

[0072] Figure 31 It is a schematic diagram of Csy4-T2A-Clo051-G4Slinker-dCas9 construct diagram (embodiment 2).

[0073] Figure 32 is a schematic diagram of the pRT1-Clo051-dCas9 double NLS construct (Example 1).

[0074] Figure 33 is a schematic diagram showing an exemplary method for producing the allogeneic CAR-T of the present disclosure.

[0075] Figure 34A Figure 2 shows efficient gene editing of endogenous TCRα in proliferating Jurkat cells and resting naive human pan T cells using Cas-CLOVER TM (RNA-guided fusion protein containing dCas9-Clo051) Exemplary method for producing allogeneic and universal CAR-T. The Cas-CLOVER system abolished TCRα expression in rapidly proliferating Jurkat T cells and non-dividing resting T cells at relatively high levels.

[0076] Figure 34Bis a series of flow cytometry images showing the use of Cas-CLOVER TM Efficient gene editing of endogenous TCRα, TCRβ, and B2M in resting naive human pan T cells. Cas-CLOVER efficiently edited TCRα, TCRβ, and B2M, important targets for mediating alloreactivity, in resting human T cells.

[0077] Figure 35 Figure 2 is a series of flow cytometry images showing that Cas-CLOVER can be multiplexed by co-delivering reagents for TCRβ and β2M into naive human T cells. TCRβ / β2M double knockout (DKO) cells were further enriched using antibody-bead-based purification, and the purified cells were analyzed by FACS for downregulation of surface-expressed CD3 and β2M.

[0078] Figure 36It is a series of graphs showing that alloreactivity is reduced after KO of TCR and MHCI. The alloreactivity of WT or DKO (TCR and MHCI) CAR-T cells was analyzed by mixed lymphocyte reaction (MLR) and IFNγ by ELISpot analysis. On the left, WT or gene-edited DKO CAR-T cells were labeled with celltrace violet (CTV) and mixed with irradiated peripheral blood mononuclear cells (PBMC) at a ratio of 1:1 and cultivated for 12 days or 20 hours, and then analyzed for proliferation or activation-induced IFNγ secretion by ELISpot analysis, respectively. WT or DKO CAR-T cells were cultivated with PBMCs from allogeneic (donor #1 PBMC and donor #2 PBMC) or autologous (autologous PBMC) donors at a ratio of 1:1. After 12 days, CTV dye dilution was assessed by FACS, and the results showed that WT CAR-T cells proliferated significantly when cultivated with allogeneic PBMCs; when cultivated with allogeneic PBMCs from two different donors, the proliferation rates of WT CAR-T cells were observed to be 40% and 39%, respectively, compared to only 2% when WT CAR-T cells were cultivated with autologous PBMCs. On the other hand, DKO CAR-T cells do not proliferate when cultivated with allogeneic PBMCs, indicating that KO of TCR and MHCI leads to the elimination of graft-versus-host allogeneic reactions. This is also true in short-term IFNγ according to ELISpo analysis (lower left figure), which shows that when cultivated with allogeneic PBMCs, only WT CAR-T cells are activated and secrete IFNγ, but this is not the case with DKO CAR-T cells. On the right, irradiated WT or DKOCAR-T cells were mixed with CFSE-labeled PBMCs at a 1:1 ratio and cultivated for 12 days or 20 hours, and then proliferation or activation-induced IFNγ secretion was analyzed by ELISpot analysis, respectively. After 12 days, CFSE dye dilution was assessed by FACS, and PBMCs (most likely T cells) were significantly proliferated when cultivated with allogeneic CAR-T cells; 37% and 9% of PBMCs proliferated, compared to only 2% when cultivated with autologous CAR-T cells. On the other hand, PBMCs did not proliferate above background when cultivated with allogeneic CAR-T cells, indicating that KO of TCR and MHCI resulted in the elimination of graft-versus-host allogeneic reactions. This was also true in short-term IFNγ according to ELISpot analysis (lower left panel), which showed that when cultivated with allogeneic CAR-T, only WT CAR-T cells caused IFNγ activation and secretion due to PBMCs, while DKO CAR-T cells were not.

[0079] Figure 37It is a series of figures showing that DKO and WT CAR-T have similar CAR expression and stem-like phenotypes.Gene editing does not affect the CAR-T cell phenotype.The phenotypes of TCRβ / β2M DKO and WT T cells expressing BCMACAR were analyzed.CAR expression is comparable in WT and DKO.The expression of CD45RA and CD62L (markers of T stem cell memory (TSCM)) in WT and DKO CAR-T cells was analyzed by FACS.These data show that gene editing of allogeneic CAR-T does not significantly reduce the composition of memory CAR-T cells, retaining an abnormally high and major Tscm phenotype.

[0080] Figure 38 It is a series of diagrams showing that DKO CAR-T has high functionality. Gene editing does not affect CAR-T cell function. The function of TCR β / β2M DKO and WT T cells expressing BCMA CAR was analyzed. The proliferation for H929 (BCMA+) tumor line was assessed as follows: by mixing CAR-T cells with H929 cells, cultivating for 7 days, and analyzing tumor-specific proliferation by FACS. The cytotoxicity and IFNg secretion for H929 (BCMA+) tumor cell line were assessed as follows: by mixing CAR-T cells with H929 cells in various ratios, cultivating for 24 hours and analyzing tumor-specific killing by FACS. The cytotoxicity data were standardized with samples of tumor cells only. These data show that the gene editing that produces DKO CAR-T cells does not significantly affect its functional capacity.

[0081] Figure 39A Schematic diagram showing the preclinical evaluation of the P-PSMA-101 transposon when delivered at a 'stress' dose using a murine xenograft model via a full-length plasmid (FLP) versus a nanotransposon (NT). A murine xenograft model using a luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSG mice was used to evaluate the in vivo anti-tumor efficacy of the P-PSMA-101 transposon, which was delivered via a full-length plasmid (FLP) or a 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 injected with the P-PSMA-101 transposon. (PB) delivery, using either FLP or NT delivery. Mice were injected with LNCaP in the axilla and stained after tumor formation (100-200 mm by caliper). 3Mice were treated with two different 'stress' doses (2.5×10^6 or 4×10^6) of P-PSMA-101 CAR-T cells via intravenous injection to detect functional differences in the efficacy of transposons delivered via FLP and transposon at greater resolution.

[0082] Figure 39B Is to show Figure 34A Figure 1 is a series of graphs showing tumor volume assessments of mice treated as described in

[0015] . Tumor volume assessments were performed by caliper measurement for control mice (black), donor #1FLP mice (red), donor #1NT mice (blue), donor #2FLP mice (orange), and donor #2NT mice (green), shown as group means (top) and individual mice (bottom) with error bars. The y-axis shows tumor volume (mm) assessed by caliper measurement. 3 ). The x-axis shows the number of days after T cell treatment. P-PSMA-101 transposon delivered by NT at a 'stress' dose demonstrated enhanced anti-tumor efficacy against established SCLNCaP.luc solid tumors compared to FLP and control mice, as measured by calipers. DETAILED DESCRIPTION

[0083] The present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[0084] The activating component may comprise, consist essentially of, or consist of one or more of a component of a human transmembrane receptor, a human cell surface receptor, a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The activating component may comprise, consist essentially of, or consist of a portion of one or more of a component of a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor to which an agonist of the activating component binds.

[0085] The extracellular domain may comprise, consist essentially of, or consist of the extracellular domain of CD2 to which the agonist binds, or a portion thereof, or the extracellular domain may comprise, consist essentially of, or consist of the extracellular domain of CD28 to which the agonist binds. The activating component may comprise, consist essentially of, or consist of the extracellular domain of CD2 to which the agonist binds, or the activating component may comprise, consist essentially of, or consist of the extracellular domain of CD28 to which the agonist binds. The extracellular domain of CD2 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 extracellular domain of CD2 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 extracellular domain of CD2 to which the agonist binds comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17111. The extracellular domain of CD28 to which the agonist binds comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17099. The extracellular domain of CD28 to which the agonist binds comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 17099. The extracellular domain of CD28 to which the agonist binds comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17099.

[0086] The signal transduction domain may comprise, consist essentially of, or consist of one or more of a human signal transduction domain, a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, or a chemokine receptor. The second protein may comprise, consist essentially of, or consist of a CD3 protein or a portion thereof. The signal transduction domain may comprise, consist essentially of, or consist of a CD3 protein or a portion thereof. The CD3 protein may 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 CD3ζ protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17102.

[0087] The intracellular domain of the CSR of the present disclosure may further comprise, consist essentially of, or consist of a cytoplasmic domain. The cytoplasmic domain may be isolated or derived from a third protein. In some aspects, the first and third proteins of the CSR of the present disclosure are the same. The cytoplasmic domain may comprise, consist essentially of, or consist of the CD2 cytoplasmic domain or a portion thereof, or the cytoplasmic domain may comprise, consist essentially of, or consist of the CD28 cytoplasmic domain or a portion thereof.

[0088] The CD2 cytoplasmic domain comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity 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 having at least 99% identity 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 having at least 95% identity 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 having at least 99% identity 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 of SEQ ID NO: 17101.

[0089] The intracellular domain of the CSR of the present disclosure may further comprise, consist essentially of, or consist of a signal peptide. The signal peptide may be isolated or derived from a fourth protein. In some aspects, the first and fourth proteins of the CSR of the present disclosure are the same. The signal peptide may comprise, consist essentially of, or consist of a CD2 signal peptide or a portion thereof; the signal peptide may comprise, consist essentially of, or consist of a CD28 signal peptide or a portion thereof; or the signal peptide may 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 having at least 95% identity 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 having at least 99% identity to the amino acid sequence of SEQ ID NO: 17110. The CD2 signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17110. The CD28 signal peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity 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 having at least 99% identity 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 having at least 95% identity 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 having at least 99% identity 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.

[0090] The transmembrane domain of the CSR of the present disclosure can be isolated or derived from a fifth protein. In some aspects, the first and fifth proteins of the CSR of the present disclosure are the same. The transmembrane domain can comprise, consist essentially of, or consist of a CD2 transmembrane domain or a portion thereof, or the transmembrane domain can comprise, consist essentially of, or consist of a CD28 transmembrane domain or a portion thereof. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO: 17112. The CD2 transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity 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 having at least 99% identity 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.

[0091] In some aspects, the activating component of a CSR of the present disclosure does not bind or is incapable of binding to a naturally occurring molecule. In some aspects, the activating component of a CSR of the present disclosure binds or is capable of binding to a naturally occurring molecule, and the CSR transduces a signal after the activating component binds to the naturally occurring molecule. In other aspects, the activating component of a CSR of the present disclosure can bind to a naturally occurring molecule, but after the activating component binds to the naturally occurring molecule, the CSR does not transduce a signal. In preferred aspects, the activating component of a CSR of the present disclosure binds or is capable of binding to a non-naturally occurring molecule. The activating component of a CSR of the present disclosure selectively transduces a signal after the non-naturally occurring molecule binds to the activating component. In one aspect, 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 activating component of the CSR can be any naturally occurring antibody or antibody fragment. The naturally occurring antibody or antibody fragment can be a naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the naturally occurring agonist / activator that can bind to the CSR activating component can be one or more of the following: 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 aspect, the non-naturally occurring molecule is a non-naturally occurring agonist / activator for the activating component of the CSR. The non-naturally occurring agonist / activator that can bind to the CSR activating component can be any non-naturally occurring antibody or antibody fragment. The non-naturally occurring antibody or antibody fragment can be a non-naturally occurring anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the non-naturally occurring agonist / activator that can bind to the CSR activating component can be one or more of the following: an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist / activator that can bind to a CSR activating component can be selected from the group consisting of: an anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and a humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab) (Bissonnette et al., Arch. Dermatol. Res. 301(6):429442, 2009).

[0092] In some aspects, the extracellular domain of the CSR of the present disclosure may comprise a modification. When compared to the wild-type amino acid sequence of the activating component or the first protein, the modification may comprise a mutation or truncation of the amino acid sequence of the activating component or the first protein. The mutation or truncation of the amino acid sequence of the activating component or the first protein may comprise a mutation or truncation of the extracellular domain of CD2 or a portion thereof to which the agonist binds. The mutation or truncation of the extracellular domain of CD2 reduces or eliminates binding to naturally occurring CD58.

[0093] Reduced binding is when the binding ability of the mutant or truncated CD2 extracellular domain 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% compared to the naturally occurring wild-type counterpart. Ablated binding is when the binding ability of the mutant or truncated CD2 extracellular domain is reduced by 100% compared to the naturally occurring wild-type CD2 extracellular domain.

[0094] The mutated or truncated CD2 extracellular domain binds to an anti-CD2 activating agonist and an anti-CD2 activating molecule, 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 having at least 80% identity 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 having at least 85% identity 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 having at least 90% identity 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 having at least 95% identity 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 having at least 99% identity 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 having at least 90% identity 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 having at least 95% identity 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 having at least 99% identity to the amino acid sequence of SEQ ID NO: 17118. The CSR comprising a mutated or truncated CD2 extracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17118.

[0095] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein and wherein the activation component binds to a non-naturally occurring molecule but does not bind to a naturally occurring molecule; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0096] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein the CSR does not transduce a signal upon binding of a naturally occurring molecule to the activating component.

[0097] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical and wherein upon binding of the non-naturally occurring molecule to the activating component, the CSR transduces a signal.

[0098] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises the extracellular domain of CD2, or a portion thereof, to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain, or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain, or a portion thereof, and wherein the at least one signaling domain comprises a CD3 zeta protein, or a portion thereof.

[0099] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activation component comprising the amino acid sequence of SEQ ID NO: 17111; (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an intracellular domain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signaling domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 17062. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17062.

[0100] The present disclosure also provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activating component comprises a mutation or truncation of a wild-type CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and wherein at least one signal transduction domain comprises a CD3 zeta protein or a portion thereof. In one aspect, the mutation or truncation of the CD2 extracellular domain reduces or eliminates binding to naturally occurring CD58. In another aspect, 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.

[0101] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising a signal peptide comprising the amino acid sequence of SEQ ID NO: 17110 and an activation component comprising the amino acid sequence of SEQ ID NO: 17119; (b) a transmembrane domain of SEQ ID NO: 17112; and (c) an intracellular domain comprising a cytoplasmic domain comprising the amino acid sequence of SEQ ID NO: 17113 and at least one signaling domain comprising the amino acid sequence of SEQ ID NO: 17102. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 17118. The non-naturally occurring chimeric stimulatory receptor (CSR) may comprise, consist essentially of, or consist of an amino acid sequence of SEQ ID NO: 17118.

[0102] The present disclosure also provides nucleic acid sequences encoding the amino acid sequences of any chimeric stimulatory receptor (CSR) disclosed herein. The present disclosure also provides transposons, vectors, donor sequences, or donor plasmids comprising, consisting essentially of, or consisting of nucleic acid sequences encoding the amino acid sequences of any chimeric stimulatory 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.

[0103] The present disclosure also provides cells comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides cells comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides cells comprising, consisting essentially of, or consisting of a transposon, a vector, a donor sequence, or a donor plasmid, each of which comprises, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. In one aspect, the vector may be a viral vector. In one aspect, the viral vector may be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector. The cells of the present disclosure comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein may be allogeneic cells or autologous cells. In some preferred embodiments, the cells are allogeneic cells.

[0104] The present disclosure also provides compositions comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a transposon, a vector, a donor sequence, or a donor plasmid, each of which comprises, consisting essentially of, or consisting of a nucleic acid sequence encoding the amino acid sequence of any chimeric stimulating receptor (CSR) disclosed herein. In one aspect, the vector may be a viral vector. In one aspect, the viral vector may be an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector. The present disclosure also provides compositions comprising, consisting essentially of, or consisting of a cell or cells, the cell or cells comprising, consisting essentially of, or consisting of any chimeric stimulating receptor (CSR) disclosed herein.

[0105] The present disclosure provides a modified cell comprising, consisting essentially of, or consisting of a chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0106] The present disclosure also provides a modified cell comprising, consisting essentially of, or consisting of: (a) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (b) an inducible pro-apoptotic polypeptide.

[0107] The present disclosure also provides a modified cell comprising, consisting essentially of, or consisting of: (a) a chimeric stimulatory receptor (CSR), comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; (b) a sequence encoding an inducible pro-apoptotic polypeptide; and wherein the cell is a T cell, (c) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR.

[0108] The present disclosure provides a modified cell comprising, consisting essentially of, or consisting of: (a) a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I); and (b) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide.

[0109] The present disclosure provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR), wherein the modifications reduce or eliminate the expression or activity level of the TCR; and (b) a chimeric stimulatory receptor (CSR), the chimeric stimulatory receptor comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0110] The present disclosure provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR), wherein the modifications reduce or eliminate the expression or activity level of the TCR; (b) a chimeric stimulatory receptor (CSR), wherein the chimeric stimulatory receptor comprises: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (c) a non-naturally occurring chimeric antigen receptor.

[0111] 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), wherein the modification reduces or eliminates the expression or activity level of the TCR; (b) a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I); and (c) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0112] 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), wherein the modification reduces or eliminates the expression or activity level of the TCR; (b) a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I); (c) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (d) a non-naturally occurring chimeric antigen receptor.

[0113] The present disclosure also 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), wherein the modification reduces or eliminates the expression or activity level of the TCR; (b) a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0114] The present disclosure also 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), wherein the modification reduces or eliminates the expression or activity level of the TCR; (b) a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I); (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E); (d) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical; and (e) a non-naturally occurring chimeric antigen receptor.

[0115] The present disclosure also provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR), wherein the modifications reduce or eliminate the expression or activity level of the TCR; (b) modifications that reduce or eliminate the expression or activity level 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 stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0116] The present disclosure also provides modified T lymphocytes (T cells) comprising, consisting essentially of, or consisting of: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR), wherein the modifications reduce or eliminate the expression or activity level of the TCR; (b) modifications that reduce or eliminate the expression or activity level 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 comprising HLA class I histocompatibility antigen, alpha chain E (HLA-E); and (d) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0117] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) may 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 having at least 95% identity 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 having at least 99% identity 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.

[0118] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) may further comprise, consist essentially of, or consist of modifications to an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modifications reduce or eliminate the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I). A reduction in the expression or activity level 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% 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 a T cell or the functional activity of MHC-I in a 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% compared to a naturally occurring wild-type T cell. An ablation of the level of expression or activity is when the expression of MHC-I in a T cell or the functional activity of MHC-I in a T cell is reduced by 100% compared to a naturally occurring wild-type counterpart of the cell. An ablation of the level of expression or activity is when the expression of MHC-I in a T cell or the functional activity of MHC-I in a T cell is reduced by 100% compared to a naturally occurring wild-type T cell.

[0119] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) may further comprise, consist essentially of, or consist of a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E). The HLA-E polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity 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 having at least 99% identity 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.

[0120] The non-naturally occurring polypeptide comprising HLA-E may 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 having at least 95% identity 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 having at least 99% identity 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.

[0121] The non-naturally occurring polypeptide comprising HLA-E may 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 having at least 95% identity to the amino acid sequence of SEQ ID NO: 17129. The B2M polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity 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.

[0122] The non-naturally occurring polypeptide comprising HLA-E may further comprise, consist essentially of, or consist of a linking molecule (referred to herein as a linker). The non-naturally occurring polypeptide comprising HLA-E may further comprise, consist essentially of, or consist of a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The linker comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 17130. The linker comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17130.

[0123] The non-naturally occurring polypeptide comprising HLA-E may 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 having at least 95% identity to the amino acid sequence of SEQ ID NO: 17127. The peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity 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.

[0124] The non-naturally occurring polypeptide comprising HLA-E may further comprise, consist essentially of, or consist of: a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the HLA-E polypeptide. The first linker comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 17128. The first linker comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17128. The second linker comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17130. The second linker comprises, consists essentially of, or consists of an amino acid sequence having at least 99% identity 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.

[0125] In one aspect, a 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 may be positioned between the B2M signal peptide and the first linker, the B2M polypeptide may be positioned between the first linker and the second linker, and the second linker may be positioned 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 having at least 95% identity 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 having at least 99% identity to the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17064. The non-naturally occurring polypeptide comprising HLA-E can be encoded by a nucleic acid having the sequence of SEQ ID NO:17065.

[0126] In one aspect, a 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 may be positioned between the B2M signal peptide and the linker, and the linker may be positioned 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 having at least 95% identity 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 having at least 99% identity 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 may be encoded by a nucleic acid having the sequence of SEQ ID NO: 17067.

[0127] In one aspect, 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 may be located before the HLA-E polypeptide (e.g., 5' in the case of a nucleic acid sequence, or amino-terminally in the case of an amino acid sequence). The non-naturally occurring polypeptide comprising HLA-E comprises, consists essentially of, or consists of an amino acid sequence having at least 95% identity 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 having at least 99% identity 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 may be encoded by a nucleic acid having the sequence of SEQ ID NO: 17069.

[0128] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) may further include, be substantially composed of, or be composed of: non-naturally occurring antigen receptors, sequences encoding therapeutic polypeptides, or combinations thereof. In preferred aspects, non-naturally occurring antigen receptors include chimeric antigen receptors (CARs), are substantially composed of, or are composed of. CAR includes, is substantially composed of, or is composed of: (a) an extracellular domain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one costimulatory domain. The extracellular domain of CAR may further include, be substantially composed of, or be composed of a signal peptide. The extracellular domain of CAR may further include, be substantially composed of, or be composed of a hinge between the antigen recognition region and the transmembrane domain. The intracellular domain of CAR may further include, be substantially composed of, or be composed of human CD3 ζ intracellular domain, be substantially composed of, or be composed of. At least one costimulatory domain of CAR can further include the following, be substantially composed of the following or be composed of the following:Human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment or its any combination.In preferred aspects, at least one costimulatory domain includes human CD28 and / or 4-1BB costimulatory domain.

[0129] The modified cells of the present disclosure may be immune cells or immune cell precursors. The immune cells may be lymphocyte 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 preferred aspects, the immune cells are T cells, early memory T cells, stem cell-like T cells, stem memory T cells (T cells), or antigen presenting cells (APCs). SCM ), central memory T cells (T CM ) or stem cell-like T cells. Immune cell precursors may be hematopoietic stem cells (HSC). The modified cells may be stem cells, differentiated cells, somatic cells, or antigen presenting cells (APC). The modified cells may be autologous cells or allogeneic cells. In one aspect, the cells are modified allogeneic T cells. On the other hand, the cells are modified allogeneic T cells expressing chimeric antigen receptors (CARs), CAR T cells.

[0130] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) can express the CSRs of the present disclosure transiently or stably. In one aspect, the CSRs of the present disclosure are transiently expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure). In one aspect, the CSRs of the present disclosure are stably expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure).

[0131] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) can transiently or stably express the non-naturally occurring polypeptide comprising the HLA-E of the present disclosure. In one aspect, the non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is transiently expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure). In one aspect, the non-naturally occurring polypeptide comprising the HLA-E of the present disclosure is stably expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure).

[0132] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) can transiently or stably express the inducible pro-apoptotic polypeptides of the present disclosure. In one aspect, the inducible pro-apoptotic polypeptides of the present disclosure are transiently expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure). In a preferred aspect, the inducible pro-apoptotic polypeptides of the present disclosure are stably expressed in the modified cells of the present disclosure (preferably the modified T cells of the present disclosure).

[0133] The modified cells of the present invention (preferably the modified T cells of the present invention) can transiently or stably express non-naturally occurring antigen receptors or sequences encoding therapeutic proteins of the present invention. In one aspect, non-naturally occurring antigen receptors or sequences encoding therapeutic proteins of the present invention are transiently expressed in the modified cells of the present invention (preferably the modified T cells of the present invention). In a preferred aspect, non-naturally occurring antigen receptors or sequences encoding therapeutic proteins of the present invention are stably expressed in the modified cells of the present invention (preferably the modified T cells of the present invention).

[0134] In one aspect, the modified cells of the present disclosure (preferably the modified T cells of the present disclosure) stably express the CSR of the present disclosure, stably express the inducible pro-apoptotic polypeptide of the present disclosure, and stably express the non-naturally occurring antigen receptor or the sequence encoding the therapeutic protein.

[0135] In one aspect, the modified cells of the present disclosure (preferably the modified T cells of the present disclosure) stably express a CSR of the present disclosure, stably express a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure, stably express an inducible pro-apoptotic polypeptide of the present disclosure, and stably express a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein.

[0136] In one aspect, the modified cells of the present disclosure (preferably the modified T cells of the present disclosure) stably express a CSR of the present disclosure, transiently express a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure, stably express an inducible pro-apoptotic polypeptide of the present disclosure, and stably express a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein.

[0137] In one aspect, a CSR of the present disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of the present disclosure is stably expressed, and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed in a modified cell of the present disclosure (preferably a modified T cell of the present disclosure).

[0138] In one aspect, a CSR of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T cell of the present disclosure), a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure is transiently expressed, an inducible pro-apoptotic polypeptide of the present disclosure is stably expressed, and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed.

[0139] In one aspect, a CSR of the present disclosure is transiently expressed in a modified cell of the present disclosure (preferably a modified T cell of the present disclosure), a non-naturally occurring polypeptide comprising an HLA-E of the present disclosure is stably expressed, an inducible pro-apoptotic polypeptide of the present disclosure is stably expressed, and a non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein is stably expressed.

[0140] The present disclosure provides modified cells (modified T cells) comprising, consisting essentially of, or consisting of: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR), wherein the modifications reduce or eliminate the expression or activity level of the TCR; and (b) a sequence encoding a chimeric stimulatory receptor (CSR), wherein the chimeric stimulatory receptor comprises: (i) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[0141] The modified cells may further comprise, consist essentially of, or consist of a sequence encoding an inducible pro-apoptotic polypeptide. The modified cells may further comprise, consist essentially of, or consist of a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor may comprise, consist essentially of, or consist of a chimeric antigen receptor (CAR).

[0142] The transposon, vector, donor sequence, or donor plasmid may comprise, consist essentially of, or consist of a sequence encoding a CSR, a sequence encoding an inducible pro-apoptotic polypeptide, or a combination thereof. The transposon, vector, donor sequence, or donor plasmid may 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 may further comprise, consist essentially of, or consist of a sequence encoding a selectable marker. The transposon may be transposons, The sequence encoding the CSR can be transiently expressed in the cell. The sequence encoding the CSR can be stably expressed in the cell. The sequence encoding the inducible pro-apoptotic polypeptide 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 aspects, 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 aspects, 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 aspects, 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 aspects, the sequence encoding the CSR can be transiently expressed in the cell, 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 aspects, the sequence encoding the CSR can be stably expressed in the cell, 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 can be stably expressed in the cell. In one aspect, the vector can be a viral vector. In one aspect, the viral vector can be an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or a chimeric viral vector.

[0143] The first transposon, the first vector, the first donor sequence, or the first donor plasmid may comprise, consist essentially of, or consist of a sequence encoding a CSR. The first transposon, the first vector, the first donor sequence, or the first donor plasmid may further comprise, consist essentially of, or consist of a sequence encoding a first selectable marker.

[0144] The second transposon, second vector, second donor sequence, or second donor plasmid may 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 may 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 the same. The first selection marker and the second selection marker are different. The selection marker may comprise, consist essentially of, or consist of a cell surface marker. The selection marker may comprise, consist essentially of, or consist of a protein that is active in dividing cells and inactive in non-dividing cells. The selection marker may comprise, consist essentially of, or consist of a metabolic marker.

[0145] In one aspect, the selectable marker can comprise, consist essentially of, or consist of a dihydrofolate reductase (DHFR) mutant protease. The DHFR mutant protease can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17012.

[0146] The DHFR mutant protease of SEQ ID NO: 17012 may 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 mutant protease of SEQ ID NO: 17012 may further comprise, consist essentially of, or consist of a substitution of phenylalanine (F) or leucine (L) at position 80; a substitution of leucine (L) or valine (V) at position 113; and a substitution of valine (V) or aspartic acid (D) at position 153.

[0147] The modified cells of the present disclosure (preferably the modified T cells of the present disclosure) may further comprise, consist essentially of, or consist of a gene editing composition. The gene editing composition may comprise, consist essentially of, or consist of a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The gene editing composition may be transiently expressed by the modified cells. The gene editing composition may be stably expressed by the modified cells.

[0148] The gene editing composition may 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 may comprise, consist essentially of, or consist of a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may 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 may comprise, consist essentially of, or consist of a nuclease-inactivated Cas (dCas) protein. The nuclease or its nuclease domain may comprise, consist essentially of, or consist of a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise, consist essentially of, or consist of a Clo051 nuclease or its nuclease domain. The gene editing composition may comprise, consist essentially of, or consist of a fusion protein. The fusion protein may comprise, consist essentially of, or consist of a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or Clo051 nuclease domain. The fusion protein may comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17013. The fusion protein is encoded by a nucleic acid comprising, consisting essentially of, or consists of the amino acid sequence of SEQ ID NO: 17014. The fusion protein may comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 17058. The fusion protein is encoded by a nucleic acid comprising, consisting essentially of, or consists of the amino acid sequence of SEQ ID NO: 17059.

[0149] The gene editing composition may further comprise, consist essentially of, or consist of a guide sequence. The guide sequence may comprise, consist essentially of, or consist of an RNA sequence. In aspects where the modified cells are T cells, the guide RNA may comprise, consist essentially of, or consist of a sequence complementary to a target sequence encoding an endogenous TCR. The guide RNA may comprise, consist essentially of, or consist of a sequence complementary to a target sequence encoding a B2M polypeptide. The guide RNA may comprise, consist essentially of, or consist of a sequence complementary to a target sequence within a safe harbor site of a genomic DNA sequence.

[0150] The transposon, vector, donor sequence, or donor plasmid may 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 a Clo051 nuclease or a nuclease domain thereof.

[0151] The first transposon, the first vector, the first donor sequence, or the first donor plasmid may 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 a Clo051 nuclease or a nuclease domain thereof.

[0152] 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 a Clo051 nuclease or a nuclease domain thereof.

[0153] The third transposon, third vector, third donor sequence, or third donor plasmid may 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 a Clo051 nuclease or a nuclease domain thereof.

[0154] The Clo051 nuclease or its nuclease domain can induce single-strand or double-strand breaks in the target sequence. The donor sequence or donor plasmid can be integrated at the site of the single-strand or double-strand break or at the site of cellular repair within the target sequence, or a combination thereof.

[0155] The present disclosure provides a composition comprising, consisting essentially of, or consisting of a modified cell of the present disclosure (preferably a modified T cell of the present disclosure).

[0156] The present disclosure provides a plurality of modified cells comprising any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein, and provides a plurality of modified cells comprising any modified cell disclosed herein. The plurality of modified cells may comprise, consist essentially of, or consist of immune cells or immune cell precursors. The plurality of immune cells may 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).

[0157] The present disclosure provides compositions comprising modified cell colonies, wherein the multiple modified cells of the colony comprise any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein, and provides compositions comprising modified cell colonies, wherein the multiple modified cells of the colony comprise any modified cells disclosed herein. The modified cell colony may comprise, consist essentially of, or consist of immune cells or immune cell precursors. The immune cell colony may comprise, consist essentially of, or consist of lymphocyte 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 may comprise a pharmaceutically acceptable carrier.

[0158] The present disclosure provides compositions comprising a modified T lymphocyte (T cell) population, wherein a plurality of the modified T cells of the population comprise any non-naturally occurring chimeric stimulatory receptor (CSR) disclosed herein, and provides compositions comprising a modified T lymphocyte (T cell) population, wherein a plurality of the modified T cells of the population comprise any modified T cell disclosed herein. The composition may comprise a pharmaceutically acceptable carrier.

[0159] Preferably, the present disclosure provides a composition comprising a population of T lymphocytes (T cells), wherein a plurality of T cells of the population comprise a non-naturally occurring chimeric stimulatory receptor (CSR) comprising, consisting essentially of, or consisting of: (a) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical. The composition may comprise a pharmaceutically acceptable carrier. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population comprises a CSR.

[0160] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population comprises an inducible pro-apoptotic polypeptide.

[0161] A plurality of T cells of the colony can further include the modification of the endogenous sequence of coding T cell receptor (TCR), wherein the modification reduces or eliminates expression or activity levels of TCR.In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the colony include the modification of the endogenous sequence of coding TCR, wherein the modification reduces or eliminates expression or activity levels of TCR.

[0162] The plurality of T cells of the population may further comprise modifications of endogenous sequences encoding beta-2 microglobulin (B2M), wherein the modifications reduce or eliminate expression or activity levels of major histocompatibility complex (MHC) class I (MHC-I). In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population comprise modifications of endogenous sequences encoding B2M, wherein the modifications reduce or eliminate expression or activity levels of MHC-I.

[0163] The plurality of T cells in the population may further comprise a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR, and a modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I).

[0164] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population comprises a modification of an endogenous sequence encoding a TCR, wherein the modification reduces or eliminates the expression or activity level of the TCR, and a modification of an endogenous sequence encoding a B2M, wherein the modification reduces or eliminates the expression or activity level of an MHC-I.

[0165] The plurality of T cells of the population may further contain a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population contain a non-naturally occurring sequence comprising an HLA-E polypeptide.

[0166] The multiple T cells of the colony may further include non-natural antigen receptors, sequences encoding therapeutic polypeptides or combinations thereof. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the colony include non-natural antigen receptors, sequences encoding therapeutic polypeptides or combinations thereof. In a preferred aspect, non-natural antigen receptors are chimeric antigen receptors (CAR).

[0167] The plurality of T cells in the population may comprise 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. In some aspects, stem cell-like T cells, stem cell memory T cells (T SCM ) and central memory T cells (T CM), 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 modified T cell population.

[0168] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising a CSR expresses stem memory T cells (T cells). SCM ) or T SCM One or more cell surface markers of leukemia-like cells; and wherein the one or more cell surface markers comprise CD45RA and CD62L.

[0169] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express central memory T cells (CMTs). CM ) or T CM One or more cell surface markers of WT-like cells; and wherein the one or more cell surface markers comprise CD45RO and CD62L.

[0170] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express one or more of the CD127, CD45RO, CD95, and IL-2Rβ cell surface markers.

[0171] The present disclosure provides compositions for treating diseases or conditions disclosed herein or compositions for treating any diseases or conditions disclosed herein. The present disclosure also provides methods for treating diseases or conditions, which comprise administering a therapeutically effective amount of a composition disclosed herein to an individual in need thereof, consisting essentially of it, or consisting of it. The composition may comprise, consist essentially of, or consist of any modified cell or modified cell population disclosed herein. Preferably, any modified T cell or CAR T cell disclosed herein.

[0172] The present disclosure provides a method for producing modified T cells, comprising, consisting essentially of, or consisting of: introducing into naive human T cells a composition comprising a chimeric stimulatory receptor (CSR) of the present disclosure or a sequence encoding the same, to produce modified T cells under conditions in which the CSR within the modified T cells is stably expressed and the desired stem-like properties of the modified T cells are retained. The naive human T cells may be resting naive human T cells. The present disclosure provides modified T cells produced by the disclosed methods. The present disclosure provides methods for administering modified T cells, the T cells comprising stably expressed CSRs produced by the disclosed methods. The present disclosure provides methods for administering modified T cells to treat a disease or condition, the T cells comprising stably expressed CSRs produced by the disclosed methods.

[0173] The present disclosure provides a method for producing a modified T cell population, comprising, consisting essentially of, or consisting of: introducing a composition comprising a chimeric stimulator receptor (CSR) of the present disclosure or a sequence encoding the same into a plurality of naive human T cells to produce a plurality of modified T cells under conditions that stably express the CSR within the plurality of modified T cells and retain the desired stem-like properties of the plurality of modified T cells. Naive human T cells may comprise resting naive human T cells. The present disclosure provides a modified T cell population produced by the disclosed method. The present disclosure provides a method for administering a modified T cell population, the T cell population comprising a stably expressed CSR produced by the disclosed method. The present disclosure provides a method for administering a modified T cell population to treat a disease or condition, the T cell population comprising a stably expressed CSR produced by the disclosed method.

[0174] The present disclosure provides a method for producing modified T cells, comprising, consisting essentially of, or consisting of: introducing into naive human T cells a composition comprising a chimeric stimulator receptor (CSR) of the present disclosure or a sequence encoding the same, to produce modified T cells under conditions in which the CSR within the modified T cells is transiently expressed and the desired stem-like properties of the modified T cells are retained. Naive human T cells may be resting naive human T cells. The present disclosure provides modified T cells produced by the disclosed methods. The present disclosure provides a method for administering modified T cells comprising transiently expressed CSRs produced by the disclosed methods. In one aspect, the present disclosure provides a method for administering modified T cells produced by the disclosed methods after the modified T cells no longer express the CSRs. The present disclosure provides a method for administering modified T cells to treat a disease or condition, wherein the T cells comprise transiently expressed CSRs produced by the disclosed methods. In one aspect, the present disclosure provides a method for administering modified T cells produced by the disclosed methods to treat a disease or condition after the modified T cells no longer express the CSRs.

[0175] The present disclosure provides a method for producing a modified T cell population, which comprises, consists essentially of, or consists of: introducing a composition comprising a chimeric stimulator receptor (CSR) of the present disclosure or a sequence encoding the same into a plurality of naive human T cells to produce a plurality of modified T cells under conditions that transiently express the CSR within the plurality of modified T cells and retain the desired stem-like properties of the plurality of modified T cells. Naive human T cells may comprise resting naive human T cells. The present disclosure provides a modified T cell population produced by the disclosed method. The present disclosure provides a method for administering a modified T cell population comprising a transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method for administering a modified T cell population produced by the disclosed method after the plurality of T cells no longer express the CSR. The present disclosure provides a method for administering a modified T cell population to treat a disease or condition, the T cell population comprising a transiently expressed CSR produced by the disclosed method. In one aspect, the present disclosure provides a method for administering a modified T cell population produced by the disclosed method to treat a disease or condition after the plurality of modified T cells no longer express the CSR.

[0176] The method for producing modified T cells or producing modified T cell colonies may further include the modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of TCR. The method for producing modified T cells or producing modified T cell colonies may further include the modification of an endogenous sequence encoding beta-2 microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-1). In some aspects, the method for producing modified T cells or producing modified T cell colonies may further include the modification of an endogenous sequence encoding TCR, wherein the modification reduces or eliminates the expression or activity level of TCR, and the modification of an endogenous sequence encoding B2M, wherein the modification reduces or eliminates the expression or activity level of MHC-1.

[0177] The method for producing modified T cells or producing modified T cell colonies may further include introducing a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding thereof into nascent human T cells or multiple nascent human T cells. In one aspect, the antigen receptor is a non-naturally occurring antigen receptor. In a preferred aspect, the method for producing modified T cells or producing modified T cell colonies may further include introducing a composition comprising a chimeric antigen receptor (CAR) or a sequence encoding thereof into nascent human T cells or multiple nascent human T cells. The method may further include introducing a composition comprising an inducible pro-apoptotic polypeptide or a sequence encoding thereof into nascent human T cells or multiple nascent human T cells. The method may further include introducing a composition comprising an inducible pro-apoptotic polypeptide or a sequence encoding thereof into nascent human T cells or multiple nascent human T cells. The method for producing modified T cells or producing modified T cell colonies may further include introducing a composition comprising an antigen receptor, a therapeutic protein or a sequence encoding thereof into nascent human T cells or multiple nascent human T cells and a composition comprising an inducible pro-apoptotic polypeptide or a sequence encoding thereof.

[0178] The method of producing modified T cells or producing modified T cell colonies may further comprise contacting the modified T cells or modified T cell colonies with an activator composition. The activator composition may comprise, consist essentially of, or consist of one or more agonists or activators that can bind to the CSR activation components of the modified T cells or multiple modified T cells. The agonist / activator may be naturally occurring or non-naturally occurring. In a preferred aspect, the agonist / activator is an antibody or antibody fragment. The agonist / activator may be one or more of the following: an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist / activator may be one or more of the following: an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. The agonist / activator may contact the modified T cells or modified T cell colonies in vitro, ex vivo, or in vivo. In preferred aspects, the agonist / activator activates the modified T cell or modified T cell population, induces cell division in the modified T cell or modified T cell population, increases cell division (e.g., cell doubling time) in the modified T cell or T cell population, increases fold expansion in the modified T cell or modified T cell population, or any combination thereof.

[0179] The present disclosure provides a method for expanding a population of modified T cells, comprising, consisting essentially of, or consisting of introducing into a plurality of naive human T cells a composition comprising a chimeric stimulatory receptor (CSR) of the present disclosure, or a sequence encoding the same, to produce a plurality of modified T cells under conditions that stably express the CSR within the plurality of modified T cells and retain the desired stem-like properties of the plurality of modified T cells, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein 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.

[0180] The present disclosure provides a method for expanding a population of modified T cells, comprising, consisting essentially of, or consisting of: introducing into a plurality of naive human T cells a composition comprising a chimeric stimulator receptor (CSR) of the present disclosure, or a sequence encoding the same, to produce a plurality of modified T cells under conditions that transiently express the CSR within the plurality of modified T cells and retain the desired stem-like properties of the plurality of modified T cells, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein 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 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 transiently express the CSR of the present disclosure under the same conditions.

[0181] The activator composition of the method for expanding a population may comprise, consist essentially of, or consist of one or more agonists or activators that can bind to the CSR activation component of the modified T cell or multiple modified T cells. The agonist / activator may be naturally occurring or non-naturally occurring. In preferred aspects, the agonist / activator is an antibody or antibody fragment. The agonist / activator may be one or more of the following: an anti-CD3 antibody or fragment thereof, an anti-CD2 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or any combination thereof. In some aspects, the agonist / activator may be one or more of the following: 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.

[0182] The conditions may comprise culturing the modified T cell or cells in a medium comprising: a sterol; an alkane; phosphorus; and one or more of octanoic acid, palmitic acid, linoleic acid, and oleic acid. The culture may be in vivo or ex vivo. The modified T cell may be an allogeneic T cell or the cells may be allogeneic T cells. The modified T cell may be an autologous T cell or the cells may be autologous T cells.

[0183] In some aspects, the culture medium may comprise 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).

[0184] In some aspects, the culture medium can comprise one or more of: octanoic acid at a concentration of about 9 mg / kg, palmitic acid at a concentration of about 2 mg / kg, linoleic acid at a concentration of about 2 mg / kg, oleic acid at a concentration of about 2 mg / kg, and sterols at a concentration of about 1 mg / kg.

[0185] In some aspects, the culture medium may comprise one or more of the following: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg (inclusive); palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg (inclusive); linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg (inclusive).

[0186] In some aspects, the culture medium can comprise 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.

[0187] The present disclosure provides compositions comprising any modified T cells produced by the methods disclosed herein. The present disclosure provides compositions comprising any modified T cell populations produced by the methods disclosed herein. The present disclosure provides compositions comprising any modified T cells amplified by the methods disclosed herein. The present disclosure provides compositions comprising any modified T cell populations amplified by the methods disclosed herein.

[0188] The present disclosure provides compositions for treating diseases or conditions disclosed herein or uses of compositions for treating any disease or condition disclosed herein. The present disclosure also provides methods for treating diseases or conditions, comprising, consisting essentially of, or consisting of administering to an individual in need thereof a therapeutically effective amount of a composition disclosed herein and at least one non-naturally occurring molecule that binds to an activating component of a CSR disclosed herein. The composition may comprise, consist essentially of, or consist of any modified cell or modified cell population disclosed herein. Preferably, any modified T cell or CAR T cell disclosed herein. Any non-naturally occurring molecule that can bind to an activating component of a CSR disclosed herein and selectively transduce a signal after binding may be administered. Preferably, the non-naturally occurring molecule is a non-natural CSR agonist / activator for the activating component. The non-naturally occurring agonist / activator that can bind to a CSR activating component may 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 aspects, the non-naturally occurring agonist / activator that can bind to the CSR activating component can be one or more of the following: an anti-human CD3 monospecific tetrameric antibody complex, an anti-human CD2 monospecific tetrameric antibody complex, an anti-human CD28 monospecific tetrameric antibody complex, or a combination thereof. In some aspects, the non-naturally occurring agonist / activator that can bind to the activating component can be selected from the group consisting of: an anti-CD2 monoclonal antibody, BTI-322 (Przepiorka et al., Blood 92(11):4066-4071, 1998) and a humanized anti-CD2 monoclonal antibody clone AFC-TAB-104 (Siplizumab) (Bissonnette et al., Arch. Dermatol. Res. 301(6):429-442, 2009). In some aspects, administration of a non-naturally occurring molecule that can bind to the activating component of the CSR stimulates the modified cells to undergo cell division in vivo. Thus, the present disclosure provides methods of stimulating modified cells of the present disclosure to undergo cell division in vivo by administering a non-natural CSR agonist / activator of an activating component to an individual having modified cells of the present disclosure.

[0189] In some aspects, the disease or condition is a cell proliferative disease or condition. In some aspects, the cell proliferative disease or condition is cancer. The cancer can be a solid tumor cancer or a blood cancer. In some aspects, the solid tumor is prostate cancer or breast cancer. In preferred aspects, the prostate cancer is castration-resistant prostate cancer. In some aspects, the blood cancer is multiple myeloma.

[0190] The modified cells or modified cell colonies contained in the disclosed compositions can be cultured in vitro or ex vivo before being administered to an individual in need thereof. The modified cells can be allogeneic modified cells or autologous modified cells. In some aspects, the cells are allogeneic modified T cells or autologous modified T cells. In some aspects, the cells are allogeneic modified CAR T cells or autologous modified CAR T cells. In some aspects, the cells are allogeneic modified CAR T cells comprising the CSRs disclosed herein or autologous modified CAR T cells comprising the CSRs disclosed herein.

[0191] The modified cell composition or a composition comprising a modified cell population can be administered to a patient by any method known in the art. In some aspects, the composition is administered by systemic administration. In some aspects, the composition is administered by intravenous administration. Intravenous administration can be intravenous injection or intravenous infusion. In some aspects, the composition is administered by topical administration. In some aspects, the composition is administered by intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0192] The therapeutically effective amount can be a single dose or multiple doses of the modified cell composition or composition comprising a modified cell population. In some aspects, the therapeutically effective dose is a single dose, and the allogeneic cells of the composition are implanted and / or maintained for a sufficient time to treat the disease or condition. In some aspects, 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 therebetween, produced simultaneously.

[0193] In some aspects, the uses and methods of treating a disease or condition further provide that following administration of a modified cell composition disclosed herein or a composition comprising a modified cell population disclosed herein, the individual does not develop graft-versus-host (GvH) disease, host-versus-graft (HvG) disease, or a combination thereof.

[0194] The allogeneic cells of the present disclosure are engineered to prevent adverse reactions to transplantation following administration to an individual.The allogeneic cells can be any type of cell.

[0195] In some embodiments of the compositions and methods of the present disclosure, the allogeneic cells are stem cells. In some embodiments, the allogeneic cells are derived from stem cells. Exemplary stem cells include, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), multipotent stem cells, pluripotent stem cells, and hematopoietic stem cells (HSCs).

[0196] In some embodiments of the disclosed compositions and methods, the allogeneic cells are differentiated somatic cells.

[0197] In some embodiments of the compositions and methods of the present disclosure, allogeneic cells are immune cells. In certain embodiments, allogeneic cells are T lymphocytes (T cells). In certain embodiments, allogeneic cells are T cells that do not express one or more components of naturally occurring T cell receptors (TCR). In certain embodiments, allogeneic cells are T cells that express non-naturally occurring antigen receptors. Alternatively or in addition, in certain embodiments, allogeneic cells are T cells that express non-naturally occurring chimeric stimulating receptors (CSR). In certain embodiments, non-naturally occurring CSRs include or consist of switch receptors. In certain embodiments, switch receptors include extracellular domains, transmembrane domains, and intracellular domains. In certain embodiments, the extracellular domains of switch receptors bind to TCR costimulatory molecules and transduce signals to the intracellular space of allogeneic cells, reproducing TCR signaling or TCR costimulatory signaling.

[0198] Chimeric stimulatory receptor (CSR)

[0199] An adoptive cell composition that is "universally" safe for administration to any patient would require significantly reduced or eliminated alloreactivity.

[0200] To this end, the allogeneic cells of the present disclosure are modified to disrupt the expression or function of the T cell receptor (TCR) and / or a class of major histocompatibility complex (MHC). TCR mediates graft-versus-host (GvH) reactions, while MHC mediates host-versus-graft (HvG) reactions. In preferred embodiments, any expression and / or function of the TCR is eliminated in the allogeneic cells of the present disclosure to prevent T cell-mediated GvH, which can cause the death of the individual. Thus, in particularly preferred embodiments, the present disclosure provides a pure TCR-negative allogeneic T cell composition (e.g., each cell of the composition expresses at a level so low as to be undetectable or absent).

[0201] In preferred embodiments, the expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated in the allogeneic cells of the present disclosure to prevent HvG and thereby improve the engraftment of the allogeneic cells of the present disclosure in an individual. The improved engraftment of the allogeneic cells of the present disclosure results in longer cell persistence and, therefore, a larger therapeutic window for the individual. Specifically, the expression and / or function of beta-2 microglobulin (B2M), a structural element of MHC-I, is reduced or eliminated in the allogeneic cells of the present disclosure.

[0202] The above strategy for producing allogeneic cells of the present disclosure has caused further challenges. T cell receptor (TCR) knockout (KO) in T cells leads to the expression loss of CD3-zeta (CD3z or CD3ζ) as a part of TCR complex. The loss of CD3ζ in TCR-KO T cells significantly reduces the ability of these cells to be optimally activated and amplified using standard stimulation / activation reagents (including but not limited to agonist anti-CD3mAb). When the expression or function of any single component of the TCR complex is interrupted, all components of the complex will be lost, including TCR-α (TCRα), TCR-β (TCRβ), CD3-γ (CD3γ), CD3-ε (CD3ε), CD3-δ (CD3δ) and CD3-ζ (CD3ζ). T cell activation and amplification require both CD3ε and CD3ζ. Agonist anti-CD3mAb typically recognizes CD3ε and possible another protein in the complex, which then sends a signal to CD3ζ. CD3ζ provides the primary stimulation (together with secondary co-stimulatory signals) of T cell activation to achieve optimal activation and amplification. Under normal circumstances, full T cell activation depends on the binding of TCR to a second signal mediated by one or more co-stimulatory receptors (e.g., CD28, CD2, 4-1BBL, etc.) that enhances the immune response. However, when TCR is absent, T cell expansion is severely reduced when stimulated using standard activation / stimulation reagents (including agonist anti-CD3 mAbs). In fact, when stimulated using standard activation / stimulation reagents (including agonist anti-CD3 mAbs), T cell expansion is reduced to only 20-40% of normal expansion levels.

[0203] The present disclosure provides chimeric stimulatory receptors (CSRs) to deliver CD3z primary stimulation to allogeneic T cells in the absence of endogenous TCR (and therefore endogenous CD3ζ) when stimulated using standard activation / stimulatory reagents, including agonist anti-CD3 mAbs.

[0204] In the absence of endogenous TCR, the chimeric stimulating receptor (CSR) of the present disclosure provides CD3 ζ stimulation to enhance the activation and amplification of allogeneic T cells. In other words, in the absence of endogenous TCR, when compared to non-allogeneic T cells expressing endogenous TCR, allogeneic cells are rescued from the shortcomings based on activation by the chimeric stimulating receptor (CSR) of the present disclosure. In certain embodiments, the CSR of the present disclosure comprises agonist mAb epitopes extracellularly and CD3 ζ stimulation domains intracellularly, and functionally converts anti-CD28 or anti-CD2 binding events on the surface into CD3z signaling events in allogeneic T cells modified to express CSR. In certain embodiments, CSR comprises wild-type CD28 or CD2 protein and CD3z intracellular stimulation domains to produce CD28z CSR and CD2z CSR respectively. In a preferred embodiment, CD28z CSR and / or CD2z CSR further express non-naturally occurring antigen receptors and / or therapeutic proteins. In a preferred embodiment, non-naturally occurring antigen receptors comprise chimeric antigen receptors.

[0205] The data presented herein demonstrate that the modified allogeneic T cells of the present disclosure comprising / expressing the CSRs of the present disclosure improve or rescue the expansion of allogeneic T cells that no longer express the endogenous TCR, compared to those cells that do not comprise / express the CSRs of the present disclosure.

[0206] The wild-type / native human CD28 protein (NCBI: CD28_HUMAN; UniProt / Swiss-Prot: P10747.1) comprises or consists of the following amino acid sequence:

[0207]

[0208] The nucleotide sequence encoding the wild-type / native CD28 protein (NCBI: CCDS2361.1) comprises or consists of the following nucleotide sequence:

[0209]

[0210] The exemplary CSR CD28z protein of the present disclosure comprises the following (CD28 signal peptide, CD28 extracellular domain, CD28 transmembrane domain , CD28 cytoplasmic domain, CD3z intracellular domain) or consists of the amino acid sequence:

[0211]

[0212]

[0213] CD28 signal peptide:

[0214]

[0215] CD28 extracellular domain:

[0216]

[0217] CD28 transmembrane domain :

[0218]

[0219] CD28 cytoplasmic domain:

[0220]

[0221] CD3z intracellular domain:

[0222]

[0223] An exemplary nucleotide sequence encoding the CSR CD28z protein of the present disclosure comprises the following (CD28 signal peptide, CD28 extracellular domain, CD28 transmembrane domain , CD28 cytoplasmic domain, CD3z intracellular domain) or consists of a nucleotide sequence thereof:

[0224]

[0225] CD28 signal peptide:

[0226]

[0227] CD28 extracellular domain:

[0228]

[0229] CD28 transmembrane domain :

[0230]

[0231]

[0232] CD28 cytoplasmic domain:

[0233]

[0234] CD3z intracellular domain:

[0235]

[0236] The wild-type / native human CD2 protein (NCBI: CD2_HUMAN; UniProt / Swiss-Prot: P06729.2) comprises or consists of the following amino acid sequence:

[0237]

[0238] The nucleotide sequence encoding the wild-type / native CD2 protein (NCBI: CCDS889.1) comprises or consists of the following nucleotide sequence:

[0239]

[0240] The exemplary CSR CD2z protein of the present disclosure comprises the following (CD2 signal peptide, CD2 extracellular domain, CD2 transmembrane domain , CD2 cytoplasmic domain, CD3z intracellular domain) or consists of the amino acid sequence:

[0241]

[0242] CD2 signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 17110)

[0243] CD2 extracellular domain:

[0244]

[0245] CD2 transmembrane domain :IYLIIGICGGSLLMVFVALLVFYIT(SEQ ID NO: 17112)

[0246] CD2 cytoplasmic domain:

[0247]

[0248] CD3z intracellular domain:

[0249]

[0250] The present disclosure provides a non-naturally occurring CSRCD2 protein comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17062. The present disclosure provides a CD2 signal peptide comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17110. The present disclosure provides a CD2 extracellular domain comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17111. The present disclosure provides a CD2 transmembrane domain comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17112. The present disclosure provides a CD2 cytoplasmic domain comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17113. The present disclosure provides a CD3z intracellular domain comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17102.

[0251] An exemplary nucleotide sequence encoding a CSR CD2z protein of the present disclosure comprises the following (CD2 signal peptide, CD2 extracellular domain, CD2 transmembrane domain , CD2 cytoplasmic domain, CD3z intracellular domain) or consists of the amino acid sequence:

[0252]

[0253]

[0254] CD2 signal peptide:

[0255]

[0256] CD2 extracellular domain:

[0257]

[0258] CD2 transmembrane domain :

[0259]

[0260] CD2 cytoplasmic domain:

[0261]

[0262] CD3z intracellular domain:

[0263]

[0264] The exemplary mutant CSR CD2z-D111H protein of the present disclosure comprises the following (CD2 signal peptide, Outside CD2 cells Within the domain have D111H mutation CD2 extracellular domain, CD2 transmembrane domain , CD2 cytoplasmic domain, CD3z intracellular domain) or consists of the amino acid sequence:

[0265]

[0266]

[0267] CD2 signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 17110)

[0268] In the CD2 extracellular domain have D111H mutation CD2 extracellular domain:

[0269]

[0270] CD2 transmembrane domain :

[0271]

[0272] CD2 cytoplasmic domain:

[0273]

[0274] CD3z intracellular domain:

[0275]

[0276] The present disclosure provides a non-naturally occurring CSRCD2 protein comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17118. The present disclosure provides a CD2 extracellular domain comprising, consisting essentially of, or consisting of an amino acid sequence having 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% identity to SEQ ID NO: 17119.

[0277] An exemplary nucleotide sequence encoding a mutant CSR CD2z-D111H protein of the present disclosure comprises the following (CD2 signal peptide, In the CD2 extracellular domain have D111H mutation CD2 extracellular domain, CD2 transmembrane domain , CD2 cytoplasmic domain, CD3z intracellular domain) or consists of the amino acid sequence:

[0278]

[0279]

[0280] CD2 signal peptide:

[0281]

[0282] In the CD2 extracellular domain have D111H mutation CD2 extracellular domain:

[0283]

[0284] CD2 transmembrane domain :

[0285]

[0286] CD2 cytoplasmic domain:

[0287]

[0288] CD3z intracellular domain:

[0289]

[0290] Endogenous TCR knockout

[0291] The 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 eliminate the expression of endogenous T cell receptors of allogeneic cells of the present disclosure. In preferred embodiments, the gene editing compositions of the present disclosure target and delete the following: a gene encoding an endogenous T cell receptor of an allogeneic cell of the present disclosure, a portion of a gene, or a regulatory element (such as a promoter) of a gene.

[0292] Non-limiting examples of primers (including T7 promoter, genomic target sequence, and gRNA backbone) used to generate guide RNA (gRNA) templates that target and delete TCR-alpha (TCR-α) are provided in Table 10.

[0293] Table 10. Underlined target sequences

[0294]

[0295]

[0296] Non-limiting examples of primers used to generate guide RNA (gRNA) templates that target and delete TCR-beta (TCR-β) are provided in Table 11.

[0297] Table 11. Underlined target sequences

[0298]

[0299]

[0300] Non-limiting examples of primers used to generate guide RNA (gRNA) templates that target and delete Beta-2 microglobulin (β2M) are provided in Table 12.

[0301] Table 12. Underlined target sequences

[0302]

[0303]

[0304] Endogenous MHC knockout

[0305] The 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 eliminate the expression of endogenous MHC I, MHC II, 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 the following: genes encoding one or more components of endogenous MHC I, MHC II, or MHC activators of allogeneic cells of the present disclosure, a portion of a gene, or a regulatory element (such as a promoter) of a gene.

[0306] Non-limiting examples of guide RNAs (gRNAs) that target and delete MHC activators are provided in Tables 13 and 14.

[0307] Table 13.

[0308]

[0309] Table 14.

[0310]

[0311]

[0312] Engineered HLA-E compositions

[0313] MHCI knockout (KO) renders cells resistant to T cell killing but also makes them susceptible to natural killer (NK) cell-mediated cytotoxicity (the "loss of self hypothesis") (see Figure 30 ). It is assumed that NK rejection will reduce the in vivo efficacy and / or persistence of these KO cells in a therapeutic setting such as allogeneic (allo) CAR-T therapy. As observed in classic mixed lymphocyte reaction (MLR) experiments, retaining MHCI on the surface of allogeneic CAR-T cells will make them susceptible to killing by host T cells. It is estimated that up to 10% of a person's T cells are specific for foreign MHC, which mediates rejection of foreign cells and tissues. Targeted KO of MHCI, especially HLA-A, B and C, can be achieved through targeted KO of B2M, causing the loss of other HLA molecules (including HLA-E). For example, due to the "loss of self hypothesis", the loss of HLA-E makes KO cells more susceptible to NK cell-mediated cytotoxicity. NK-mediated cytotoxicity against cells that have lost their self is a defense mechanism against pathogens that downregulate the MHC on the surface of infected cells, thereby escaping detection and killing by cells of the adaptive immune system.

[0314] The present disclosure contemplates two strategies for engineering allogeneic (MHCI-negative) T cells (including CAR-T cells) that are more resistant to NK cell-mediated cytotoxicity. In some embodiments, sequences encoding molecules that reduce or prevent NK killing (e.g., single-chain HLA-E) are introduced or delivered to allogeneic cells. Alternatively or in addition, the gene editing methods of the present disclosure retain certain endogenous HLA molecules (e.g., endogenous HLA-E). For example, the first method involves the introduction of single-chain (sc) HLA-E molecules into B2M KO T cells. (PB) delivery.

[0315] The second approach uses a gene-editing composition with guide RNA that is selective for HLA-A, HLA-B, and HLA-C, but not for, for example, HLA-E or other molecules that protect MHC IKO cells from natural killer cell-mediated cytotoxicity.

[0316] 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 (FNAR2), 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 hemagglutinins, CD48, LLT1 (also known as C-type lectin domain family 2 member (CLC2D)), ULBP2, ULBP3, and sMICA or variants thereof.

[0317] Exemplary CD47 proteins of the present disclosure include the following ( signal peptide , extracellular, TM, cytoplasmic) amino acid sequence or consisting of:

[0318]

[0319] Exemplary INFAR1 proteins of the present disclosure include the following ( signal peptide , extracellular, TM, cytoplasmic) amino acid sequence or consisting of:

[0320]

[0321]

[0322] Exemplary INFAR2 proteins of the present disclosure include the following ( signal peptide , extracellular, TM, cytoplasmic) amino acid sequence or consisting of:

[0323]

[0324] Exemplary HLA-G1 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 ) or consisting of the amino acid sequence:

[0325]

[0326] Exemplary HLA-G2 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 ) or consisting of the amino acid sequence:

[0327]

[0328] Exemplary HLA-G3 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 ) or consisting of the amino acid sequence:

[0329]

[0330] Exemplary HLA-G4 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 ) or consisting of the amino acid sequence:

[0331]

[0332] Exemplary HLA-G5 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 , intron 4) or consisting of the amino acid sequence:

[0333]

[0334]

[0335] Exemplary HLA-G5 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 , intron 4) or consisting of the amino acid sequence:

[0336]

[0337] Exemplary HLA-G5 proteins of the present disclosure comprise the following (α chain 1, α chain 2, α chain 3 , intron 2) or consisting of the amino acid sequence:

[0338]

[0339] Exemplary CEACAM1 proteins of the present disclosure include the following (extracellular, TM, cytoplasm ) or consisting of the amino acid sequence:

[0340]

[0341] Exemplary viral hemagglutinin proteins of the present disclosure comprise or consist of the amino acid sequence of HA of influenza A virus (A / New Caledonia / 20 / 1999 (H1N1); TM):

[0342]

[0343] Exemplary CD48 proteins of the present disclosure include the following ( signal peptide , chains, and propeptide removed in their mature form) or consisting of an amino acid sequence:

[0344]

[0345] Exemplary LLT1 proteins of the present disclosure comprise the following (cytoplasmic, TM, Extracellular ) or consisting of the amino acid sequence:

[0346]

[0347]

[0348] An exemplary ULBP2 protein of the present disclosure comprises or consists of the amino acid sequence of:

[0349]

[0350] An exemplary ULBP3 protein of the present disclosure comprises or consists of the amino acid sequence of:

[0351]

[0352] Exemplary sMICA proteins of the present disclosure include the following ( signal peptide , the extracellular domain, TM and cytoplasmic domains ) or consisting of the amino acid sequence of (GenBank Accession No. Q29983):

[0353]

[0354] Exemplary sMICA proteins of the present disclosure include the following ( α-1 , α-2, α-3) amino acid sequence or consisting thereof:

[0355]

[0356] Exemplary sMICA proteins of the present disclosure comprise the following (signal peptide; α-1, α-2, α-3) amino acid sequence or consisting thereof:

[0357]

[0358] Exemplary sMICA proteins of the present disclosure comprise or consist of the following amino acid sequence (signal peptide):

[0359]

[0360] Exemplary bGBE trimers of the present disclosure ( 270G and 484S) proteins comprising or consisting of the following amino acid sequence:

[0361]

[0362]

[0363] Exemplary bGBE trimers of the present disclosure ( 270G and 484S) protein comprises or consists of the following nucleic acid sequence:

[0364]

[0365] Exemplary bGBE trimers of the present disclosure ( 270R and 484S) proteins comprising or consisting of the following amino acid sequence:

[0366]

[0367] Exemplary bGBE trimers of the present disclosure ( 270R and 484S) protein comprises or consists of the following nucleic acid sequence:

[0368]

[0369]

[0370] Exemplary gBE dimers of the present disclosure ( R and S) protein comprises or consists of the following amino acid sequence:

[0371]

[0372] Exemplary gBE dimers of the present disclosure ( R and S) protein comprises or consists of the following nucleic acid sequence:

[0373]

[0374]

[0375] Exemplary gBE dimer (G and S) proteins of the present disclosure comprise or consist of the following amino acid sequence:

[0376]

[0377] Exemplary gBE dimers of the present disclosure ( G and S) protein comprises or consists of the following amino acid sequence:

[0378]

[0379] 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:

[0380]

[0381] The nucleotide sequence encoding the wild-type / natural HLA-E protein (NCBI: CCDS34379.1) comprises or consists of the following nucleotide sequence:

[0382]

[0383]

[0384] Exemplary WT HLA-E monomers of the present disclosure ( R and S) protein comprises or consists of the following amino acid sequence:

[0385]

[0386] Exemplary WT HLA-E monomers of the present disclosure ( R and S) protein comprises or consists of the following nucleic acid sequence:

[0387]

[0388] Exemplary WT HLA-E monomers of the present disclosure ( G and S) protein comprises or consists of the following nucleic acid sequence:

[0389]

[0390] Exemplary WT HLA-E monomers of the present disclosure ( G and S) protein comprises or consists of the following nucleic acid sequence:

[0391]

[0392]

[0393] The wild-type / native human B2M protein (NCBI: B2MG_HUMAN; UniProt / Swiss-Prot: P61769.1) comprises or consists of the following amino acid sequence:

[0394]

[0395] The nucleotide sequence encoding the wild-type / native B2M protein (NCBI: CCDS10113.1) comprises or consists of the following nucleotide sequence:

[0396]

[0397] The exemplary HLA-bGBE (single chain trimer) protein of the present disclosure comprises the following (B2M signal peptide, peptide, Linker , B2M domain, connector, HLA-E peptide ) or consisting of the amino acid sequence:

[0398]

[0399] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126)

[0400] Peptide: VMAPRTLIL (SEQ ID NO: 17127)

[0401] Linker :GGGGSGGGGSGGGGS (SEQ ID NO: 17128)

[0402] B2M domain:

[0403]

[0404] Linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130)

[0405] HLA-E peptides:

[0406]

[0407]

[0408] An exemplary nucleotide sequence encoding the HLA-bGBE (single-chain trimer) protein of the present disclosure comprises the following (B2M signal peptide, peptide, Linker , B2M domain, connector, HLA-E peptide ) or consists of a nucleotide sequence thereof:

[0409]

[0410] B2M signal peptide:

[0411]

[0412] Peptide: GTGATGGCCCCCCGGACCCTGATCCTG (SEQ ID NO: 17133)

[0413] Linker : GGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCT (SEQ ID NO: 17134)

[0414] B2M domain

[0415]

[0416] Linker:

[0417]

[0418] HLA-E peptide :

[0419]

[0420]

[0421] The exemplary HLA-gBE (single chain dimer) protein of the present disclosure comprises the following (B2M signal peptide, B2M domain, connect The amino acid sequence of a peptide (e.g., HLA-E peptide) or consisting of:

[0422]

[0423] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126)

[0424] B2M domain:

[0425]

[0426] Linker :GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17130)

[0427] HLA-E peptides:

[0428]

[0429] An exemplary nucleotide sequence encoding the HLA-gBE (single-chain dimer) protein of the present disclosure comprises the following (B2M signal peptide, B2M domain, Linker , HLA-E peptide) or consists of the nucleotide sequence:

[0430]

[0431]

[0432] B2M signal peptide:

[0433]

[0434] B2M domain:

[0435]

[0436] Linker :

[0437] GGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCT (SEQ IDNO: 17136)

[0438] HLA-E peptides:

[0439]

[0440] An exemplary HLA-bE (monomeric) protein of the present disclosure comprises or consists of the following amino acid sequence (B2M signal peptide, HLA-E peptide):

[0441]

[0442] B2M signal peptide: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 17126)

[0443] HLA-E peptides:

[0444]

[0445] An exemplary nucleotide sequence encoding the HLA-bE (monomeric) protein of the present disclosure comprises or consists of the following nucleotide sequence (B2M signal peptide, HLA-E peptide):

[0446]

[0447] B2M signal peptide:

[0448]

[0449] HLA-E peptides:

[0450]

[0451] Immune and immune precursor cells

[0452] In certain embodiments, the immune cells of the present disclosure include lymphocyte progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (T cells), SCM cells), central memory T cells (T CM ), stem-like T cells, B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[0453] In certain embodiments, immune precursor cells include any cells 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 (HSC) or their offspring. In certain embodiments, immune precursor cells include precursor cells that can self-develop into immune cells. In certain embodiments, immune precursor cells include hematopoietic progenitor cells (HPC).

[0454] Hematopoietic stem cells (HSC)

[0455] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages originate from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0456] The HSCs of the present disclosure may be isolated or derived from primary or cultured stem cells. The HSCs of the present disclosure may be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0457] Immune precursor cells of the present disclosure may comprise 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 lymphocytes (T cells), B lymphocytes (B cells), bone marrow progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0458] HSCs produced by the methods of the present disclosure can retain the characteristics of "primitive" stem cells: despite being isolated or derived from adult stem cells and despite being committed to a single lineage, they share characteristics of embryonic stem cells. For example, "primitive" HSCs produced by the methods of the present disclosure retain their "stemness" after division and do not differentiate. Therefore, as an adoptive cell therapy, "primitive" HSCs produced by the methods of the present disclosure not only replenish their numbers but also expand in vivo. When administered as a single dose, "primitive" HSCs produced by the methods of the present disclosure can be therapeutically effective. 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, a majority of primitive HSCs of the present disclosure are CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0459] In some embodiments of the present disclosure, the original HSC, HSC and / or HSC progeny cells can be modified according to the methods of the present disclosure to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). In some embodiments of the present disclosure, the modified original HSC, modified HSC and / or modified HSC progeny cells can be forward differentiated to produce the modified immune cells of the present disclosure, including but not limited to modified T cells, modified natural killer cells and / or modified B cells.

[0460] T cells

[0461] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0462] Unlike traditional biological agents and chemotherapeutic agents, the modified T cells of the present disclosure have the ability to rapidly reproduce after antigen recognition, thereby potentially avoiding the need for repeated treatment. To achieve this, in some embodiments, the modified T cells of the present disclosure not only drive the initial response, but also persist in the patient as a stable population of surviving memory T cells to prevent potential relapse. Alternatively, in some embodiments, the modified T cells of the present disclosure do not persist in the patient's body when not needed.

[0463] Efforts have focused on developing antigen receptor molecules that do not induce T cell exhaustion through antigen-dependent (tonic) signaling, and on the development of novel targets for early memory T cells, particularly stem cell memory (T SCM) or modified T cell products of stem cell-like T cells. The stem cell-like modified T cells of the present disclosure exhibit the greatest self-renewal capacity and multipotency to derive central memory (T CM )T cells or T CM Like cells, effector memory (T EM ) and effector T cells (TE), resulting in better tumor eradication and long-term modified T cell engraftment. A linear pathway of differentiation may be responsible for the generation of these cells: naive T cells (T N )>T sCM >T CM >T EM >T E >T TE , where T N To directly generate T SCM parental precursor cells, which in turn directly give rise to T CM The composition of the T cells of the present disclosure may include one or more of each parent T cell subset, wherein T sCM cells are the most abundant (e.g. T SCM >T CM >T EM >T E＞ T TE ).

[0464] In some embodiments of the methods of the present disclosure, immune cell precursors differentiate into or are capable of differentiating into early memory T cells, stem cell-like T cells, naive T cells (T cells), 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.

[0465] In some embodiments of the methods of the present disclosure, the immune cells are early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM 、T CM ,TEM,T E or T TE .

[0466] In some embodiments of the disclosed methods, the immune cells are early memory T cells.

[0467] In some embodiments of the disclosed methods, the immune cells are stem-like T cells.

[0468] In some embodiments of the methods of the present disclosure, the immune cells are T SCM .

[0469] In some embodiments of the methods of the present disclosure, the immune cells are T CM .

[0470] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of modified T cells express one or more cell surface markers of early memory T cells. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified stem cell-like T cell. In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T cell. SCM In certain embodiments, the plurality of modified early memory T cells comprises at least one modified T CM .

[0471] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween of the plurality of modified T cells express one or more cell surface markers of stem-like T cells. In certain embodiments, the plurality of modified stem-like T cells comprises at least one modified T cell. SCM In certain embodiments, the plurality of modified stem-like T cells comprises at least one modified T CM .

[0472] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of modified T cells express stem memory T cells (T SCM ). In certain embodiments, the cell surface markers include one or more cell surface markers of IL-2Rβ. 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.

[0473] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of modified T cells express central memory T cells (T CM ). In certain embodiments, the cell surface markers comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0474] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of modified T cells express a naive T cell (T N ). In certain embodiments, the cell surface markers comprise one or more of CD45RA, CCR7, and CD62L.

[0475] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween of the plurality of modified T cells express effector T cells (modified T cells). EFF In certain embodiments, the cell surface markers comprise one or more of CD45RA, CD95, and IL-2Rβ.

[0476] In some embodiments of the methods of the present disclosure, the methods modify and / or the methods generate a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage therebetween of the plurality of modified T cells express stem cell-like T cells, stem memory T cells (T cells), or any combination thereof. SCM ) or central memory T cells (T CM )'s cell surface markers.

[0477] In some embodiments of the methods of the present disclosure, the buffer comprises an immune cell or a precursor thereof. The buffer maintains or enhances the cell viability and / or stem-like phenotype level of the immune cell or its precursor (including T cells). In certain embodiments, before nuclear transfection, the buffer maintains or enhances the cell viability and / or stem-like phenotype level of nascent human T cells. In certain embodiments, during nuclear transfection, the buffer maintains or enhances the cell viability and / or stem-like phenotype level of nascent human T cells. In certain embodiments, after nuclear transfection, the buffer maintains or enhances the cell viability and / or stem-like phenotype level of nascent human T cells. In certain embodiments, the buffer comprises any absolute or relative abundance or concentration of KCl, MgCl2, ClNa, glucose, and Ca(NO3)2, and optionally, the buffer further comprises a supplement selected from the group consisting of: HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer comprises 5mM KCl, 15mM MgCl2, 90mM ClNa, 10mM glucose, and 0.4mM Ca(NO3)2. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, and a supplement comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl2, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO3)2, and a supplement comprising 40 mM Na2HPO4 / NaH2PO4 at a pH of 7.2. In certain embodiments, the composition comprising primary human T cells comprises 100 μl of buffer and 5×10 6 to 25×10 6 In certain embodiments, during the introducing step, the composition comprises a scalable 250×10 6 100 primary human T cells / mL buffer or other culture medium.

[0478] In some embodiments of the methods of the present disclosure, the methods include contacting the 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 embodiments in which the introducing step of the method includes an electroporation or nucleofection step, the electroporation or nucleofection step may be performed while the immune cells are in contact with the T cell expansion composition of the present disclosure.

[0479] In some embodiments of the methods of the present disclosure, 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.

[0480] In certain embodiments of the method for producing the modified T cells of the present disclosure, the expansion supplement comprises one or more cytokines. One or more cytokines may comprise 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-γ (INFγ). One or more cytokines may comprise IL-2.

[0481] In some embodiments of the methods of the present disclosure, the T cell expansion composition comprises human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement. In certain embodiments of this method, the T cell expansion composition further comprises one or more of the following: octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-phthalic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterols, and alkanes. In certain embodiments of this method, the T cell expansion composition further comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols. In certain embodiments of this method, the T cell expansion composition further comprises one or more of the following: 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). In certain embodiments of this method, the T cell expansion composition further comprises one or more of the following: 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. In certain embodiments of this method, the T cell expansion composition further comprises one or more of the following: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg (inclusive); palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg (inclusive); linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg (inclusive). In certain embodiments of this method, the T cell expansion composition further comprises one or more of the following: 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.

[0482] In certain embodiments, the T cell expansion composition comprises one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement to produce a plurality of expanded modified T cells, wherein at least 2% of the plurality of modified T cells express early memory T cells, stem cell-like T cells, stem memory T cells (T cells), or a combination of these. 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 the following: octanoic acid, nicotinamide, 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD), diisopropyl adipate (DIPA), n-butyl-benzenesulfonamide, 1,2-phthalic acid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid, stearic acid hydrazide, oleamide, sterols, and alkanes. In certain embodiments, the T cell expansion composition comprises one or more of octanoic acid, palmitic acid, linoleic acid, oleic acid, and sterols (e.g., cholesterol). In certain embodiments, the T cell expansion composition comprises one or more of the following: 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). In certain embodiments, the T cell expansion composition comprises one or more of the following: 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 (where mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises one or more of the following: 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 (where mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises 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 (where mg / kg = parts per million). In certain embodiments, the T cell expansion composition comprises one or more of the following: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg (inclusive); palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg (inclusive); linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg (inclusive).In certain embodiments, the T cell expansion composition comprises one or more of the following: 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. In certain embodiments, the T cell expansion composition comprises one or more of the following: 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 sterols 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 about 7.56 μmol / kg, and sterols at a concentration of about 2.61 μmol / kg.

[0483] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement at 37°C. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of phosphorus, octanoic fatty acid, palmitic fatty acid, linoleic fatty acid, and oleic acid. In certain embodiments, the culture medium comprises phosphorus in an amount 10-fold greater than that found, for example, in Iscove's Modified Dulbecco's Medium (IMDM; available from ThermoFisher Scientific as catalog number 12440053).

[0484] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Igcove'g IMDM, and an expansion supplement at 37°C. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of the following elements: boron, sodium, magnesium, phosphorus, potassium, and calcium. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising 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.

[0485] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement at 37° C. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622- 84-2), 1,2-phthalic 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), sterols (such as cholesterol) (CAS No. 57-88-5) and alkanes (such as nonadecane) (CAS No. 629-92-5). In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1, 2-Benzenedicarboxylic acid, bis(2-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), 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" may be used interchangeably with a culture medium comprising one or more of the following ingredients: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9), n-butyl-benzene Sulfonamide (CAS No. 3622-84-2), 1,2-phthalic 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.

[0486] In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement at 37° C. Alternatively or additionally, the terms "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of the following ions: sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate, and phosphate.

[0487] As used herein, the terms "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, and an expansion supplement at 37° C. Alternatively or additionally, the terms "supplemented T cell expansion composition" or "T cell expansion composition" may be used interchangeably with a culture medium comprising one or more of the following free amino acids: histidine, asparagine, serine, glutamic acid, 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" are used interchangeably with culture medium comprising the corresponding average molar percentages of one or more of the following free amino acids: histidine (about 1%), asparagine (about 0.5%), serine (about 1.5%), glutamate (about 67%), arginine (about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamate (about 2%), threonine (about 2%), alanine (about 1%), proline (about 1.5%), cysteine ​​(about 1.5%), lysine (about 3%), tyrosine (about 1.5%), methionine (about 1%), valine (about 3.5%), isoleucine (about 3%), leucine (about 3.5%), phenylalanine (about 1.5%), and tryptophan (about 0.5%). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" are used interchangeably with culture medium comprising corresponding average molar percentages of one or more of the following free amino acids: histidine (about 0.78%), asparagine (about 0.4%), serine (about 1.6%), glutamate (about 67.01%), arginine (about 1.67%), glycine (about 1.72%), aspartic acid (about 1.00%), glutamate (about 1.93%), threonine (about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine ​​(about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine (about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine (about 3.3%), phenylalanine (about 1.64%), and tryptophan (about 0.37%).

[0488] As used herein, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of human serum albumin, recombinant human insulin, human transferrin, 2-mercaptoethanol, Iscove's IMDM, and an expansion supplement at 37°C. Alternatively or additionally, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture medium comprising one or more of phosphorus, octanoic fatty acid, palmitic fatty acid, linoleic fatty acid, and oleic acid. In certain embodiments, the culture medium comprises phosphorus in an amount 10-fold greater than that found, for example, in Iscove's Modified Dulbecco's Medium (IMDM; available at ThermoFisher Scientific as catalog number 12440053).

[0489] In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" can be used interchangeably with a culture 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" can be used interchangeably with a culture medium comprising one or more of: octanoic acid at a concentration of 0.9 mg / kg to 90 mg / kg (inclusive); palmitic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); linoleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); oleic acid at a concentration of 0.2 mg / kg to 20 mg / kg (inclusive); and sterols at a concentration of about 0.1 mg / kg to 10 mg / kg (inclusive) (where 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 culture medium comprising one or more of the following: 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 (wherein 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 culture medium comprising one or more of the following: 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 (wherein mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" are used interchangeably with a culture medium comprising one or more of the following: 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 (where mg / kg = parts per million). In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" are used interchangeably with a culture medium comprising one or more of: octanoic acid at a concentration of 6.4 μmol / kg to 640 μmol / kg (inclusive); palmitic acid at a concentration of 0.7 μmol / kg to 70 μmol / kg (inclusive); linoleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); oleic acid at a concentration of 0.75 μmol / kg to 75 μmol / kg (inclusive); and sterols at a concentration of 0.25 μmol / kg to 25 μmol / kg (inclusive).In certain embodiments, the terms "supplemented T cell expansion composition" or "T cell expansion composition" are used interchangeably with a culture medium comprising one or more of the following: 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.

[0490] In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" is used interchangeably with a culture medium comprising one or more of the following: 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 sterols at a concentration of about 2.61 μmol / kg. In certain embodiments, the term "supplemented T cell expansion composition" or "T cell expansion composition" is used interchangeably with a culture medium comprising one or more of the following: 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 sterols at a concentration of 2.61 μmol / kg.

[0491] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the method of the present invention, the method comprises contacting the modified T cells with an inhibitor of the P13K-Akt-mTOR pathway. The modified T cells of the present invention include the modified stem cell-like T cells, T cells, and T cells of the present invention. sCM and / or T CM The cells may be incubated, cultured, grown, stored, or otherwise combined with a growth medium containing one or more inhibitors of components of the PI3K pathway at any step in the method of the procedure. Exemplary inhibitors of components of the PI3K pathway include, but are not limited to, GSK3β inhibitors, such as TWS119 (also known as GSK 3B inhibitor XII; CAS No. 601514-19-6, having the chemical formula C 18 H 14 N4O2). Exemplary inhibitors of components of the PI3K pathway include, but are not limited to, bb007 (BLUEBIRDBIO TMAdditional exemplary inhibitors of components of the PI3K pathway include, but are not limited to, allosteric Akt inhibitor VIII (also known as Akti-1 / 2, with 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 (-)-barano), 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-674563, and A-443654), phenylpyrazole derivatives (AT7867 and AT13148), thiophene carboxamide derivatives (Afuresertib (GSK2110183), 2-pyrimidinyl-5-amidothiophene derivatives (DC120), uprosertib (GSK2141795)), allosteric inhibitors (superior to orthosteric inhibitors, providing greater specificity, reduced side effects and lower toxicity), 2,3-diphenylquinoxaline analogs (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivatives (MK-2206)), alkyl phospholipids (Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3), imofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphocholine), indole-3-carbinol analogs (indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindole[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 analogs (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivatives API-1,3-phenyl-3H-imidazo[4,5-b]pyridine derivatives, ARQ 092), BAY1125976, 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucallicacid, 3α- and 3β-acetoxy-tirucallicacid, acetoxy-tirucallicacid and irreversible inhibitors (antibiotics, lactoquinomycin, frenolicin B, carafungin, medamicin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives and imidazo-1,2-pyridine derivatives). .

[0492] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the method of claim 1, the method comprises contacting the modified T cell with an inhibitor of T cell effector differentiation. Exemplary inhibitors of T cell effector differentiation include, but are not limited to, BET inhibitors (e.g., JQ1, thienotriazolidine) and / or inhibitors of the BET protein family (e.g., BRD2, BRD3, BRD4, and BRDT).

[0493] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the method of claim 1 , the method comprises contacting the modified T cell with an agent that reduces nucleoplasmic acetyl-CoA. Exemplary agents that reduce nucleoplasmic acetyl-CoA include, but are not limited to, 2-hydroxycitrate (2-HC) and agents that increase Acss1 expression.

[0494] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the method of claim 1, the method comprises contacting the modified T cells with 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).

[0495] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM ), the activation supplement may comprise one or more cytokines. The one or more cytokines may comprise 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-γ (INFγ). The one or more cytokines may comprise IL-2.

[0496] In the present disclosure, modified T cells (e.g., stem cell-like T cells, T SCM and / or T CM In certain embodiments of the methods of the present invention, the activation supplement may comprise one or more activator complexes. Exemplary and non-limiting activator complexes may comprise monomeric, dimeric, trimeric, or tetrameric antibody complexes that bind to one or more of CD3, CD28, and CD2. In some embodiments, the activation supplement comprises or consists of an activator complex comprising a human, humanized, recombinant, or chimeric antibody. In some embodiments, the activation supplement comprises or consists of an activator complex that binds to CD3 and CD28. In some embodiments, the activation supplement comprises or consists of an activator complex that binds to CD3, CD28, and CD2.

[0497] Natural killer (NK) cells

[0498] In certain embodiments, the modified immune or immune precursor cells of the present disclosure are natural killer (NK) cells. In certain embodiments, NK cells are cytotoxic lymphocytes other than lymphocyte progenitor cells.

[0499] The modified NK cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0500] In certain embodiments, non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0501] In certain embodiments, NK cells are electroporated using a Lonza 4D Nucleofector or a BTX ECM 830 (500 V, 700 usec pulse length, 0.2 mm electrode gap, one pulse). All Lonza 4D Nucleofector procedures are considered to be within the scope of the methods of the present disclosure.

[0502] In certain embodiments, 5×10E6 cells were electroporated per electroporation in 100 μL of P3 buffer in a cuvette. However, this cell / volume ratio is scalable for commercial manufacturing processes.

[0503] In certain embodiments, NK cells are stimulated by co-culturing with another cell line. In certain embodiments, the other cell line comprises artificial antigen presenting cells (aAPC). In certain embodiments, stimulation occurs on day 1, 2, 3, 4, 5, 6, or 7 after electroporation. In certain embodiments, stimulation occurs on day 2 after electroporation.

[0504] In certain embodiments, NK cells express CD56.

[0505] B cells

[0506] In certain embodiments, the modified immune or immune precursor cells of the present disclosure are B cells. B cells are a type of lymphocyte that express a B cell receptor on the cell surface. The B cell receptor binds to a specific antigen.

[0507] The modified B cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0508] 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.

[0509] In certain embodiments, after initiation, the modified HSPC cells are transferred to a feeder cell layer and fed once every two weeks while being transferred to a fresh feeder layer once a week. In certain embodiments, the feeder cells are MS-5 feeder cells.

[0510] 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.

[0511] Inducible apoptosis-promoting peptide

[0512] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides disclosed herein are much less immunogenic. Although the inducible pro-apoptotic polypeptides disclosed herein are recombinant polypeptides and therefore non-naturally occurring, the sequences recombined to produce the inducible pro-apoptotic polypeptides disclosed herein do not contain non-human sequences that the host human immune system can recognize as "non-self" and therefore induce an immune response in an individual that receives the inducible pro-apoptotic polypeptides disclosed herein, cells comprising the inducible pro-apoptotic polypeptides, or compositions comprising the inducible pro-apoptotic polypeptides or cells comprising the inducible pro-apoptotic polypeptides.

[0513] The present disclosure provides inducible pro-apoptotic polypeptides comprising a ligand binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not contain non-human sequences. In certain embodiments, the non-human sequence contains restriction sites. 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 polypeptides of the present disclosure may be non-naturally occurring.

[0514] The 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. The caspase polypeptides of the present disclosure include, but are not limited to, those caspase polypeptides associated with apoptosis, including caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. The caspase polypeptides of the present disclosure include, but are not limited to, those caspase polypeptides that trigger apoptosis, including caspase 2, caspase 8, caspase 9, and caspase 10. The caspase polypeptides of the present disclosure include, but are not limited to, those caspase polypeptides that execute apoptosis, including caspase 3, caspase 6, and caspase 7.

[0515] The caspase polypeptides of the present invention 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 polypeptides of the present invention may be codon-optimized. One or more modifications to the amino acid and / or nucleic acid sequence of the caspase polypeptides of the present invention may increase interaction, cross-linking, cross-activation, or activation of the caspase polypeptides of the present invention compared to the wild-type amino acid or nucleic acid sequence. Alternatively or additionally, one or more modifications to the amino acid and / or nucleic acid sequence of the caspase polypeptides of the present invention may reduce the immunogenicity of the caspase polypeptides of the present invention compared to the wild-type amino acid or nucleic acid sequence.

[0516] Compared to wild-type caspase polypeptides, the caspase polypeptides of the present invention may be truncated. For example, the caspase polypeptides may be truncated to eliminate sequences encoding the caspase activation and recruitment domain (CARD) to eliminate or minimize the possibility of activating a local inflammatory response and inducing apoptosis in cells containing the inducible caspase polypeptides of the present invention. The nucleic acid sequence encoding the caspase polypeptides of the present invention may be spliced ​​to form variant amino acid sequences of the caspase polypeptides of the present invention compared to the wild-type caspase polypeptide. The caspase polypeptides of the present invention may be encoded by recombinant and / or chimeric sequences. The recombinant and / or chimeric caspase polypeptides of the present invention may include sequences from one or more different caspase polypeptides. Alternatively or in addition, the recombinant and / or chimeric caspase polypeptides of the present invention may include sequences from one or more species (e.g., human and non-human sequences). The caspase polypeptides of the present invention may be non-naturally occurring.

[0517] The ligand binding region of the inducible pro-apoptotic polypeptides of the present disclosure may include any polypeptide sequence that facilitates or promotes the dimerization of the first inducible pro-apoptotic polypeptide of the present disclosure and the second inducible pro-apoptotic polypeptide of the present disclosure, said dimerization activating or inducing cross-linking of the pro-apoptotic polypeptides and initiation of cell apoptosis.

[0518] The ligand binding ("dimerization") region may comprise any polypeptide or functional domain thereof that will allow induction using an endogenous or non-naturally occurring ligand (i.e., and inducer), such as a non-naturally occurring synthetic ligand. Depending on the nature of the inducible pro-apoptotic polypeptide and the choice of ligand (i.e., inducer), the ligand binding region may be inside or outside the cell membrane. A variety of ligand-binding polypeptides and their functional domains are known, including receptors. The ligand binding region of the present disclosure may include one or more sequences from a receptor. Of particular interest are ligand binding regions for which ligands (e.g., small organic ligands) are known or can be readily produced. These ligand binding regions or receptors may include, but are not limited to, FKBP and cyclophilin receptors, steroid receptors, tetracycline receptors, and the like, as well as "non-naturally occurring" receptors that may be obtained from antibodies, particularly heavy or light chain subunits, mutant sequences thereof, random amino acid sequences obtained by random procedures, combinatorial synthesis, and the like. 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.

[0519] The ligand binding region comprising one or more receptor domains can be at least about 50 amino acids, and less than about 350 amino acids, typically less than 200 amino acids, as an active portion of an endogenous domain or a truncation thereof. The binding region can, for example, be small (<25 kDa, to allow efficient transfection in viral vectors), monomeric, non-immunogenic, with a synthetically accessible, cell-permeable, non-toxic ligand that can be configured for dimerization.

[0520] Depending on the design of the inducible pro-apoptotic polypeptide and the availability of an appropriate ligand (i.e., an inducer), the ligand binding region comprising one or more receptor domains may be intracellular or extracellular. For hydrophobic ligands, the binding region may be on either side of the membrane, but for hydrophilic ligands, particularly protein ligands, the binding region will generally be outside the cell membrane unless there is a transport system for internalizing the ligand in a form in which the ligand is available for binding. For intracellular receptors, the inducible pro-apoptotic polypeptide or a transposon or vector comprising the inducible pro-apoptotic polypeptide may encode a signal peptide and a transmembrane domain at 5' or 3' of the receptor domain sequence, or may have a lipid attachment signal sequence at 5' of the receptor domain sequence. When the receptor domain is between the signal peptide and the transmembrane domain, the receptor domain will be extracellular.

[0521] Antibodies and antibody subunits, such as heavy or light chains, specifically fragments, more specifically all or part of the variable region, or fusions of heavy and light chains to produce high-affinity binding can be used as ligand binding regions of the present disclosure. Contemplated antibodies include antibodies that are ectopically expressed human products, such as extracellular domains that do not trigger an immune response and are not typically 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). In addition, antibodies can be prepared against physiologically acceptable hapten molecules, and individual antibody subunits can be screened for binding affinity. The cDNA encoding the subunit can be isolated and modified by deleting a portion of the constant region, a portion of the variable region, mutagenizing the variable region, etc., to obtain a binding protein domain with appropriate affinity for the ligand. In this way, almost any physiologically acceptable hapten compound can be used as a ligand or provide an epitope for the ligand. Instead of an antibody unit, an endogenous receptor can be used, where the binding region or binding domain is known and there is an applicable or known binding ligand.

[0522] In order to allow the receptor to multimerize, the ligand of the ligand binding region / receptor domain of the inducible pro-apoptotic polypeptide can be a multimer, because the ligand can have at least two binding sites, each of which can bind to the ligand receptor region (i.e., a ligand having a first binding site and a second binding site, wherein the first binding site can bind to the ligand binding region of the first inducible pro-apoptotic polypeptide, and the second binding site can bind to the ligand binding region of the second inducible pro-apoptotic polypeptide, wherein the ligand binding regions of the first and second inducible pro-apoptotic polypeptides are the same or different). Therefore, as used herein, the term "multimeric ligand binding region" refers to the ligand binding region of the inducible pro-apoptotic polypeptide of the present invention that binds to the multimeric ligand. The multimeric ligands of the present invention include dimeric ligands. The dimeric ligands of the present invention may have two binding sites that can bind to the ligand receptor domain. In certain embodiments, the multimeric ligands of the present invention are dimers or higher order oligomers of small synthetic organic molecules, typically no larger than about a tetramer, with individual molecules typically being at least about 150 Da and less than about 5 kDa, typically less than about 3 kDa. Multiple pairs of synthetic ligands and receptors can be used. For example, in embodiments involving endogenous receptors, dimeric FK506 can be used with FKBP12 receptors, dimeric cyclosporin A can be used with cyclophilin receptors, dimeric estrogen can be used with estrogen receptors, dimeric glucocorticoids can be used with glucocorticoid receptors, dimeric tetracycline can be used with tetracycline receptors, dimeric vitamin D can be used with vitamin D receptors, etc. Alternatively, higher-order ligands can be used, such as trimers. For embodiments involving non-naturally occurring receptors, such as antibody subunits, modified antibody subunits, single-chain antibodies composed of tandem heavy and light chain variable regions separated by flexible linkers, or modified receptors and mutant sequences thereof, etc., any of a variety of compounds can be used. A notable feature of the units comprising the multimeric ligands disclosed herein is that each binding site is capable of binding to a receptor with high affinity, and preferably, it is capable of being chemically dimerized. Likewise, methods can be used to balance the hydrophobicity / hydrophilicity of a ligand so that it is soluble in serum at functional levels, yet diffuses across the plasma membrane for most applications.

[0523] The activation of the inducible pro-apoptotic polypeptides of the present disclosure can be accomplished, for example, by chemically induced dimerization (CID) mediated by an inducer to produce conditionally controlled proteins or polypeptides. Due to degradation of unstable dimerizers or administration of monomeric competitive inhibitors, the pro-apoptotic polypeptides of the present disclosure are not only inducible, but the induction of these polypeptides is also reversible.

[0524] In certain embodiments, the ligand binding region comprises a FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the ligand binding region comprises a FKBP12 polypeptide having a valine (V) substituted for a phenylalanine (F) at position 36 (F36V). In certain embodiments wherein the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) substituted for a phenylalanine (F) at position 36 (F36V), the inducer can comprise AP1903, a synthetic drug (CAS Index Name: 2-piperidinic 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*]]]]]-(9C1) CAS Registry Number: 195514-63-7; Molecular Formula: C78H98N4020; Molecular Weight: 1411.65)). In certain embodiments wherein the ligand-binding region comprises an FKBP12 polypeptide having a valine (V) substituted for a phenylalanine (F) at position 36 (F36V), the inducer may comprise AP20187 (CAS Registry Number: 195514-80-8 and Molecular Formula: C82H107N5020). In certain embodiments, the inducer is an AP20187 analog, such as AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 are used interchangeably.

[0525] AP1903 API is manufactured by Alphora Research Inc., and AP1903 injection is manufactured by Formatech Inc. It is formulated as a 5 mg / mL solution of AP1903 in a 25% solution of the nonionic solubilizer Solutol HS 15 (250 mg / mL, BASF). At room temperature, this formulation is a clear, slightly yellow solution. Upon freezing, the formulation undergoes a reversible phase transition, resulting in a milky white solution. This phase transition is reversed upon warming to room temperature. The fill volume in a 3 mL glass vial is 2.33 mL (a total of approximately 10 mg of AP1903 is injected per vial). Once the need for AP1903 is confirmed, a single fixed dose of AP1903 injection (0.4 mg / kg) can be administered to the patient via intravenous infusion over 2 hours, for example, using a non-DEHP, non-ethylene oxide sterile infusion set. The dose of AP1903 was calculated individually for all patients and was not recalculated unless there was a ≥10% body weight fluctuation. Prior to infusion, the calculated dose was diluted in 100 mL of 0.9% normal saline. In a previous Phase I study of AP1903, 24 healthy volunteers were treated with a single dose of AP1903 injection at dose levels of 0.01, 0.05, 0.1, 0.5, and 1.0 mg / kg, infused intravenously over 2 hours. AP1903 plasma levels were dose-proportional, with mean Cmax values ​​ranging from approximately 10 to 1275 ng / mL across the 0.01-1.0 mg / kg dose range. Following the initial infusion period, blood concentrations exhibited a rapid distribution phase, with plasma levels decreasing to 18%, 7%, and 1% of the maximum concentration at 0.5, 2, and 10 hours post-dose, respectively. AP1903 for injection was safe and well-tolerated at all dose levels and exhibited a favorable pharmacokinetic profile. Iuliucci JD et al., J Clin Pharmacol. 41: 870-9, 2001.

[0526] The fixed dose of AP1903 for injection used can be, for example, 0.4 mg / kg intravenously infused over 2 hours. The amount of AP1903 required for effective cell signaling in vitro is 10-100 nM (1600 Da MW). This is equivalent to 16-160 μg / L or ~0.016-1.6 μg / kg (1.6-160 μg / kg). In the aforementioned Phase I study of AP1903, a dose of up to 1 mg / kg was tolerated. Therefore, for this Phase I study, 0.4 mg / kg may be a safe and effective dose of AP1903 in combination with therapeutic cells.

[0527] Compared to the wild-type amino acid or nucleic acid sequence, the amino acid and / or nucleic acid sequence encoding the ligand binding of the present invention may contain one or more modified sequences. For example, the amino acid and / or nucleic acid sequence encoding the ligand binding region of the present invention may be a codon-optimized sequence. Compared to the wild-type polypeptide, one or more modifications may increase the binding affinity of the ligand (e.g., inducer) to the ligand binding region of the present invention. Alternatively or additionally, compared to the wild-type polypeptide, one or more modifications may reduce the immunogenicity of the ligand binding region of the present invention. The ligand binding region of the present invention and / or the inducer of the present invention may be non-naturally occurring.

[0528] The modified cells, transposons and / or vectors of the present disclosure may comprise an inducible pro-apoptotic polypeptide comprising (a) a ligand binding region, (b) a linker and (c) a pro-apoptotic polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. The inducible pro-apoptotic polypeptide of the present disclosure may also be referred to as an "iC9 safety switch". In certain embodiments, the modified cells and / or transposons of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the modified cells and / or transposons of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the transposon of the present disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments of the inducible pro-apoptotic polypeptide, inducible caspase polypeptide, or truncated caspase 9 polypeptide of the present disclosure, the ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. In certain embodiments, the amino acid sequence of the ligand binding region comprising the FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V).

[0529] In certain embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising GVQVETISPGDGRTFPKRGQTCVVHYGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 14635).

[0530] In certain embodiments, the FKBP12 polypeptide consists of GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGGCCAGACTTGCGTCGTGCATTACAACCGGGATGCTGGAGGACGGGAAGAAAGTGGACAGCTCCAGGGATCGCAACAAGCCCTTCAA GTTCATGCTGGGAAAGCAGGAAGTGATCCGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGACCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTCATGCCACCCTGGTCTTCGAT The nucleic acid sequence encoding GTGGAACTGCTGAAGCTGGAG (SEQ ID NO: 14636). In certain embodiments, the inducer specific for a ligand binding region comprising an FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and / or AP1903 (both synthetic drugs).

[0531] In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase-9 polypeptides disclosed herein, 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.

[0532] In certain embodiments of the truncated caspase-9 polypeptides disclosed herein, the truncated caspase-9 polypeptide is encoded by an amino acid sequence that does not comprise arginine (R) at position 87 of the sequence. Alternatively or additionally, in certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides, or truncated caspase-9 polypeptides disclosed herein, the truncated caspase-9 polypeptide is encoded by an amino acid sequence that does not comprise alanine (A) at position 282 of the sequence. In certain embodiments of the inducible pro-apoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides disclosed herein, the truncated caspase 9 polypeptide comprises a polypeptide comprising GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ IDThe amino acid of NO: 14639) or the nucleic acid sequence encoding TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGGATCTGACCGCCAAGAAAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGCTGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGAGCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTCGTGTCTTACAGTACCTTCCCAGGCTI TGTCTCATGGCGCGATCCCAAGTCAGGGAGCTGGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 14640).

[0533]

[0534] The inducible pro-apoptotic polypeptides of the present invention can be expressed in the cells under the transcriptional regulation of any promoter capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present invention in the cells. As used herein, the term "promoter" refers to a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene. For example, the inducible pro-apoptotic polypeptides of the present invention can be expressed in mammalian cells under the transcriptional regulation of any promoter (including but not limited to natural, endogenous, exogenous and heterologous promoters) capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present invention in mammalian cells. Preferred mammalian cells include human cells. Therefore, the inducible pro-apoptotic polypeptides of the present invention can be expressed in human cells under the transcriptional regulation of any promoter (including but not limited to human promoters or viral promoters) capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptides of the present invention in human cells. Exemplary promoters expressed in human cells include, but are not limited to, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, the β-actin promoter, the rat insulin promoter, and the glyceraldehyde-3-phosphate dehydrogenase promoter, each of which can be used to obtain high levels of expression of the inducible pro-apoptotic polypeptides of the present invention. It is also contemplated to use 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 invention, provided that the expression level is sufficient to induce apoptosis. By employing promoters with well-known properties, the expression level and pattern of the protein of interest after transfection or transformation can be optimized.

[0535] The selection of promoters that are regulated in response to specific physiological or synthetic signals allows for inducible expression of the inducible pro-apoptotic polypeptides of the present invention. The ecdysone system (Invitrogen, Carlsbad, Calif.) is such a system. This system is designed to allow for the regulation of expression of genes of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows almost no basal level expression of transgenes, but with an induction rate exceeding 200-fold. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog (muristerone A) binds to the receptor, the receptor activates the promoter to turn on expression of the downstream transgene, thereby achieving high levels of mRNA transcripts. In this system, the two monomers of the heterodimeric receptor are constitutively expressed from one vector, while the ecdysone-responsive promoter that drives the expression of the gene of interest is on another plasmid. Therefore, it may be useful to engineer this type of system into a vector of interest. Another inducible system that may be useful is the Tet-Off system 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). TM or Tet-On TM System (Clontech, Palo Alto, Calif.). This system also allows for regulation of high-level gene expression in response to tetracycline or tetracycline derivatives such as doxycycline. TM In the Tet-Off TM In the Tet-Off system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements of the tetracycline resistance operon derived from Escherichia coli: the tetracycline operator sequence (a sequence that binds to the tetracycline repressor) and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that contains a tetracycline response element. The second plasmid contains a regulatory element called a tetracycline-controlled transactivator, which is activated in the Tet-Off system. TM The system consists of the VP16 domain from herpes simplex virus and the wild-type tetracycline repressor. Thus, transcription is constitutively active in the absence of doxycycline. TM In this system, the tetracycline repressor is not wild type and transcription is activated in the presence of doxycycline. For gene therapy vector production, Tet-Off TMsystem so that production cells can be grown in the presence of tetracycline or doxycycline and expression of potentially toxic transgenes is prevented, but when the vector is introduced into a patient, gene expression will occur constitutively.

[0536] In some cases, it is desirable to regulate the expression of a transgene in a gene therapy vector. For example, different viral promoters with different activity strengths are utilized depending on the desired expression level. In mammalian cells, the CMV immediate early promoter is often used to provide strong transcriptional activation. The CMV promoter is reviewed in Donnelly, JJ et al., 1997. Annu. Rev. Immunol. 15: 617-48. When it is necessary to reduce the expression level of a transgene, modified forms of the less potent CMV promoter are also used. When it is necessary to express a transgene in hematopoietic cells, a retroviral promoter, such as the LTR from MLV or MMTV, is often used. Other viral promoters used depending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters (e.g., from E1A, E2A, or MLP regions), AAV LTR, HSV-TK, and avian sarcoma virus.

[0537] In other examples, a promoter can be selected that is developmentally regulated and active in specific differentiated cells. Thus, for example, a promoter may not be active in pluripotent stem cells, but may then be activated, for example, when the pluripotent stem cells differentiate into more mature cells.

[0538] Similarly, tissue-specific promoters are used to transcribe in specific tissues or cells, thereby reducing potential toxicity or undesirable effects on non-target tissues. Compared to stronger promoters such as the CMV promoter, these promoters may result in reduced expression, but may also result in more limited expression and immunogenicity (Bojak, A. et al., 2002. Vaccine. 20: 1975-79; Cazeaux, N. et al., 2002. Vaccine 20: 3322-31). For example, where appropriate, tissue-specific promoters such as the PSA-associated promoter or the prostate-specific glandular kallikrein, or the muscle creatine kinase gene, may be used.

[0539] Examples of tissue-specific or differentiation-specific promoters include, but are not limited to, the following: B29 (B cells); CD14 (monocytes); CD43 (leukocytes and platelets); CD45 (hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1 (pancreatic acinar cells); endoglin (endothelial cells); fibronectin (differentiated cells, callus); and Flt-1 (endothelial cells); GFAP (astrocytes).

[0540] In some indications, it is necessary to activate transcription at a specific time after administering the gene therapy vector. This is accomplished by, for example, those promoters of adjustable hormones or cytokines. Cytokine and inflammatory protein responsive promoters include K and T kininogen (Kageyama et al., (1987) "Journal of Biological Chemistry (J.Biol.Chem.), 262, 2345-2351), c-fos, TNF-α, C-reactive protein (Arcone et al., (1988) "Nucl.Acids Res.", 16 (8), 3195-3207), haptoglobulin (Oliviero et al., (1987) "European Journal of Molecular Biology (EMBO J.)", 6, 1905-1912), serum amyloid A2, C / EBPα, IL-1, IL-6 (Poli and Cortese, (1989) "Proceedings of the National Academy of Sciences of the United States of America (Proc.Nat'l Acad. Sci. USA, 86, 8202-8206), complement C3 (Wilson et al. (1990) Mol. Cell. Biol., 6181-6191), IL-8, α-1 acid glycoprotein (Prowse and Baumann (1988) Mol. Cell. Biol., 8, 42-51), α-1 antitrypsin, lipoprotein lipase (Zechner et al., Mol. Cell. Biol., 239 4-2401, 1988), angiotensinogen (Ron et al. (1991) Mol. Cell Biol., 2887-2895), fibrinogen, c-jun (inducible by phorbol esters, TNF-α, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (inducible by phorbol esters and retinoic acid), metallothioneins (inducible by heavy metals and glucocorticoids), stromelysin (inducible by phorbol esters, interleukin-1, and EGF), α-2 macroglobulin, and α-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.

[0541] It is contemplated that any of the above promoters may be useful, alone or in combination with another promoter, depending on the desired effect. The promoter and other regulatory elements are selected so that it is functional in the desired cell or tissue. In addition, this list of promoters should not be interpreted as exhaustive or limiting; other promoters are used in conjunction with the promoters and methods disclosed herein.

[0542] Antigen receptors

[0543] In some embodiments of the compositions and methods of the present disclosure, the modified autologous cells of the present disclosure comprise an antigen receptor.

[0544] In some embodiments of the compositions and methods of the present disclosure, the vector comprises a sequence encoding a chimeric antigen receptor or a portion thereof. Exemplary vectors of the present disclosure include, but are not limited to, viral vectors, non-viral vectors, plasmids, nanoplasmids, minicircles, transposition systems, liposomes, polymersomes, micelles, and nanoparticles.

[0545] 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. In some embodiments, the transposon is integrated into the genomic sequence of an autologous cell by a transposase.

[0546] 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. In some embodiments, after single-strand or double-strand breaks and optionally cell-mediated repair, the donor oligonucleotide or donor plasmid is fully or partially integrated into the chromosomal sequence of the autologous cell.

[0547] Exemplary antigen receptors include non-naturally occurring transmembrane proteins that bind antigen at a site in the extracellular domain and transduce or induce an intracellular signal through the intracellular domain.

[0548] In certain embodiments, non-natural antigen receptors include but are not limited to recombinant, variant, chimeric or synthetic T cell receptors (TCR). In some embodiments, compared with wild-type TCR, variant TCR contains one or more sequence variations in the nucleotide or amino acid sequence encoding TCR. In certain embodiments, synthetic TCR comprises at least one synthetic or modified nucleic acid or amino acid encoding TCR. In certain embodiments, recombinant and / or chimeric TCR is encoded by non-natural nucleic acid or amino acid sequence over its entire length or a portion thereof because the sequence is separated or derived from one or more source sequences.

[0549] In some embodiments, non-naturally occurring antigen receptors include, but are not limited to, chimeric antigen receptors.

[0550] Chimeric antigen receptor

[0551] 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.

[0552] In some embodiments of the disclosed compositions and methods, the transposon comprises a sequence encoding a chimeric antigen receptor or a portion thereof.

[0553] Chimeric antigen receptor (CAR) of the present disclosure may include (a) an extracellular domain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an intracellular domain comprising at least one costimulatory domain. In another embodiment, the extracellular domain may further include a signal peptide. Alternatively or additionally, in certain embodiments, the extracellular domain may further include a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments of CAR of the present disclosure, the signal peptide may include a sequence encoding human CD2, CD3δ, CD3ε, CD3y, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptides. In certain embodiments of CAR of the present disclosure, the signal peptide may include a sequence encoding human CD8α signal peptides. In certain embodiments, the transmembrane domain may include a sequence encoding human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domains. In certain embodiments of CARs of the present disclosure, the transmembrane domain may comprise a sequence encoding a human CD8 alpha transmembrane domain. In certain embodiments of CARs of the present disclosure, the intracellular domain may comprise a human CD3 zeta intracellular domain.

[0554] In certain embodiments of CAR of the present disclosure, at least one costimulatory domain may include human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment or any combination thereof. In certain embodiments of CAR of the present disclosure, at least one costimulatory domain may include CD28 and / or 4-1BB costimulatory domain. In certain embodiments of CAR of the present disclosure, the hinge may include a sequence derived from human CD8 α, IgG4 and / or CD4 sequence. In certain embodiments of CAR of the present disclosure, the hinge may include a sequence derived from human CD8 α sequence.

[0555] The CD28 costimulatory domain can contain an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14477), or a sequence at least 70%, 80%, 90%, 95%, or 99% identical to an amino acid sequence comprising RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14477). The CD28 costimulatory domain can be composed of a protein containing cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctca ggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg(SEQ ID NO: 14478) nucleic acid sequence encoding. The 4-1BB costimulatory domain can contain 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.

[0556] In certain embodiments of the CARs of the present disclosure, the hinge may comprise a sequence derived from a human CD8 alpha, 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 CD8 alpha sequence. The hinge may contain a human CD8 alpha 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 (SEQ ID NO: 14482).

[0557] ScFv

[0558] The present disclosure provides single-chain variable fragment (scFv) compositions and methods of using these compositions to identify and bind to specific target proteins. ScFv compositions comprise the heavy chain variable region and light chain variable region 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 chain (VH) chain and light chain (VL) of an immunoglobulin, and the VH and VL domains are connected with a short peptide linker. Despite the removal of the constant region and the introduction of a linker, scFV retains the specificity of the original immunoglobulin. An exemplary linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 14483).

[0559] Centyrins

[0560] The qinturin proteins of the present disclosure specifically bind to antigens. The chimeric antigen receptors of the present disclosure comprising one or more qinturin proteins that specifically bind to an antigen can be used to direct the specificity of cells (eg, cytotoxic immune cells) toward a specific antigen.

[0561] The qinturin protein of the present disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding to an antigen. The qinturin protein of the present disclosure may contain a protein scaffold comprising a consensus sequence of at least one type III fibronectin (FN3) domain, wherein the scaffold is capable of specifically binding to an antigen. The at least one type III fibronectin (FN3) domain may be derived from a human protein. The human protein may be tenascin-C. The consensus sequence may comprise LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence can comprise an amino sequence at least 74% identical to LPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14488) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 14489). The consensus sequence can be obtained by containing the ccattgtcctgacagtgccagggtccgaacgctcttatgacctgacagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcaccaca(SEQ ID NO: 14490) nucleic acid sequence encoding.The consensus sequence may be modified at one or more positions within the following: (a) an AB loop comprising or consisting of the amino acid residue TEDS (SEQ ID NO: 14491) at positions 13-16 of the consensus sequence; (b) a BC loop comprising or consisting of the amino acid residues TAPDAAF (SEQ ID NO: 14492) at positions 22-28 of the consensus sequence; (c) a CD loop comprising or consisting of the amino acid residues SEKVGE (SEQ ID NO: 14493) at positions 38-43 of the consensus sequence; (d) a DE loop comprising or consisting of the amino acid residues GSER (SEQ ID NO: 14494) at positions 51-54 of the consensus sequence; (e) an EF loop comprising or consisting of the amino acid residues GLKPG (SEQ ID NO: 14495) at positions 60-64 of the consensus sequence; (f) a loop comprising or consisting of the amino acid residues KGGHRSN (SEQ ID NO: 14496) at positions 75-81 of the consensus sequence. NO: 14496) or an FG loop consisting thereof; or any combination of (g)(a)-(f). The qinturin protein of the present disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains, or at least 15 fibronectin type III (FN3) domains. The backbone may be selected from at least one of the following: less than or equal to 10 affinity binding antigens. -9 M, less than or equal to 10 -10 M, less than or equal to 10 -11 M, less than or equal to 10 - 12 M, less than or equal to 10 -13 M, less than or equal to 10 -14 M and less than or equal to 10 -15 M's K D .K D It can be determined by surface plasmon resonance.

[0562] The term "antibody mimics" is intended to describe organic compounds that specifically bind to a target sequence and have a structure different from that of naturally occurring antibodies. Antibody mimics may comprise 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 provide properties superior to antibodies, including but not limited to superior solubility, tissue permeability, stability to heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimics include but are not limited to affibodies, human ubiquitin (afflilin), affibodies, affitins (affitins), alpha antibodies, anticalins, and high-affinity multimers (also referred to as avidity multimers), DARPins (designed ankyrin repeat proteins), Fynomers, Kunitz domain peptides, and monofunctional antibodies (monobodies).

[0563] The affibody molecules of the present disclosure comprise a protein backbone comprising or consisting of one or more α-helices without any disulfide bridges. Preferably, the affibody molecules of the present disclosure comprise or consist of three α-helices. For example, the affibody molecules of the present disclosure may comprise an immunoglobulin binding domain. The affibody molecules of the present disclosure may comprise the Z domain of protein A.

[0564] The human ubiquitin molecules disclosed herein comprise a protein backbone generated by modifying exposed amino acids of, for example, γ-B crystallin or ubiquitin. Human ubiquitin molecules functionally mimic the affinity of antibodies for antigens, but do not structurally mimic antibodies. In any protein backbone used to make human ubiquitin, amino acids that are accessible to solvent or potential binding partners in a properly folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids can be modified to specifically bind to a target sequence or antigen.

[0565] Affibody molecules of the present disclosure comprise a protein backbone comprising a highly stable protein engineered to display a peptide loop that provides a high affinity binding site for a specific target sequence. Exemplary affibody molecules of the present disclosure comprise a protein backbone based on a cystatin protein or its tertiary structure. Exemplary affibody molecules of the present disclosure may share a common tertiary structure comprising an α-helix on top of an antiparallel β-sheet.

[0566] The avidin molecule of the present disclosure comprises an artificial protein backbone, the structure of which can be, for example, derived from a DNA binding protein (e.g., DNA binding protein Sac7d). The avidin of the present disclosure selectively binds to a target sequence, which can be all or part of an antigen. Exemplary avidins of the present disclosure are manufactured by randomizing one or more amino acid sequences on the binding surface of the DNA binding protein and subjecting the resulting protein to ribosome display and selection. The target sequence of the avidin of the present disclosure can be, for example, found in a genome or on the surface of a peptide, protein, virus, or bacterium. In certain embodiments of the present disclosure, the avidin molecule can be used as a specific inhibitor of an enzyme. The avidin molecule of the present disclosure can include a heat-resistant protein or a derivative thereof.

[0567] The alpha antibody molecules of the present disclosure may also be referred to as cell-penetrating alpha antibodies (CPABs). The alpha antibody 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 antibody molecules are able to reach and bind to intracellular target sequences. Structurally, the alpha antibody molecules of the present disclosure comprise artificial sequences that form single-stranded alpha helices (similar to naturally occurring coiled-coil structures). The alpha antibody molecules of the present disclosure may comprise a protein backbone comprising one or more amino acids modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the alpha antibody molecules of the present disclosure maintain proper folding and thermal stability.

[0568] The anticalin molecules of the present disclosure comprise artificial proteins that bind to a target sequence or site in a protein or small molecule. The anticalin molecules of the present disclosure may comprise artificial proteins derived from human lipocalin. 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 tissue penetration and thermal stability that is superior to monoclonal antibodies or fragments thereof. An exemplary anticalin molecule of the present disclosure may comprise approximately 180 amino acids and have a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure comprise a tubular structure comprising antiparallel β-strands connected in pairs by loops and connecting α-helices. In a preferred embodiment, the anticalin molecules of the present disclosure comprise a tubular structure comprising eight antiparallel β-strands connected in pairs by loops and connecting α-helices.

[0569] The high-affinity multimer molecules disclosed herein comprise artificial proteins that specifically bind to a target sequence (which may also be an antigen). The high-affinity multimers disclosed herein can recognize multiple binding sites within the same target or within different targets. When the high-affinity multimers disclosed herein recognize more than one target, the high-affinity multimers simulate the function of bispecific antibodies. The artificial protein high-affinity multimer may comprise two or more peptide sequences, each of approximately 30-35 amino acids. These peptides may be connected by one or more linker peptides. The amino acid sequence of one or more peptides of the high-affinity multimer may be derived from the A domain of a membrane receptor. The high-affinity multimer has a rigid structure, which may optionally contain disulfide bonds and / or calcium. Compared to antibodies, the high-affinity multimers disclosed herein can exhibit higher thermal stability.

[0570] The DARPins (designed ankyrin repeat proteins) disclosed herein comprise genetically engineered, recombinant, or chimeric proteins with high specificity and high affinity for a target sequence. In certain embodiments, the DARPins disclosed herein are derived from ankyrin and optionally comprise at least three repeating motifs (also referred to as repeating structural units) of ankyrin. Ankyrin mediates high-affinity protein-protein interactions. The DARPins disclosed herein comprise large target interaction surfaces.

[0571] The Fynomers of the present disclosure comprise small binding proteins (approximately 7 kDa) derived from the human Fyn SH3 domain and engineered to bind to target sequences and molecules with the same affinity and specificity as antibodies.

[0572] The Kunitz domain peptides disclosed herein contain a protein backbone comprising a Kunitz domain. The Kunitz domain contains an active site that inhibits protease activity. Structurally, the Kunitz domains disclosed herein comprise an α+β fold rich in disulfide bonds. This structure is exemplified by bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and act as competitive protease inhibitors. The Kunitz domains disclosed herein may comprise ecallantide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

[0573] The monofunctional antibodies disclosed herein are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) of a size comparable to that of single-chain antibodies. These genetically engineered proteins specifically bind to target sequences, including antigens. The monofunctional antibodies disclosed herein can specifically target one or more different proteins or target sequences. In a preferred embodiment, the monofunctional antibodies disclosed herein comprise a structure that mimics the structure of human fibronectin, and more preferably, a protein skeleton that mimics the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin and its monofunctional antibody mimics contain seven β-folds that form a barrel, and three exposed loops corresponding to the three complementary determining regions (CDRs) of the antibody on each side. Compared to the structure of the variable domain of an antibody, the monofunctional antibodies lack any binding sites for metal ions and a central disulfide bond. Multispecific monofunctional antibodies can be optimized by modifying loops BC and FG. The monofunctional antibodies disclosed herein may comprise fibronectin.

[0574] VHH

[0575] In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VHH.

[0576] The present disclosure provides a chimeric antigen receptor (CAR) (VCAR) comprising at least one VHH. The chimeric antigen receptor of the present disclosure may comprise more than one VHH. For example, a bispecific VCAR may comprise two VHHs that specifically bind to two different antigens.

[0577] The VHH proteins of the present disclosure specifically bind to antigens. The chimeric antigen receptors of the present disclosure comprising one or more VHHs that specifically bind to an antigen can be used to direct the specificity of cells (eg, cytotoxic immune cells) toward a specific antigen.

[0578] As is well known in the art, the at least one VHH protein or VCAR of the present disclosure may optionally be produced from a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells. See, e.g., 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 ed., 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).

[0579] Amino acids from a VHH protein may be altered, 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 characteristic, as known in the art.

[0580] Optionally, VHH proteins can be engineered while retaining high affinity for antigens and other favorable biological properties. To achieve this goal, three-dimensional models of parental and engineered sequences can be optionally used to prepare VH proteins through analytical processes of parental sequences and various conceptual engineered products. Three-dimensional models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., the Immunofilter program of Xencor, Inc. of Monrovia, Calif.). Inspection of these displays allows analysis of the possible role of residues in the function of the candidate sequence, i.e., analysis of residues that affect the ability of the candidate VHH protein to bind to its antigen. In this way, residues can be selected from parental and reference sequences and combined to obtain desired characteristics, such as affinity for the target antigen. As an alternative or supplement to the above-mentioned procedures, other suitable engineering methods can be used.

[0581] Screening VHHs for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries (e.g., in vitro display). This method involves screening a large number of peptides for a single member with the desired function or structure. The length of the displayed nucleotide or peptide sequence can be 3 to 5000 nucleotides or amino acids or more, often 5-100 amino acids long, and usually 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 displaying peptide sequences on the surface of phages or cells. Each phage or cell contains a nucleotide sequence encoding a specific displayed peptide sequence. The VHH proteins of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one VHH of the present disclosure can optionally bind to a target protein with high affinity, for example, with an affinity equal to or less than about 10 -7 KD of M, for example but not limited to 0.1-9.9 (or any range or value thereof)×10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 or any range or value therein, as determined by surface plasmon resonance or Kinexa methods, as practiced by one skilled in the art.

[0582] 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," Fundamental Immunology, Paul, W E., ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992); and methods described herein). If measured under different conditions (e.g., salt concentration, pH), the affinity of the specific VHH-antigen or VCAR-antigen interaction measured may vary. Therefore, the measurement of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) is preferably performed with a standardized solution of VHH or VCAR and antigen, and a standardized buffer (such as a buffer described herein).

[0583] Competitive analysis can be carried out with VHH or VCAR disclosed herein to determine which proteins, antibodies and other antagonists compete with VHH or VCAR disclosed herein for binding to target protein and / or shared epitope regions. Competition for a limited number of binding sites on protein is evaluated between these analytical assessment antagonists or ligands that are readily known to those skilled in the art. Before or after competition, protein and / or antibody are fixed or insoluble, and the sample bound to the target protein is separated from the unbound sample, for example, by decantation (wherein protein / antibody is pre-dissolved) or by centrifugation (wherein protein / antibody is precipitated after competitive reaction). In addition, competitive binding can be determined by whether or not to change function by VHH or VCAR and target protein binding or not, such as whether VCAR molecules suppress or enhance the enzymatic activity of, for example, labeling. As well known in the art, ELISA and other functional analyses can be used.

[0584] VH

[0585] In certain embodiments, the CAR comprises a single domain antibody (SdAb). In certain embodiments, the SdAb is a VH.

[0586] The present disclosure provides chimeric antigen receptors (CARs) (VCARs) comprising single domain antibodies. In certain embodiments, single domain antibodies comprise VH. In certain embodiments, VH is isolated or derived from human sequences. In certain embodiments, VH comprises human CDR sequences and / or human framework sequences and non-human or humanized sequences (e.g., rat Fc domains). In certain embodiments, VH is a fully humanized VH. In certain embodiments, VH is neither a naturally occurring antibody nor a fragment of a naturally occurring antibody. In certain embodiments, VH is not a fragment of a monoclonal antibody. In certain embodiments, VH is a UniDab TM Antibodies (TeneoBio).

[0587] In certain embodiments, VH is expressed using UniRat TM (TeneoBio) system and "NGS-based Discovery" are fully engineered to generate VH. Using this approach, specific VHs are not naturally occurring and are produced using a fully engineered system. VHs are not derived from naturally occurring monoclonal antibodies (mAbs), which are directly isolated from a host (e.g., mouse, rat, or human), or directly isolated from a single cell or cell line clone (hybridoma). These VHs are not subsequently cloned from the cell line. Instead, UniRat TM The system fully engineered the VH sequence as a transgene that contains a human variable region (VH domain) and a rat Fc domain, and is therefore a human / rat chimera without a light chain, and unlike standard mAb formats. The native rat gene was knocked out, and the only antibodies expressed in rats were from transgenics with a VH domain linked to a rat Fc (UniAbs). These are exclusive Abs expressed in UniRat. Next generation sequencing (NGS) and bioinformatics were used to identify antibodies expressed by UniRat after immunization. TM The complete antigen-specific spectrum of the heavy chain antibodies produced. A unique 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 for multiple functions in vitro. In certain embodiments, fully humanized VH is produced by fusing the human VH domain with the human Fc in vitro (to produce non-naturally occurring recombinant VH antibodies). In certain embodiments, the VH is fully humanized, but it is expressed in vivo as a human / rat chimera without a light chain (human VH, rat Fc). The fully humanized VH expressed in vivo as a human / rat chimera without a light chain (human VH, rat Fc) is approximately 80 kDa (relative to 150 kDa).

[0588] 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 the Fc domain or a portion thereof. In certain embodiments, the framework sequence of the VH of the present disclosure may be modified to, for example, improve expression, reduce immunogenicity, or improve function.

[0589] As used throughout this disclosure, the singular forms "a," "and," and "the" include plural referents 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 skilled in the art, and so forth.

[0590] 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, which will depend in part on how the value is measured or determined, e.g., limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations. Alternatively, "about" can mean a range of at most 20%, or at most 10%, or at most 5%, or at most 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value. Where specific values ​​are described in the application and claims, it should be assumed that the term "about" means within an acceptable error range for the specific value unless otherwise indicated.

[0591] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. "Isolated" or "purified" polynucleotides or proteins or their biologically active portions are substantially or substantially free of the components that typically accompany the polynucleotides or proteins or interact with them (as found in their naturally occurring environment). Therefore, isolated or purified polynucleotides or proteins are substantially free of other cellular materials or culture media when produced by recombinant technology, or are substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, "isolated" polynucleotides do not contain sequences (optimally, protein coding sequences) that naturally flank the polynucleotides in the genomic DNA of the source organism of the polynucleotides (i.e., sequences at the 5' and 3' ends of the polynucleotides). For example, in various embodiments, the isolated polynucleotides can contain a nucleotide sequence that is less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or 0.1kb, and the nucleotide sequence naturally flanks the polynucleotides in the genomic DNA of the source cell of the polynucleotides. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure, or a biologically active portion thereof, is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest.

[0592] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence and therefore refers to a protein encoded thereby. A fragment of a DNA sequence comprising a coding sequence can encode a protein fragment that retains the biological activity of the native protein and therefore retains DNA recognition or binding activity for a target DNA sequence as described herein. Alternatively, a fragment of a DNA sequence suitable for use as a hybridization probe generally does not encode a protein that retains biological activity or does not retain promoter activity. Therefore, a fragment of a DNA sequence can be within the range of at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the present disclosure.

[0593] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach comprising preassembling monomeric units and / or repeating units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the present disclosure can comprise repeating monomers of the present disclosure and can be constructed by a modular approach by preassembling repeating units in a target vector, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced by this modular approach.

[0594] The term "antibody" is used in the broadest sense, and specifically, covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with multiple epitope specificities. Natural or synthetic analogs, mutants, variants, alleles, homologues and orthologues (collectively referred to as "analogues" in this article) of antibodies as defined herein are also used within the scope of this article. Therefore, according to one embodiment of this article, the term "antibody herein" also covers such analogues in its broadest sense. In general, in such analogues, one or more amino acid residues may have been replaced, deleted and / or added compared to the antibodies of the present invention as defined herein.

[0595] As used herein, "antibody fragment" and all grammatical variations thereof are defined as a portion of an intact antibody that contains the antigen binding site or variable region of the intact antibody, wherein the portion does not contain the constant heavy chain domains (i.e., CH2, CH3, and CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of continuous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing the three CDRs of a light chain variable domain without the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable domain, or fragments thereof containing the three CDRs of a heavy chain variable domain without the associated light chain portion; and multispecific or multivalent structures formed by antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CHI in an IgG isotype), and / or may contain any hinge region sequence found in an intact antibody, and / or may contain a leucine zipper sequence fused to or within the hinge region sequence or constant domain sequence of the heavy chain. The term further includes single-domain antibodies ("sdABs"), which generally refer to antibody fragments having a single monomeric variable antibody domain (e.g., from a camelid). Such antibody fragment types will be readily understood by those skilled in the art.

[0596] "Binding" refers to sequence-specific, non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids). Not all components of the binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone), as long as the overall interaction is sequence-specific.

[0597] The term "comprising" is intended to mean that compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements that are of any significance to the combination when used for its intended purpose. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0598] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope may comprise three amino acids in a spatial conformation that is unique to that epitope. Generally, an epi...

Claims

1. A non-naturally occurring chimeric stimulatory receptor (CSR), comprising: (a) an extracellular domain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

2. The CSR of claim 1, wherein the activation component comprises a portion of one or more of the following: a component of a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activation component binds.

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

4. The CSR of claim 1, wherein the signal transduction domain comprises one or more of: components of a human signal transduction domain, a T cell receptor (TCR), components of a TCR complex, components of a TCR co-receptor, components of a TCR co-stimulatory protein, components of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor.

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

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

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

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

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

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

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