Allogeneic cell compositions and methods of use

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 896,495, filed September 5, 2019, and U.S. Provisional Application No. 62 / 976,536, filed February 14, 2020. The contents of each of these applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to molecular biology and, more particularly, to chimeric receptors, allogeneic cell compositions, methods of making and using the same.

[0004] Sequence Listing Incorporated by Reference

[0005] The contents of the file named “POTH-055_001WO_SequenceListing_ST25.txt”, created on August 21, 2020 and 291 KB in size, are hereby incorporated by reference in their entirety. Background Art

[0006] There has been a long-felt but unmet need in the art for allogeneic cell compositions that overcome the challenges presented by eliminating genes involved in graft-versus-host and host-versus-graft responses. The present disclosure provides allogeneic cell compositions, methods of making these compositions, and methods of using these compositions that contain non-naturally occurring structural improvements to restore allogeneic cell responsiveness to environmental stimuli and reduce or prevent rejection through natural killer cell-mediated cytotoxicity. Summary of the Invention

[0007] The present disclosure provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide, and wherein the activation component comprises an agonist-binding CD2 extracellular domain, or a portion thereof; (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 a signaling domain, wherein the cytoplasmic domain is a CD28 intracellular domain, a 4-1BB intracellular domain, an IL17RA intracellular domain, an IL15RA intracellular domain, an IL21R intracellular domain, an ICOS intracellular domain, a CD27 intracellular domain, an OX40 intracellular domain, or a GITR intracellular domain, or any combination thereof, and wherein the signaling domain comprises a CD3 zeta protein, or a portion thereof; and wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0008] In some aspects, the CD2 signal peptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 5. In a preferred aspect, the CD2 signal peptide comprises the amino acid sequence of SEQ ID NO: 5.

[0009] The present disclosure also 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 CD8α signal peptide, and wherein the activation component comprises an agonist-binding CD2 extracellular domain, or a portion thereof; (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 a signaling domain, wherein the cytoplasmic domain is a CD2 intracellular domain, a CD28 intracellular domain, a 4-1BB intracellular domain, an IL17RA intracellular domain, an IL15RA intracellular domain, an IL21R intracellular domain, an ICOS intracellular domain, a CD27 intracellular domain, an OX40 intracellular domain, or a GITR intracellular domain, or any combination thereof, and wherein the signaling domain comprises a CD3ζ protein, or a portion thereof; and wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0010] In some aspects, the CD8α signal peptide comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 7. In a preferred aspect, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0011] The present disclosure also provides non-naturally occurring chimeric stimulating receptors (CSRs), wherein the activating component comprises a modification. In some aspects, the modification comprises a mutation or a truncation of the amino acid sequence of the CD2 extracellular domain or a portion thereof to which the agonist binds compared to the wild-type sequence of the CD2 extracellular domain or a portion thereof. In some aspects, the non-naturally occurring CSR comprising a mutation or a truncation of the CD2 extracellular domain or a portion thereof to which the agonist binds does not bind CD58. In some aspects, the CD2 extracellular domain or a portion thereof comprising a mutation or a truncation comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 3. In a preferred aspect, the CD2 extracellular domain or a portion thereof comprising a mutation or a truncation comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 3.

[0012] In some aspects, the CD2 transmembrane domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 9. In a preferred aspect, the CD2 transmembrane domain or a portion thereof comprises the amino acid sequence of SEQ ID NO: 9.

[0013] In some aspects, the CD2 intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 13. In a preferred aspect, the CD2 intracellular domain comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, the CD28 intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 15. In a preferred aspect, the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 15. In some aspects, the 4-1BB intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 17. In a preferred aspect, the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some aspects, the IL17RA intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 19. In a preferred aspect, the IL17RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some aspects, the IL15RA intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 21. In a preferred aspect, the IL15RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. In some aspects, the IL21R intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: XX. In a preferred aspect, the IL21R intracellular domain comprises the amino acid sequence of SEQ ID NO: 23. In some aspects, the ICOS intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 25. In a preferred aspect, the ICOS intracellular domain comprises the amino acid sequence of SEQ ID NO: 25. In some aspects, the CD27 intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 27. In a preferred aspect, the CD27 intracellular domain comprises the amino acid sequence of SEQ ID NO: 27. In some aspects, the OX40 intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 29. In a preferred aspect, the OX40 intracellular domain comprises the amino acid sequence of SEQ ID NO: 29.In some aspects, the GITR intracellular domain comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 31. In a preferred aspect, the GITR intracellular domain comprises the amino acid sequence of SEQ ID NO: 31.

[0014] In some aspects, the signaling domain comprising a CD3 zeta protein or a portion thereof comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11. In a preferred aspect, the signaling domain comprising a CD3 zeta protein or a portion thereof comprises the amino acid sequence of SEQ ID NO: 11.

[0015] In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 39. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 39. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 43. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 43. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 47. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 47. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 51. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 51. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 55. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 55. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 59. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 59. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 63. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 63. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 67. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 67. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 71. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 71.

[0016] In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 37. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 37. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 41. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 41. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 45. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 45. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 49. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 49. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 53. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 53. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 57. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 57. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 61. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 61. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 65. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 65. In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 69. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 69.In some aspects, the non-naturally occurring CSR comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 73. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 73.

[0017] The present disclosure provides nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides vectors comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides transposons comprising nucleic acid sequences encoding any of the CSRs disclosed herein.

[0018] The present disclosure provides cells comprising any of the CSRs disclosed herein. The present disclosure provides cells comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides cells comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides cells comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0019] The present disclosure also provides modified T lymphocytes (T cells), comprising: (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) any chimeric stimulating receptor (CSR) disclosed herein. The modified T 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 CAR T cells.

[0020] The present disclosure provides compositions comprising any of the CSRs disclosed herein. The present disclosure provides compositions comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising vectors comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising transposons comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising modified cells disclosed herein, or compositions comprising multiple modified cells disclosed herein.

[0021] 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) 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.

[0022] 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).

[0023] 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.

[0024] The modified T cell 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).

[0025] 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.

[0026] 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.

[0027] 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 in 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 in the population comprise a modified T cell disclosed herein.

[0028] The present disclosure provides a method for treating a disease or condition comprising administering to a subject in need thereof a therapeutically effective amount of any composition disclosed herein; or a composition for use in 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 a subject 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.

[0029] The present disclosure provides a method for producing a modified T cell colony, which includes the following, is essentially composed of or is composed of: a composition comprising a CSR or its coding sequence of the present disclosure is introduced into multiple primary human T cells, so as to stably express CSR in multiple modified T cells and preserve the desired stem-like properties of multiple modified T cells under the conditions of producing multiple modified T cells. 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 CSR expresses stem memory T cells (T SCM ) or T SCMIn some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population expresses 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 in the treatment of 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 further provides a method for treating a disease or condition, which comprises administering a therapeutically effective amount of the composition produced by the method to a subject in need. The treatment method may further comprise administering an activator composition to the subject to activate the modified T cell population in vivo, induce cell division of the modified T cell population in vivo, or a combination thereof.

[0030] The present disclosure provides a method for producing a modified T cell colony, which includes the following, is essentially composed of or is composed of: a composition comprising a CSR or its coding sequence of the present disclosure is introduced into multiple primary human T cells, to transiently express CSR in multiple modified T cells and preserve the desired stem-like properties of multiple modified T cells under conditions of, produce multiple modified T cells. 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 CSR expresses stem memory T cells (T SCM ) or T SCMIn some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population expresses 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 in the treatment of 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 further provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of the composition produced by the method to a subject in need. In some aspects, the modified T cells within the modified T cell population administered to the subject no longer express CSR.

[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 its coding sequence into a plurality of primary human T cells to stably express the CSR in the plurality of modified T cells and preserve the desired stem-like properties of the plurality of modified T cells, producing a plurality of modified T cells, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least 2-fold compared to the expansion of a plurality of wild-type T cells that 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 the CSR expresses stem memory T cells (T cells). SCM ) or T SCMIn some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population expresses 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 in the treatment of 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 further provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of a composition expanded by the method to a subject in need. The treatment method may further include administering an activator composition to the subject to activate the modified T cell population in vivo, induce cell division of the modified T cell population in vivo, or a combination thereof.

[0032] 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 its coding sequence into a plurality of primary human T cells to produce a plurality of modified T cells under conditions that transiently express the CSR in the plurality of modified T cells and preserve 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 2-fold compared to 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 a CSR expresses stem memory T cells (T SCM ) or T SCMIn some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the population expresses 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 in the treatment of a disease or condition. The present disclosure also provides the use of a composition amplified by the method for treating a disease or condition. The present disclosure further provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of a composition amplified by the method to a subject in need. In some aspects, the modified T cells within the modified T cell population administered to the subject no longer express CSR.

[0033] Any aspect above may be combined with any other aspect.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. In this specification, the singular also includes the plural, unless the context clearly dictates otherwise; as an example, the terms "a", "an", and "the / said" are to be understood as singular or plural, and the term "or" is to be understood as inclusive. For example, "element" means one or more elements. Throughout the specification, the word "comprising" or variations such as "comprises" or "comprising" are to be understood as implying the inclusion of the element, integer, or step, or a group of elements, integers, or steps, but not excluding any other element, integer, or step, or a group of elements, integers, or steps. Approximately can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the value. Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0035] Although suitable methods and materials are described below, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art of the present invention. In the event of a conflict, the present specification, including definitions, shall prevail. In addition, the materials, methods, and examples are illustrative only 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

[0036] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0037] Figure 1 Schematic diagram showing exemplary CSR CD2z-D111H mutants for enhanced manufacturing of allogeneic or autologous CAR-T cells. The CSR CD2z-D111H mutant can be delivered to allogeneic or autologous CAR-T cells during manufacturing to enhance cell growth and expansion, quality, survival, phenotype, function, subpopulation composition, gene editing efficiency, etc. These mutant CSRs can be delivered transiently, as encoded in mRNA, or stably, as encoded in transposons.

[0038] Figure 2 Schematic diagram showing exemplary CSRCD2z-D111H mutants with CD8a signal peptides for enhanced manufacturing of allogeneic or autologous CAR-T cells. The CSR CD2z-D111H mutant can be delivered to allogeneic or autologous CAR-T cells during manufacturing to enhance cell growth and expansion, quality, survival, phenotype, function, subset composition, gene editing efficiency, etc. These mutant CSRs can be delivered transiently, as encoded in mRNA, or stably, as encoded in transposons.

[0039] Figure 3It is a diagram showing that the delivery of CSR enhances CAR-T cell expansion during production. Using the piggyBac® DNA modification system combined with Cas-CLOVER™ gene editing system, pan-T cells isolated from normal donor blood are genetically modified. In a single reaction, with transposons encoding at least CAR and selection genes, mRNA encoding CSR, super piggyBac® transposase mRNA, encoding Cas-CLOVER™ mRNA and multiple guide RNAs (gRNA) targeting TCRb and b2M, cells are electroporated to knock out TCR and MHCI (double knockout; DKO). Cells are subsequently stimulated with agonist mAb anti-CD2, anti-CD3 and anti-CD28, and are subsequently selected for genetic modification during a 14-day culture period. At the end of the initial culture period, all T cells express CAR, indicating the successful selection of genetically modified cells. In samples expressing CSR, a greater amplification of DKO cells was observed.

[0040] Figure 4 Schematic diagram showing the experimental scheme for evaluating in vivo tumor control by using CAR-T cells produced using different CSRs. Figure 1 and Figure 3 As described in Tables 1 and 2, allogeneic CAR-T cells were produced using different boosters. A mouse xenograft model of multiple myeloma was used to evaluate the in vivo anti-tumor efficacy of allogeneic CAR-T cells produced with 10 different boosters. Specifically, the RPMI-8226 cell line was cultured at 1x10 7 A dose of 10 cells was injected subcutaneously (SC) into female NSG mice (day -7) and subsequently inoculated after tumors were established (75-125 mm by caliper). 3 [Target average value ~100 mm 3 ]) on day 0 by administering a 'stress' dose (5x10 6 ) were treated with allogeneic CAR-T cells via intravenous (IV) injection of 100 mg / kg CAR-T cells. The 'stress' dose was used to allow for greater resolution in detecting possible functional differences in efficacy among CAR-T cells produced with different booster molecules.

[0041] Figure 5 This graph shows the tumor volume over time after allogeneic CAR-T cell therapy. Figure 4 The protocol shown in and in vivo tumor control was evaluated by using a 'stress' dose of CAR-T cells produced using different CSRs. Tumor volume evaluation by caliper measurement for all animals is shown as group mean, with error bars as SEM (standard error of the mean).

[0042] Figure 6 This graph shows the total T cells in the blood over time after allogeneic CAR-T cell therapy. Figure 4 In vivo tumor control was evaluated using the protocol shown in and by using a 'stress' dose of CAR-T cells produced using different CSRs. For all animals, total T cells in the blood were measured by TruCount staining for human CD45+ cells / µl (hCD45+ / µL), which are shown as group means with error bars as SEM.

[0043] Figure 7 It is a graph showing the peak T cells (T cell Cmax) in the blood. Figure 4 In vivo tumor control was evaluated using the protocol shown in and by using a 'stress' dose of CAR-T cells produced using different CSRs. For all animals, peak T cell levels in the blood, as measured by TruCount staining of human CD45+ (hCD45+) cells, are shown as group means, with error bars as SEM.

[0044] Figure 8 It is a graph showing the area under the curve of T cells (hCD45+) in blood. Figure 4 In vivo tumor control was evaluated using the protocol shown in and using a 'stress' dose of CAR-T cells produced using different CSRs. For all animals, T cell AUC in blood is shown as group mean, as calculated from TruCount staining of human CD45+ cells, with error bars as SEM.

[0045] Figure 9 is a series of graphs showing the phenotype of CD8+ T cells in the blood. Figure 4 The protocol shown in and in vivo tumor control was evaluated by using 'stress' doses of CAR-T cells produced using different CSRs. On days 14 and 35 after CAR-T treatment, the CD8+ T cell phenotype in the blood as measured by FACS staining for all animals was shown as group mean values, and the error bars were used as SEM. Cells were stained for the expression of surface CD45RA, CD45RO, and CD62L to define TSCM, TCM, TEM, and TEFF cells; TSCM (CD45RA+CD45RO-CD62L+; blue), TCM (CD45RA-CD45RO+CD62L+; red), TEM (CD45RA-CD45RO+CD62L-; green), TEFF (CD45RA+CD45RO-CD62L-; purple).

[0046] All documents cited herein, including any cross-referenced or related patents or applications, are hereby incorporated by reference in their entirety for all purposes unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, nor is it an admission that it alone or in any combination with any other reference or references teaches, suggests, or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control. DETAILED DESCRIPTION

[0047] The present disclosure provides allogeneic cell compositions, methods of making these compositions, and methods of using these compositions that contain non-naturally occurring structural improvements to restore allogeneic cell responsiveness to environmental stimuli and reduce or prevent rejection through natural killer cell-mediated cytotoxicity.

[0048] Chimeric stimulatory receptor (CSR) and recombinant HLA-E peptide

[0049] For adoptive cell compositions that are "universally" safe to be administered to any patient, a significant reduction or elimination of alloreactivity is required. To this end, the cells of the present disclosure (e.g., allogeneic cells) can be modified to interrupt 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 a preferred aspect, any expression and / or function of the TCR is eliminated to prevent T cell-mediated GvH that can cause death in the subject. Therefore, in a preferred aspect, the present disclosure provides a pure TCR-negative allogeneic T cell composition (e.g., each cell in the composition expresses at such a low level that it is either undetectable or absent).

[0050] The expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated to prevent HvG and thereby improve the engraftment of cells in a subject. Improved engraftment results in longer persistence of cells and, therefore, a larger therapeutic window for the subject. Specifically, the expression and / or function of beta-2-microglobulin (B2M), a structural element of MHC-I, is reduced or eliminated.

[0051] The above strategy causes further challenge.T cell receptor (TCR) knocks out (KO) in T cell and causes the expression loss of CD3-ζ (CD3z or CD3ζ), which is the part of TCR complex.CD3ζ in TCR-KO T cell loses and sharply reduces the ability of using standard stimulation / activation reagent (including but not limited to agonist anti-CD3 mAb), optimally activates and amplifies these cells.When the expression or function of any one component of TCR complex are interrupted, all components of the complex are lost, including TCR-α (TCRα), TCR-β (TCRβ), CD3-γ (CD3γ), CD3-ε (CD3ε), CD3-δ (CD3δ) and CD3-ζ (CD3ζ).CD3ε and CD3ζ are both needed for T cell activation and amplification.Anti-CD3 mAb of agonist usually identifies another possible protein in CD3ε and complex, which in turn signals CD3ζ.CD3ζ provides the main stimulation (together with secondary costimulatory signals) for T cell activation, for optimal activation and amplification. Under normal conditions, complete T cell activation depends on the engagement of the TCR combined with the second signal, which is mediated by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL), which enhance the immune response. However, when TCR is not present, when standard activation / stimulation reagents (including agonist anti-CD3 mAb) are used to stimulate, T cell expansion is severely reduced. In fact, when standard activation / stimulation reagents (including agonist anti-CD3 mAb) are used to stimulate, T cell expansion is reduced to only 20-40% of normal expansion levels.

[0052] Accordingly, the present disclosure provides non-naturally occurring chimeric stimulatory receptors (CSRs) comprising: (a) an extracellular domain comprising a signal peptide and an activation component; (b) a transmembrane domain; and (c) an intracellular domain comprising a cytoplasmic domain and a signal transduction domain; and wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0053] The activation component can include 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 costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which the agonist of the activation component is bound. The activation component can include an agonist-bound CD2 extracellular domain or a portion thereof.

[0054] The signal transduction domain can include 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 can include a CD3 protein or a portion thereof. The CD3 protein can include a CD3 zeta protein or a portion thereof.

[0055] The activation domain can be isolated or derived from a first protein. The signal peptide can be isolated or derived from a second protein. The transmembrane domain can be isolated or derived from a third protein. The cytoplasmic domain can be isolated or derived from a fourth protein. The signaling domain can be isolated or derived from a fifth protein. The first and second proteins can be identical. The first and third proteins can be identical. The first and fourth proteins cannot be identical. The first and fifth proteins cannot be identical. The second and third proteins can be identical. The second and fourth proteins cannot be identical. The second and fifth proteins cannot be identical. The third and fourth proteins cannot be identical. The third and fifth proteins cannot be identical. The fourth and fifth proteins cannot be identical.

[0056] In some aspects, the activation component does not bind to a naturally occurring molecule. In some aspects, the activation component binds to a naturally occurring molecule, but the CSR does not transduce a signal after the activation component binds to the naturally occurring molecule. In some aspects, the activation component binds to a non-naturally occurring molecule. In some aspects, the activation component does not bind to a naturally occurring molecule, but instead binds to a non-naturally occurring molecule. The CSR can selectively transduce a signal after the activation component binds to the non-naturally occurring molecule.

[0057] The present disclosure provides nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides transposons or vectors comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0058] The present disclosure provides cells comprising any of the CSRs disclosed herein. The present disclosure provides cells comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides cells comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides cells comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0059] The modified cells disclosed herein can be allogeneic cells or autologous cells. In some preferred aspects, the modified cells are allogeneic cells. In some aspects, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred aspects, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0060] The present disclosure provides compositions comprising any of the CSRs disclosed herein. The present disclosure provides compositions comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising vectors comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising transposons comprising nucleic acid sequences encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising modified cells disclosed herein, or compositions comprising multiple modified cells disclosed herein.

[0061] 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) 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.

[0062] 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).

[0063] 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.

[0064] The modified T cell 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).

[0065] 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.

[0066] As described in detail herein, gene editing compositions, 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. In a preferred aspect, the gene editing composition targets and deletes a gene encoding an endogenous T cell receptor, a portion of a gene, or a regulatory element (e.g., a promoter) of a gene. Non-limiting examples of primers (including a T7 promoter, a genomic target sequence, and a gRNA scaffold) for generating guide RNA (gRNA) templates are disclosed in PCT Application No. PCT / US2019 / 049816, wherein the guide RNA template is used to target and delete TCR-α (TCR-α), target and delete TCR-β (TCR-β), and target and delete β-2-microglobulin (β2M).

[0067] Gene editing compositions, 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. In preferred aspects, the gene editing composition targets and deletes genes, portions of genes, or regulatory elements (e.g., promoters) encoding one or more components of endogenous MHC I, MHC II, or MHC activators. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT Application No. PCT / US2019 / 049816 (incorporated herein by reference in its entirety).

[0068] Detailed descriptions of non-naturally occurring chimeric stimulatory receptors, genetic modifications of endogenous sequences encoding TCR-alpha (TCR-α), TCR-beta (TCR-β) and / or beta-2-microglobulin (β2M), and non-naturally occurring polypeptides comprising an HLA class I histocompatibility antigen alpha chain E (HLA-E) polypeptide are disclosed in PCT Application No. PCT / US2019 / 049816 (incorporated herein by reference in its entirety).

[0069] Chimeric Stimulatory Receptors of the Disclosure

[0070] The present disclosure provides chimeric stimulatory receptors (CSRs) comprising an activating component comprising, consisting essentially of, or consisting of an agonist-binding extracellular domain of CD2, or a portion thereof. The agonist-binding extracellular domain of CD2, or a portion thereof, comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to:

[0071] KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (SEQ ID NO: 1). In a preferred aspect, the agonist-binding CD2 extracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1.

[0072] In some aspects, the agonist-binding extracellular domain of CD2, or a portion thereof, is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACATCTATAAGGTGTCCATCTACGACACCAAGGG In a preferred aspect, the agonist-bound CD2 extracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 2.

[0073] The present disclosure provides a chimeric stimulatory receptor (CSR) comprising an extracellular domain comprising an activation component, the activation component comprising, essentially consisting of, or consisting of a non-naturally occurring CD2 extracellular domain. In some aspects, the extracellular domain of the CSR of the present disclosure may comprise a modification. The modification may comprise a mutation or truncation in the amino acid sequence of the activation component compared to the wild-type amino acid sequence of the activation component. The mutation or truncation in the amino acid sequence of the activation component may comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. 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. In some aspects, the mutation present in the CD2 extracellular domain that binds to anti-CD2 activating agonists but does not bind to CD58 is a D111H mutation. The CD2 extracellular structure having the D111H mutation comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the following: KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (SEQ ID NO: 3). In a preferred aspect, the CD2 extracellular domain having the D111H mutation comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3.

[0074] In some aspects, the extracellular domain of CD2 having a D111H mutation is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to: (SEQ ID NO: 4). In a preferred aspect, the extracellular domain of CD2 having a D111H mutation is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 4.

[0075] The intracellular domain of the CSR of the present disclosure may further comprise, consist essentially of, or consist of a signal peptide. In some aspects, the signal peptide may comprise, consist essentially of, or consist of a CD2 signal peptide or a portion thereof. In some aspects, the signal peptide may comprise, consist essentially of, or consist of a CD8a signal peptide or a portion thereof.

[0076] In some aspects, the CD2 signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 5). In a preferred aspect, the CD2 signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5.

[0077] In some aspects, the CD2 signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAGGGCGCCGTGTCC (SEQ ID NO: 6). In a preferred aspect, the CD2 signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 6.

[0078] In some aspects, the CD8a signal peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to: MALPVTALLLPLALLLHAARP (SEQ ID NO: 7). In a preferred aspect, the CD8a signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7.

[0079] In some aspects, the CD8a signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to: ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT (SEQ ID NO: 8). In a preferred aspect, the CD8a signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 8.

[0080] The present disclosure provides CSRs comprising a transmembrane domain. In some aspects, the transmembrane domain can comprise, consist essentially of, or consist of the CD2 transmembrane domain, or a portion thereof. In some aspects, the CD2 transmembrane domain, or a portion thereof, comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 9). In a preferred aspect, the CD2 transmembrane domain, or a portion thereof, comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9.

[0081] In some aspects, the CD2 transmembrane domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACATCACC (SEQ ID NO: 10). In a preferred aspect, the CD2 transmembrane domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 10.

[0082] The present disclosure provides a CSR comprising an intracellular domain comprising at least one signal transduction domain. In some aspects, the signal transduction domain can comprise, consist essentially of, or consist of the CD3 zeta intracellular domain or a portion thereof. In some aspects, the CD3 zeta intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11). In a preferred aspect, the CD3 zeta intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11.

[0083] In some aspects, the CD3 zeta intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to In one preferred aspect, the CD3 zeta intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 12.

[0084] The intracellular domain of the CSR of the present disclosure can further include a cytoplasmic domain, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include a CD2 intracellular domain (ICD) or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include a CD28 intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include a 4-1BB intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include an IL17RA intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include an IL15RA intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include an IL21R intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can include an ICOS intracellular domain or a portion thereof, be essentially composed of, or be composed of. In some aspects, the cytoplasmic domain can comprise, consist essentially of, or consist of the CD27 intracellular domain, or a portion thereof. In some aspects, the cytoplasmic domain can comprise, consist essentially of, or consist of the OX40 intracellular domain, or a portion thereof. In some aspects, the cytoplasmic domain can comprise, consist essentially of, or consist of the GITR intracellular domain, or a portion thereof.

[0085] In some aspects, the CD2 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the following: KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 13). In a preferred aspect, the CD2 intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13.

[0086] In some aspects, the CD2 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCACCTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAAC (SEQ ID NO: 14). In a preferred aspect, the CD2 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 14.

[0087] In some aspects, the CD28 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 15). In a preferred aspect, the CD28 intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15.

[0088] In some aspects, the CD28 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: AGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCC (SEQ ID NO: 16). In a preferred aspect, the CD28 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 16.

[0089] In some aspects, the 4-1BB intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 17). In a preferred aspect, the 4-1BB intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17.

[0090] In some aspects, the 4-1BB intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to: AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTG (SEQ ID NO: 18). In a preferred aspect, the 4-1BB intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 18.

[0091] In some aspects, the IL17RA intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ (SEQ ID NO: 19). In a preferred aspect, the IL17RA intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 19.

[0092] In some aspects, the IL17RA intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: AAGAAGCGGATCAAGCCCATCGTGTGGCCCAGCCTGCCTGACCACAAGAAAACCCTGGAACACCTGTGCAAGAAGCCCCGGAAGAACCTGAATGTGTCCTTCAATCCCGAGAGCTTCCTGGACTGCCAGATCCACAGAGTGGACGACATCCAGGCCAGAGATGAGGTGGAAGGCTTTCTGCAGGACACCTTTCCACAGCAGCTGGAAGAGAGCGAGAAGCAGAGACTCGGCGGAGATGTGCAGAGCCCTAATTGCCCTAGCGAGGACGTGGTCATCACCCCTGA (SEQ ID NO: 20). In a preferred aspect, the IL17RA intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 20.

[0093] In some aspects, the IL15RA intracellular domain, or a portion thereof, comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 21). In a preferred aspect, the IL15RA intracellular domain, or a portion thereof, comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21.

[0094] In some aspects, the IL15RA intracellular domain, or a portion thereof, is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: AAGAGCCCGGCAGACACCTCCTCTGGCCAGCGTGGAAATGGAAGCCATGGAAGCTCTGCCTGTGACCTGGGGCACAAGCAGCAGAGATGAGGACCTGGAAAACTGCAGCCACCACCTG (SEQ ID NO: 22). In a preferred aspect, the IL15RA intracellular domain, or a portion thereof, is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 22.

[0095] In some aspects, the IL21R intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (SEQ ID NO: 23). In a preferred aspect, the IL21R intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23.

[0096] In some aspects, the IL21R intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (or any percentage in between) Equivalent to: AGCCTGAAAACACACCCACTCTGGCGGCTGTGAAGAAAATCTGGGCCGTGCCATCTCCTGAGCGGTTCTTCATGCCTCTGTACAAGGGCTGCAGCGGCGACTTCAAGAAATGGGTCGGAGCCCCTTTTACCGGCAGCTCTCTGGAACTTGGACCTTGGAGCCCTGAGGTGCCCAGCACACTGGAAGTGTACAGCTGTCAC CCTCCTAGAAGCCCCGCCAAGAGACTGCAGCTGACAGAGCTGCAAGAGCCTGCCGAGCTGGTGGAATCTGATGGCGTGCCCAAGCCTAGCTTCTGGCCCACAGCTCAGAATAGCGGCGGCTCTGCCTACAGCGAGGAAAGGGATAGACCTTACGGCCTGGTGTCTATCGACACCGTGACCGTGCTGGATGCCGAGGGACCTTGTACATGGCCTTGCAG CTGCGAGGACGATGGCTACCCTGCTCTGGATCTGGACGCAGGCCTTGAGCCTTCTCCAGGACTGGAAGATCCTCTGCTGGACGCCGGAACAACCGTGCTGTCTTGTGGCTGTGTGTCTGCCGGATCTCCTGGACTTGGAGGCCCTCTGGGAAGCCTGCTGGATAGACTGAAACCTCCTGGCCGACGGCGAAGATTGGGCTGGTGGACTTCCTTGGG GCGGAAGATCTCCAGGCGGAGTGTCTGAGTCTGAAGCCGGTTCTCCACTGGCCGGCCTGGACATGGATACCTTCGATTCTGGCTTCGTGGGCAGCGACTGTAGCAGCCCTGTGGAATGCGACTTCACAAGCCCTGGCGACGAGGGCCCACCTAGAAGCTATCTGAGACAGTGGGTCGTGATCCCTCCACCTCTGTCTAGTCCTGGACCTCAGGCCAGC (SEQ ID NO: 24).In a preferred aspect, the IL21R intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 24.

[0097] In some aspects, the ICOS intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 25). In a preferred aspect, the ICOS intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

[0098] In some aspects, the ICOS intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: TGTTGGCTGACCAAGAAAAAGTACAGCAGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTG (SEQ ID NO: 26). In a preferred aspect, the ICOS intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 26.

[0099] In some aspects, the CD27 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 27). In a preferred aspect, the CD27 intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 27.

[0100] In some aspects, the CD27 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: CAGCGGCGGAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAACCTGCCGAGCCTTGTCACTACAGCTGCCCCAGAGAGGAAGAGGGCAGCACAATCCCCATCCAAGAGGACTACAGAAAGCCCGAGCCTGCCTGCTCTCCC (SEQ ID NO: 28). In a preferred aspect, the CD27 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 28.

[0101] In some aspects, the OX40 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 29). In a preferred aspect, the OX40 intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 29.

[0102] In some aspects, the OX40 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: GCCCTGTACCTGCTGCGGCGGGATCAAAGATTGCCTCCTGACGCTCACAAGCCTCCAGGCGGAGGCAGCTTTAGAACCCCTATCCAAGAGGAACAGGCTGACGCCCACAGCACCCTGGCCAAGATC (SEQ ID NO 30). In a preferred aspect, the OX40 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO 30.

[0103] In some aspects, the GITR intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 31). In a preferred aspect, the GITR intracellular domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 31.

[0104] In some aspects, the GITR intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to: CAGCTGGGACTGCACATCTGGCAGCTGAGAAGCCAGTGCATGTGGCCCAGAGAGACACAGCTGCTGCTGGAAGTGCCTCCTAGCACCGAGGATGCCAGAAGCTGTCAGTTCCCCGAGGAAGAGAGGCGAGAGATCCGCCGAGGAAAAAGGCAGACTGGGCGACCTGTGGGTCCGAGTG (SEQ ID NO: 32). In a preferred aspect, the GITR intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 32.

[0105] Exemplary "CSR CD2z" polypeptides of the present disclosure comprise (CD2 signal peptide, CD2 extracellular domain 、 CD2 transmembrane domain 、 CD2 cytoplasmic domain , CD3ζ intracellular domain) amino acid sequence:

[0106] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHR PPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 33)

[0107] CD2 signal peptide: SEQ ID NO: 5

[0108] CD2 extracellular domain :SEQ ID NO: 1

[0109] CD2 transmembrane domain :SEQ ID NO: 9

[0110] CD2 cytoplasmic domain :SEQ ID NO: 13

[0111] CD3ζ intracellular domain: SEQ ID NO: 11

[0112] An exemplary polynucleotide sequence encoding a "CSR CD2z" polypeptide of the present disclosure comprises (CD2 signal peptide, CD2 Extracellular domain 、 CD2 transmembrane domain 、 CD2 cytoplasmic domain , CD3ζ intracellular domain) nucleic acid sequence:

[0113] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAAC ACGGGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCT GCCACCTCTCAACACCCTCCACCTCCACCTGGACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTC ATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCC ACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAACAGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 34)

[0114] CD2 signal peptide: SEQ ID NO: 6

[0115] CD2 extracellular domain :SEQ ID NO: 2

[0116] CD2 transmembrane domain :SEQ ID NO: 10

[0117] CD2 cytoplasmic domain :SEQ ID NO: 14

[0118] CD3ζ intracellular domain: SEQ ID NO: 12

[0119] An exemplary "mutant CSR CD2z-D111H" or "CD2.DH.z" or "CSR 01CD2.DH.z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular structure domain 、 CD2 transmembrane domain 、 CD2 cytoplasmic domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0120] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHR PPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 35)

[0121] CD2 signal peptide: SEQ ID NO: 5

[0122] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain:

[0123] SEQ ID NO: 3

[0124] CD2 transmembrane domain :SEQ ID NO: 9

[0125] CD2 cytoplasmic domain :SEQ ID NO: 13

[0126] CD3ζ intracellular domain: SEQ ID NO: 11

[0127] An exemplary polynucleotide sequence encoding a "mutant CSR CD2z-D111H" or "CD2.DH.z" or "CSR 01CD2.DH.z" polypeptide of the present disclosure comprises (CD2 signal peptide, D111H in the CD2 extracellular domain Mutated CD2 extracellular domain 、 CD2 transmembrane domain 、 CD2 cytoplasmic domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0128] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAAC ACGGGCCCATAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCT GCCACCTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTC ATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCC ACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAACAGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 36)

[0129] CD2 signal peptide: SEQ ID NO: 6

[0130] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain:

[0131] SEQ ID NO: 4

[0132] CD2 transmembrane domain :SEQ ID NO: 10

[0133] CD2 cytoplasmic domain :SEQ ID NO: 14

[0134] CD3ζ intracellular domain: SEQ ID NO: 12

[0135] An exemplary "mutant CSR 01b CD2.8.DH.z" or "CD2.8.DH.z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 CD2 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0136] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPP PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 37)

[0137] CD8a signal peptide: SEQ ID NO: 7

[0138] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0139] CD2 transmembrane domain :SEQ ID NO: 9

[0140] CD2 intracellular domain :SEQ ID NO: 13

[0141] CD3ζ intracellular domain: SEQ ID NO: 11

[0142] An exemplary polynucleotide sequence encoding a "mutant CSR 01b CD2.8.DH.z" or "CD2.8.DH.z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular structure domain 、 CD2 transmembrane domain 、 CD2 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0143] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCA TAGAGTGGCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTGCCACCTCT CAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTCATCGGGTGC AGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAGGTGCACCAGCAAAAAGGACCTCCACTGCCTCG GCCTAGAGTGCAGCCTAAACCTCCTCATGGCGCCGCTGAGAACAGCCTGTCTCCAAGCAGCAACAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 38)

[0144] CD8a signal peptide: SEQ ID NO: 8

[0145] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0146] CD2 transmembrane domain :SEQ ID NO: 10

[0147] CD2 intracellular domain :SEQ ID NO: 14

[0148] CD3ζ intracellular domain: SEQ ID NO: 12

[0149] An exemplary "mutant CSR 02 CD2.DH.28z" or "CD2.DH.28.z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 CD28 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0150] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 39)

[0151] CD2 signal peptide: SEQ ID NO: 5

[0152] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 3

[0153] CD2 transmembrane domain :SEQ ID NO: 9

[0154] CD28 intracellular domain :SEQ ID NO: 15

[0155] CD3ζ intracellular domain: SEQ ID NO: 11

[0156] An exemplary polynucleotide sequence encoding a "mutant CSR 02 CD2.DH.28z" or "CD2.DH.28.z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 CD28 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0157] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGAC CCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 40)

[0158] CD2 signal peptide: SEQ ID NO: 6

[0159] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 4

[0160] CD2 transmembrane domain :SEQ ID NO: 10

[0161] CD28 intracellular domain :SEQ ID NO: 16

[0162] CD3ζ intracellular domain: SEQ ID NO: 12

[0163] An exemplary "mutant CSR 02b CD2.8.DH.28z" or "CD2.8.DH.28z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 CD28 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0164] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 41)

[0165] CD8a signal peptide: SEQ ID NO: 7

[0166] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 3

[0167] CD2 transmembrane domain :SEQ ID NO: 9

[0168] CD28 intracellular domain :SEQ ID NO: 15

[0169] CD3ζ intracellular domain: SEQ ID NO: 11

[0170] An exemplary polynucleotide sequence encoding a "mutant CSR 02b CD2.8.DH.28z" or "CD2.8.DH.28z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 CD28 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0171] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC AGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACG GCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 42)

[0172] CD8a signal peptide: SEQ ID NO: 8

[0173] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 4

[0174] CD2 transmembrane domain :SEQ ID NO: 10

[0175] CD28 intracellular domain :SEQ ID NO: 16

[0176] CD3ζ intracellular domain: SEQ ID NO: 12

[0177] An exemplary "mutant CSR 03 CD2.DH.BBz" or "CD2.DH.BBz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 4- 1BB intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0178] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 43)

[0179] CD2 signal peptide: SEQ ID NO: 5

[0180] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 3

[0181] CD2 transmembrane domain :SEQ ID NO: 9

[0182] 4-1BB intracellular domain :SEQ ID NO: 17

[0183] CD3ζ intracellular domain: SEQ ID NO: 11

[0184] An exemplary polynucleotide sequence encoding a "mutant CSR 03 CD2.DH.BBz" or "CD2.DH.BBz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 4-1BB intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0185] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCAT GCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAG CTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 44)

[0186] CD2 signal peptide: SEQ ID NO: 6

[0187] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain: SEQ ID NO: 4

[0188] CD2 transmembrane domain :SEQ ID NO: 10

[0189] 4-1BB intracellular domain :SEQ ID NO: 18

[0190] CD3ζ intracellular domain: SEQ ID NO: 12

[0191] An exemplary "mutant CSR 03 CD2.8.DH.BBz" or "CD2.8.DH.BBz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 4-1BB intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0192] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 45)

[0193] CD8a signal peptide: SEQ ID NO: 7

[0194] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0195] CD2 transmembrane domain :SEQ ID NO: 9

[0196] 4-1BB intracellular domain :SEQ ID NO: 17

[0197] CD3ζ intracellular domain: SEQ ID NO: 11

[0198] An exemplary polynucleotide sequence encoding a "mutant CSR 03b CD2.8.DH.BBz" or "CD2.8.DH.BBz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 4-1BB intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0199] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGT GCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 46)

[0200] CD8a signal peptide: SEQ ID NO: 8

[0201] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0202] CD2 transmembrane domain :SEQ ID NO: 10

[0203] 4-1BB intracellular domain :SEQ ID NO: 18

[0204] CD3ζ intracellular domain: SEQ ID NO: 12

[0205] An exemplary "mutant CSR 04 CD2.DH.7z" or "CD2.DH.7z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL7RA Intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0206] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT KKRIKPIVWPSLLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQ DTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSSRSLDCRESGKNGPHVY QDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 47)

[0207] CD2 signal peptide: SEQ ID NO: 5

[0208] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0209] CD2 transmembrane domain :SEQ ID NO: 9

[0210] IL7RA intracellular domain :SEQ ID NO: 19

[0211] CD3ζ intracellular domain: SEQ ID NO: 11

[0212] An exemplary polynucleotide sequence encoding a "mutant CSR 04 CD2.DH.7z" or "CD2.DH.7z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL7RA intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0213] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AAGAAGCGGATCAAGCCCATCGTGTGGCCCAGCCTGCCTGACCACAA GAAAACCCTGGAACACCTGTGCAAGAAGCCCCGGAAGAACCTGAATGTGTCCTTCAATCCCGAGAGCTTCCTGGAC TGCCAGATCCACAGAGTGGACGACATCCAGGCCAGAGATGAGGTGGAAGGCTTTCTGCAGGACACCTTTCCACAGC AGCTGGAAGAGAGCGAGAAGCAGAGACTCGGCGGAGATGTGCAGAGCCCTAATTGCCCTAGCGAGGACGTGGTCAT CACCCCTGAGAGCTTCGGCAGAGATAGCAGCCTGACATGTCTGGCCGGCAATGTGTCCGCCTGTGATGCCCCTATC CTGAGCAGCAGCAGAAGCCTGGATTGCAGAGAGAGCGGAAAGAACGGCCCTCATGTGTATCAGGACCTGCTGCTGA GCCTGGGCACCACCAATTCTACACTGCCTCCACCATTCAGCCTGCAGAGCGGCATCCTGACACTGAACCCTGTTGC TCAGGGCCAGCCAATCCTGACAAGCCTGGGCTCCAATCAAGAAGAGGCCTACGTCACCATGTCCAGCTTCTACCAG AACCAGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 48)

[0214] CD2 signal peptide: SEQ ID NO: 6

[0215] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0216] CD2 transmembrane domain :SEQ ID NO: 10

[0217] IL7RA intracellular domain :SEQ ID NO: 20

[0218] CD3ζ intracellular domain: SEQ ID NO: 12

[0219] An exemplary "mutant CSR 04b CD2.8.DH.7z" or "CD2.8.DH.7z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 IL7RA intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0220] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTF PQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDL LLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 49)

[0221] CD8a signal peptide: SEQ ID NO: 7

[0222] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0223] CD2 transmembrane domain :SEQ ID NO: 9

[0224] IL7RA intracellular domain :SEQ ID NO: 19

[0225] CD3ζ intracellular domain: SEQ ID NO: 11

[0226] An exemplary polynucleotide sequence encoding a "mutant CSR 04b CD2.8.DH.7z" or "CD2.8.DH.7z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular structure domain 、 CD2 transmembrane domain 、 IL7RA intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0227] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTTGTGGCTC TGCTGGTGTTCTACATCACC AAGAAGCGGATCAAGCCCATCGTGTGGCCCAGCCTGCCTGACCACAAGAAAACCCT GGAACACCTGTGCAAGAAGCCCCGGAAGAACCTGAATGTGTCCTTCAATCCCGAGAGCTTCCTGGACTGCCAGATC CACAGAGTGGACGACATCCAGGCCAGAGATGAGGTGGAAGGCTTTCTGCAGGACACCTTTCCACAGCAGCTGGAAG AGAGCGAGAAGCAGAGACTCGGCGGAGATGTGCAGAGCCCTAATTGCCCTAGCGAGGACGTGGTCATCACCCCTGA GAGCTTCGGCAGAGATAGCAGCCTGACATGTCTGGCCGGCAATGTGTCCGCCTGTGATGCCCCTATCCTGAGCAGC AGCAGAAGCCTGGATTGCAGAGAGAGCGGAAAGAACGGCCCTCATGTGTATCAGGACCTGCTGCTGAGCCTGGGCA CCACCAATTCTACACTGCCTCCACCATTCAGCCTGCAGAGCGGCATCCTGACACTGAACCCTGTTGCTCAGGGCCA GCCAATCCTGACAAGCCTGGGCTCCAATCAAGAAGAGGCCTACGTCACCATGTCCAGCTTCTACCAGAACCAGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 50)

[0228] CD8a signal peptide: SEQ ID NO: 8

[0229] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0230] CD2 transmembrane domain :SEQ ID NO: 10

[0231] IL7RA intracellular domain :SEQ ID NO: 20

[0232] CD3ζ intracellular domain: SEQ ID NO: 12

[0233] An exemplary "mutant CSR 05 CD2.DH.15z" or "CD2.DH.15z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL15RA intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0234] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 51)

[0235] CD2 signal peptide: SEQ ID NO: 5

[0236] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0237] CD2 transmembrane domain :SEQ ID NO: 9

[0238] IL15RA intracellular domain :SEQ ID NO: 21

[0239] CD3ζ intracellular domain: SEQ ID NO: 11

[0240] An exemplary polynucleotide sequence encoding a "mutant CSR 05 CD2.DH.15z" or "CD2.DH.15z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL15RA intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0241] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AAGAGCCGGCAGACACCTCCTCTGGCCAGCGTGGAAATGGAAGCCAT GGAAGCTCTGCCTGTGACCTGGGGCACAAGCAGCAGAGATGAGGACCTGGAAAACTGCAGCCACCACCTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 52)

[0242] CD2 signal peptide: SEQ ID NO: 6

[0243] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0244] CD2 transmembrane domain :SEQ ID NO: 10

[0245] IL15RA intracellular domain :SEQ ID NO: 22

[0246] CD3ζ intracellular domain: SEQ ID NO: 12

[0247] An exemplary "mutant CSR 05b CD2.8.DH.15z" or "CD2.8.DH.15z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 IL15RA intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0248] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 53)

[0249] CD8a signal peptide: SEQ ID NO: 7

[0250] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0251] CD2 transmembrane domain :SEQ ID NO: 9

[0252] IL15RA intracellular domain :SEQ ID NO: 21

[0253] CD3ζ intracellular domain: SEQ ID NO: 11

[0254] An exemplary polynucleotide sequence encoding a "mutant CSR 05b CD2.8.DH.15z" or "CD2.8.DH.15z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 IL15RA intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0255] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC AAGAGCCGGCAGACACCTCCTCTGGCCAGCGTGGAAATGGAAGCCATGGAAGCTCT GCCTGTGACCTGGGGCACAAGCAGCAGAGATGAGGACCTGGAAAACTGCAGCCACCACCTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 54)

[0256] CD8a signal peptide: SEQ ID NO: 8

[0257] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0258] CD2 transmembrane domain :SEQ ID NO: 10

[0259] IL15RA intracellular domain :SEQ ID NO: 22

[0260] CD3ζ intracellular domain: SEQ ID NO: 12

[0261] An exemplary "mutant CSR 06 CD2.DH.21z" or "CD2.DH.21z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL21R intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0262] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSLELGPWSPEVPSTL EVYSCHPPSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTW PCSCEDDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWG GRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQASRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 55)

[0263] CD2 signal peptide: SEQ ID NO: 5

[0264] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0265] CD2 transmembrane domain :SEQ ID NO: 9

[0266] IL21R intracellular domain :SEQ ID NO: 23

[0267] CD3ζ intracellular domain: SEQ ID NO: 11

[0268] An exemplary polynucleotide sequence encoding a "mutant CSR 06 CD2.DH.21z" or "CD2.DH.21z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 IL21R intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0269] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC AGCCTGAAAACACACCCACTCTGGCGGCTGTGGAAGAAAATCTGGGC CGTGCCATCTCCTGAGCGGTTCTTCATGCCTCTGTACAAGGGCTGCAGCGGCGACTTCAAGAAATGGGTCGGAGCC CCTTTTACCGGCAGCTCTCTGGAACTTGGACCTTGGAGCCCTGAGGTGCCCAGCACACTGGAAGTGTACAGCTGTC ACCCTCCTAGAAGCCCCGCCAAGAGACTGCAGCTGACAGAGCTGCAAGAGCCTGCCGAGCTGGTGGAATCTGATGG CGTGCCCAAGCCTAGCTTCTGGCCCACAGCTCAGAATAGCGGCGGCTCTGCCTACAGCGAGGAAAGGGATAGACCT TACGGCCTGGTGTCTATCGACACCGTGACCGTGCTGGATGCCGAGGGACCTTGTACATGGCCTTGCAGCTGCGAGG ACGATGGCTACCCTGCTCTGGATCTGGACGCAGGCCTTGAGCCTTCTCCAGGACTGGAAGATCCTCTGCTGGACGC CGGAACAACCGTGCTGTCTTGTGGCTGTGTGTCTGCCGGATCTCCTGGACTTGGAGGCCCTCTGGGAAGCCTGCTG GATAGACTGAAACCTCCTCTGGCCGACGGCGAAGATTGGGCTGGTGGACTTCCTTGGGGCGGAAGATCTCCAGGCG GAGTGTCTGAGTCTGAAGCCGGTTCTCCACTGGCCGGCCTGGACATGGATACCTTCGATTCTGGCTTCGTGGGCAG CGACTGTAGCAGCCCTGTGGAATGCGACTTCACAAGCCCTGGCGACGAGGGCCCACCTAGAAGCTATCTGAGACAG TGGGTCGTGATCCCTCCACCTCTGTCTAGTCCTGGACCTCAGGCCAGCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 56)

[0270] CD2 signal peptide: SEQ ID NO: 6

[0271] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0272] CD2 transmembrane domain :SEQ ID NO: 10

[0273] IL21R intracellular domain :SEQ ID NO: 24

[0274] CD3ζ intracellular domain: SEQ ID NO: 12

[0275] An exemplary "mutant CSR 06b CD2.8.DH.21z" or "CD2.8.DH.21z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 IL21R intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0276] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSLELGPWSPEVPSTLEVY SCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCS CEDDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRS PGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQASRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 57)

[0277] CD8a signal peptide: SEQ ID NO: 7

[0278] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0279] CD2 transmembrane domain :SEQ ID NO: 9

[0280] IL21R intracellular domain :SEQ ID NO: 23

[0281] CD3ζ intracellular domain: SEQ ID NO: 11

[0282] An exemplary polynucleotide sequence encoding a "mutant CSR 06b CD2.8.DH.21z" or "CD2.8.DH.21z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 IL21R intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0283] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTTGTGGCTC TGCTGGTGTTCTACATCACC AGCCTGAAAACACACCCACTCTGGCGGCTGTGGAAGAAAATCTGGGCCGTGCCATC TCCTGAGCGGTTCTTCATGCCTCTGTACAAGGGCTGCAGCGGCGACTTCAAGAAATGGGTCGGAGCCCCTTTTACC GGCAGCTCTCTGGAACTTGGACCTTGGAGCCCTGAGGTGCCCAGCACACTGGAAGTGTACAGCTGTCACCCTCCTA GAAGCCCCGCCAAGAGACTGCAGCTGACAGAGCTGCAAGAGCCTGCCGAGCTGGTGGAATCTGATGGCGTGCCCAA GCCTAGCTTCTGGCCCACAGCTCAGAATAGCGGCGGCTCTGCCTACAGCGAGGAAAGGGATAGACCTTACGGCCTG GTGTCTATCGACACCGTGACCGTGCTGGATGCCGAGGGACCTTGTACATGGCCTTGCAGCTGCGAGGACGATGGCT ACCCTGCTCTGGATCTGGACGCAGGCCTTGAGCCTTCTCCAGGACTGGAAGATCCTCTGCTGGACGCCGGAACAAC CGTGCTGTCTTGTGGCTGTGTGTCTGCCGGATCTCCTGGACTTGGAGGCCCTCTGGGAAGCCTGCTGGATAGACTG AAACCTCCTCTGGCCGACGGCGAAGATTGGGCTGGTGGACTTCCTTGGGGCGGAAGATCTCCAGGCGGAGTGTCTG AGTCTGAAGCCGGTTCTCCACTGGCCGGCCTGGACATGGATACCTTCGATTCTGGCTTCGTGGGCAGCGACTGTAG CAGCCCTGTGGAATGCGACTTCACAAGCCCTGGCGACGAGGGCCCACCTAGAAGCTATCTGAGACAGTGGGTCGTG ATCCCTCCACCTCTGTCTAGTCCTGGACCTCAGGCCAGCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 58)

[0284] CD8a signal peptide: SEQ ID NO: 8

[0285] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0286] CD2 transmembrane domain :SEQ ID NO: 10

[0287] IL21R intracellular domain :SEQ ID NO: 24

[0288] CD3ζ intracellular domain: SEQ ID NO: 12

[0289] An exemplary "mutant CSR 07 CD2.DH.Iz" or "CD2.DH.Iz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 ICOS Intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0290] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 59)

[0291] CD2 signal peptide: SEQ ID NO: 5

[0292] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0293] CD2 transmembrane domain :SEQ ID NO: 9

[0294] ICOS intracellular domain :SEQ ID NO: 25

[0295] CD3ζ intracellular domain: SEQ ID NO: 11

[0296] An exemplary polynucleotide sequence encoding a "mutant CSR 07 CD2.DH.Iz" or "CD2.DH.Iz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 ICOS intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0297] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC TGTTGGCTGACCAAGAAAAAGTACAGCAGCAGCGTGCACGACCCCAA CGGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 60)

[0298] CD2 signal peptide: SEQ ID NO: 6

[0299] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0300] CD2 transmembrane domain :SEQ ID NO: 10

[0301] ICOS intracellular domain :SEQ ID NO: 26

[0302] CD3ζ intracellular domain: SEQ ID NO: 12

[0303] An exemplary "mutant CSR 07b CD2.8.DH.Iz" or "CD2.8.DH.Iz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 ICOS intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0304] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61)

[0305] CD8a signal peptide: SEQ ID NO: 7

[0306] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0307] CD2 transmembrane domain :SEQ ID NO: 9

[0308] ICOS intracellular domain : SEQ ID NO: 25

[0309] CD3ζ intracellular domain: SEQ ID NO: 11

[0310] An exemplary polynucleotide sequence encoding a "mutant CSR 07b CD2.8.DH.Iz" or "CD2.8.DH.Iz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular structure domain 、 CD2 transmembrane domain 、 ICOS intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0311] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC TGTTGGCTGACCAAGAAAAAGTACAGCAGCAGCGTGCACGACCCCAACGGCGAGTA CATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTGAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 62)

[0312] CD8a signal peptide: SEQ ID NO: 8

[0313] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0314] CD2 transmembrane domain :SEQ ID NO: 10

[0315] ICOS intracellular domain :SEQ ID NO: 26

[0316] CD3ζ intracellular domain: SEQ ID NO: 12

[0317] An exemplary "mutant CSR 08 CD2.DH.27z" or "CD2.DH.27z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 CD27 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0318] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 63)

[0319] CD2 signal peptide: SEQ ID NO: 5

[0320] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0321] CD2 transmembrane domain :SEQ ID NO: 9

[0322] CD27 intracellular domain :SEQ ID NO: 27

[0323] CD3ζ intracellular domain: SEQ ID NO: 11

[0324] An exemplary polynucleotide sequence encoding a "mutant CSR 08 CD2.DH.27z" or "CD2.DH.27z" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 CD27 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0325] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC CAGCGGCGGAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAACC TGCCGAGCCTTGTCACTACAGCTGCCCCAGAGAGGAAGAGGGCAGCACAATCCCCATCCAAGAGGACTACAGAAAG CCCGAGCCTGCCTGCTCTCCCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 64)

[0326] CD2 signal peptide: SEQ ID NO: 6

[0327] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0328] CD2 transmembrane domain :SEQ ID NO: 10

[0329] CD27 intracellular domain :SEQ ID NO: 28

[0330] CD3ζ intracellular domain: SEQ ID NO: 12

[0331] An exemplary "mutant CSR 08b CD2.8.DH.27z" or "CD2.8.DH.27z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 CD27 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0332] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 65)

[0333] CD8a signal peptide: SEQ ID NO: 7

[0334] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0335] CD2 transmembrane domain :SEQ ID NO: 9

[0336] CD27 intracellular domain :SEQ ID NO: 27

[0337] CD3ζ intracellular domain: SEQ ID NO: 11

[0338] An exemplary polynucleotide sequence encoding a "mutant CSR 08b CD2.8.DH.27z" or "CD2.8.DH.27z" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 CD27 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0339] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC CAGCGGCGGAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAACCTGCCGAGCC TTGTCACTACAGCTGCCCCAGAGAGGAAGAGGGCAGCACAATCCCCATCCAAGAGGACTACAGAAAGCCCGAGCCT GCCTGCTCTCCCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 66)

[0340] CD8a signal peptide: SEQ ID NO: 8

[0341] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0342] CD2 transmembrane domain :SEQ ID NO: 10

[0343] CD27 intracellular domain :SEQ ID NO: 28

[0344] CD3ζ intracellular domain: SEQ ID NO: 12

[0345] An exemplary "mutant CSR 09 CD2.DH.Oxz" or "CD2.DH.Oxz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 OX40 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0346] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 67)

[0347] CD2 signal peptide: SEQ ID NO: 5

[0348] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0349] CD2 transmembrane domain :SEQ ID NO: 9

[0350] OX40 intracellular domain :SEQ ID NO: 29

[0351] CD3ζ intracellular domain: SEQ ID NO: 11

[0352] An exemplary polynucleotide sequence encoding a "mutant CSR 09 CD2.DH.Oxz" or "CD2.DH.Oxz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 OX40 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0353] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACC GCCCTGTACCTGCTGCGGCGGGATCAAAGATTGCCTCCTGACGCTCA CAAGCCTCCAGGCGGAGGCAGCTTTAGAACCCCTATCCAAGAGGAACAGGCTGACGCCCACAGCACCCTGGCCAAG ATCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 68)

[0354] CD2 signal peptide: SEQ ID NO: 6

[0355] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0356] CD2 transmembrane domain :SEQ ID NO: 10

[0357] OX40 intracellular domain :SEQ ID NO: 30

[0358] CD3ζ intracellular domain: SEQ ID NO: 12

[0359] An exemplary "mutant CSR 09b CD2.8.DH.Oxz" or "CD2.8.DH.Oxz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 OX40 intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0360] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 69)

[0361] CD8a signal peptide: SEQ ID NO: 7

[0362] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0363] CD2 transmembrane domain :SEQ ID NO: 9

[0364] OX40 intracellular domain :SEQ ID NO: 29

[0365] CD3ζ intracellular domain: SEQ ID NO: 11

[0366] An exemplary polynucleotide sequence encoding a "mutant CSR 09b CD2.8.DH.Oxz" or "CD2.8.DH.Oxz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular binding domain 、 CD2 transmembrane domain 、 OX40 intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0367] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACC GCCCTGTACCTGCTGCGGCGGGATCAAAGATTGCCTCCTGACGCTCACAAGCCTCC AGGCGGAGGCAGCTTTAGAACCCCTATCCAAGAGGAACAGGCTGACGCCCACAGCACCCTGGCCAAGATCAGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 70)

[0368] CD8a signal peptide: SEQ ID NO: 8

[0369] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0370] CD2 transmembrane domain :SEQ ID NO: 10

[0371] OX40 intracellular domain :SEQ ID NO: 30

[0372] CD3ζ intracellular domain: SEQ ID NO: 12

[0373] An exemplary "mutant CSR10 CD2.DH.Gz" or "CD2.DH.Gz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 GITR cells Intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0374] MSFPCKFVASFLLIFNVSSKGAVS KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKI AQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTL TCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGG SLLMVFVALLVFYIT QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 71)

[0375] CD2 signal peptide: SEQ ID NO: 5

[0376] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0377] CD2 transmembrane domain :SEQ ID NO: 9

[0378] GITR intracellular domain :SEQ ID NO: 31

[0379] CD3ζ intracellular domain: SEQ ID NO: 11

[0380] An exemplary polynucleotide sequence encoding a "mutant CSR10 CD2.DH.Gz" or "CD2.DH.Gz" polypeptide of the present disclosure comprises (CD2 signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane domain 、 GITR intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0381] ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTTCTAAGGGCGCCGTGTCC AAAGAAATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTC CAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAG AGAAAGAGACATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAAC CGACGACCAGGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AAGATCCAAGAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAA GTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCC GTGTCTTGCCCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGT TTGTGGCTCTGCTGGTGTTCTACATCACA CAGCTGGGACTGCACATCTGGCAGCTGAGAAGCCAGTGCATGTGGCC CAGAGAGACACAGCTGCTGCTGGAAGTGCCTCCTAGCACCGAGGATGCCAGAAGCTGTCAGTTCCCCGAGGAAGAG AGAGGCGAGAGATCCGCCGAGGAAAAAGGCAGACTGGGCGACCTGTGGGTCCGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGC CTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 72)

[0382] CD2 signal peptide: SEQ ID NO: 6

[0383] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0384] CD2 transmembrane domain :SEQ ID NO: 10

[0385] GITR intracellular domain :SEQ ID NO: 32

[0386] CD3ζ intracellular domain: SEQ ID NO: 12

[0387] An exemplary "mutant CSR 10b CD2.8.DH.Gz" or "CD2.8.DH.Gz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain 、 CD2 transmembrane structure domain 、 GITR intracellular domain , CD3ζ intracellular domain) amino acid sequence or consisting thereof:

[0388] MALPVTALLLPLALLLHAARP KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQF RKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIY H TKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCE VMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD IYLIIGICGGGSLL MVFVALLVFYIT QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 73)

[0389] CD8a signal peptide: SEQ ID NO: 7

[0390] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 3

[0391] CD2 transmembrane domain :SEQ ID NO: 9

[0392] GITR intracellular domain :SEQ ID NO: 31

[0393] CD3ζ intracellular domain: SEQ ID NO: 11

[0394] An exemplary polynucleotide sequence encoding a "mutant CSR 10b CD2.8.DH.Gz" or "CD2.8.DH.Gz" polypeptide of the present disclosure comprises (CD8a signal peptide, The CD2 extracellular domain has a D111H mutation. CD2 extracellular structure domain 、 CD2 transmembrane domain 、 GITR intracellular domain , CD3ζ intracellular domain) nucleic acid sequence or consisting thereof:

[0395] ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCT AAAGA AATCACGAATGCATTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGACATCCCCAGCTTCCAGATGAGC GACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGA CATTCAAAGAGAAGGACACCTACAAGCTGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCA GGACATCTATAAGGTGTCCATCTAC CAC ACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAA GAGCGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGGCACAG ACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATCACCCACAAGTGGACAAC AAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGC CCTGAAAAAGGACTGGAC ATCTACCTGATCATCGGCATCTGTGGCGGCGGATCCCTGCTGATGGTGTTGTGGCTC TGCTGGTGTTCTACATCACA CAGCTGGGACTGCACATCTGGCAGCTGAGAAGCCAGTGCATGTGGCCCAGAGAGAC ACAGCTGCTGCTGGAAGTGCCCCTAGCACCGAGGATGCCAGAAGCTGTCAGTTCCCCGAGGAAGAGAGAGGCGAG AGATCCGCCGAGGAAAAAGGCAGACTGGGCGACCTGTGGGTCCGAGTGAAATTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCT GTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA(SEQ ID NO: 74)

[0396] CD8a signal peptide: SEQ ID NO: 8

[0397] The CD2 extracellular domain has a D111H mutation. CD2 extracellular domain :SEQ ID NO: 4

[0398] CD2 transmembrane domain :SEQ ID NO: 10

[0399] GITR intracellular domain :SEQ ID NO: 32

[0400] CD3ζ intracellular domain: SEQ ID NO: 12

[0401] The compositions of the present disclosure (eg, CSR) bind to an anti-CD2 activating agonist and an anti-CD2 activating molecule, but do not bind to naturally occurring CD58.

[0402] Compositions comprising the CSRs of the present disclosure can be incorporated into a cell delivery composition (eg, a transposon or vector) as described in detail herein, and optionally can be incorporated into a cell.

[0403] Cells and modified cells of the disclosure

[0404] The cells and modified cells of the present disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure may be immune cells. The immune cells of the present disclosure may include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (T cells), and SCM cells), central memory T cells (T CM), stem-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[0405] Immune precursor cells can include any cells that can differentiate into one or more types of immune cells. Immune precursor cells can include pluripotent stem cells that can self-renew and develop into immune cells. Immune precursor cells can include hematopoietic stem cells (HSC) or their progeny. Immune precursor cells can include precursor cells that can develop into immune cells. Immune precursor cells can include hematopoietic progenitor cells (HPC).

[0406] 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.

[0407] HSCs can be isolated or derived from primary or cultured stem cells.HSCs can be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0408] Immune precursor cells can comprise HSC or HSC progeny cells. Non-limiting examples of HSC progeny cells include pluripotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes and macrophages.

[0409] The HSCs produced by the disclosed methods can retain the characteristics of "primitive" stem cells, which, when isolated or derived from adult stem cells and committed to a single lineage, share the properties of embryonic stem cells. For example, the "primitive" HSCs produced by the disclosed methods retain their "stemness" after division and do not differentiate. Thus, as an adoptive cell therapy, the "primitive" HSCs produced by the disclosed methods not only replenish their numbers but also expand in vivo. When administered as a single dose, the "primitive" HSCs produced by the disclosed methods can be therapeutically effective.

[0410] A primitive HSC may be CD34+. A primitive HSC may be CD34+ and CD38-. A primitive HSC may be CD34+, CD38-, and CD90+. A primitive HSC may be CD34+, CD38-, CD90+, and CD45RA-. A primitive HSC may be CD34+, CD38-, CD90+, CD45RA-, and CD49f+. A primitive HSC may be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0411] The original HSC, HSC and / or HSC progeny cells can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). The modified original HSC, modified HSC and / or modified HSC progeny cells can be further differentiated to produce modified immune cells, including but not limited to modified T cells, modified natural killer cells and / or modified B cells.

[0412] The immune or immune precursor cells of modification can be NK cells.NK cells can be cytotoxic lymphocytes differentiated from lymphoid progenitor cells.The NK cells of modification can be derived from the hematopoietic stem cells and progenitor cells (HSPC) or the HSC of modification. In some aspects, unactivated NK cells are derived from the leukocyte removal (leukapheresis) (containing CD14 / CD19 / CD56+ cells) of CD3 exhaustion.

[0413] The modified immune or immune precursor cells can be B cells. B cells are a type of lymphocyte that expresses B cell receptors on the cell surface. B cell receptors bind to specific antigens. Modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0414] The T cells of the modification of the present disclosure can be derived from the hematopoietic stem cells and progenitor cells (HSPC) or modified HSC of modification. Different from traditional biological products and chemotherapeutic agents, the T cells of the modification disclosed can reproduce rapidly after antigen recognition, thereby potentially avoiding the need for repeated treatment. To achieve this, in some embodiments, the T cells of modification not only drive the initial response, but also persist in the patient as a stable living memory T cell colony, to prevent potential recurrence. Alternatively, in some aspects, when not needed, the T cells of modification do not persist in the patient.

[0415] Considerable effort has been focused on the development of antigen receptor molecules that do not lead to T cell exhaustion through antigen-independent (tonic) signaling, and on the development of novel targets containing early memory T cells, particularly stem cell memory (T SCM) or development of modified T cell products of stem cell-like T cells. The stem cell-like modified T cells of the present disclosure show 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 (T E ), resulting in better tumor eradication and long-term engraftment of modified T cells. 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 , so T N It directly generates T SCM parental precursor cells, which in turn directly give rise to T CM The composition of T cells of the present disclosure may comprise one or more of each parental T cell subset, wherein T SCM cells are the most abundant (e.g., T SCM > T CM > T EM > T E > T TE ).

[0416] Immune cell precursors can differentiate into or are capable of differentiating into early memory T cells, stem cells such as T cells, naive T cells (T N ), T SCM 、T CM 、T EM 、T E or T TE The immune cell precursor may be the original HSC, HSC or HSC progeny cell of the present disclosure. The immune cell may be an early memory T cell, a stem cell such as a T cell, a naive T cell (T N ), T SCM 、T CM 、T EM 、T E or T TE .

[0417] The methods of the present disclosure can modify and / or generate a modified T cell population 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 a plurality of modified T cells in the population express one or more cell surface markers of early memory T cells. The modified early memory T cell population comprises a plurality of modified stem cell-like T cells. The modified early memory T cell population comprises a plurality of modified T cells. SCM The modified early memory T cell population contains multiple modified T CM cell.

[0418] The methods of the present disclosure can modify and / or generate a modified T cell population 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 a plurality of modified T cells in the population express one or more cell surface markers of stem-like T cells. The modified stem-like T cell population comprises a plurality of modified T cells. SCM The modified stem cell-like T cell population contains multiple modified T CM cell.

[0419] 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%, or any percentage therebetween, of the plurality of modified T cells in a population express stem memory T cells (T SCM ) or T SCM One or more cell surface markers of leukemia-like cells; and wherein the one or more cell surface markers include CD45RA and CD62L. The cell surface markers may include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. The cell surface markers may include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0420] 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 plurality of modified T cells in a population express central memory T cells (T CM ) or T CM One or more cell surface markers of CRISPR-like cells; and wherein the one or more cell surface markers include CD45RO and CD62L. The cell surface markers may include one or more of CD45RO, CD95, IL-2Rβ, CCR7 and CD62L.

[0421] The methods of the present disclosure can modify and / or generate a population 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 a plurality of modified T cells in the population express a naive T cell (T N ). The cell surface markers may include one or more of CD45RA, CCR7 and CD62L.

[0422] The methods of the present disclosure can modify and / or generate a population 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 a plurality of modified T cells in the population express an effector T cell (modified T) EFF ). The cell surface markers may include one or more of CD45RA, CD95 and IL-2Rβ.

[0423] The methods of the present disclosure can modify and / or generate a population 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 a plurality of modified T cells in the population express stem cell-like T cells, stem memory T cells (T SCM ) or central memory T cells (T CM )'s cell surface markers.

[0424] A plurality of modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 70%, at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the modified cell population express one or more cell surface markers comprising CD34, or wherein at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% of the modified cell population express one or more cell surface markers comprising CD34 (e.g., comprise the cell surface marker phenotype CD34+).

[0425] A plurality of the modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein 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 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, In some embodiments, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the modified cell population express one or more cell surface markers comprising CD34 and do not express one or more cell surface markers comprising CD38, or wherein at least about 45% to about 90%, about 50% to about 80%, or about 65% to about 75% of the modified cell population express one or more cell surface markers comprising CD34 and do not express one or more cell surface markers comprising CD38 (e.g., comprise the cell surface marker phenotype CD34+ and CD38-).

[0426] A plurality of the modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, 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 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0 At least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 (e.g., comprise the cell surface marker phenotype CD34+, CD38-, and CD90+).

[0427] A plurality of the modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, 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 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%, at least 99.5%, at least 99.9%, or 100% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 and CD45RA, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 and CD45RA (e.g., comprise the cell surface marker phenotype CD34+, CD38-, CD90+, CD45RA).

[0428] A plurality of modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, 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 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%, at least 99.5%, at least 99.9%, or 100% of the modified cell population expresses one or more cell surface markers comprising CD34, CD90, and CD49f, and does not express one or more cell surface markers comprising CD38 and CD45RA. One or more cell surface markers, or a population of cells in which at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2%, or about 0.04% to about 1% of the modified cells express one or more cell surface markers comprising CD34, CD90, and CD49f and do not express one or more cell surface markers comprising CD38 and CD45RA (e.g., comprising the cell surface marker phenotype CD34+, CD38-, CD90+, CD45RA, and CD49f+).

[0429] A plurality of the modified cells in the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, 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%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells in the population comprises a transgene or a sequence encoding a transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 3 In some embodiments, the modified cell population expresses one or more cell surface markers comprising CD34 and CD90 and does not express one or more cell surface markers comprising CD45RA, or wherein at least about 0.2% to about 5%, about 0.2% to about 3%, or about 0.4% to about 3% of the modified cell population expresses one or more cell surface markers comprising CD34 and CD90 and does not express one or more cell surface markers comprising CD45RA (e.g., comprises the cell surface marker phenotype CD34+, CD90+, and CD45RA-).

[0430] Compositions and methods for generating and / or expanding immune cells or immune precursor cells (e.g., the disclosed modified T cells), and buffers for maintaining or enhancing the cell viability level and / or stem-like phenotype of immune cells or immune precursor cells (e.g., the disclosed modified T cells), are disclosed elsewhere herein and in more detail in U.S. Patent No. 10,329,543 and PCT Publication No. WO 2019 / 173636.

[0431] The cells and modified cells of the present disclosure can be somatic cells. The cells and modified cells of the present disclosure can be differentiated cells. The cells and modified cells of the present disclosure can be autologous cells or allogeneic cells. Allogeneic cells are modified to prevent adverse reactions to transplantation after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0432] Methods for expressing chimeric antigen receptors

[0433] The present disclosure provides a method for expressing CAR on the surface of a cell. The method includes (a) obtaining a cell colony; (b) under conditions sufficient to transfer CAR across the cell membrane of at least one cell in the cell colony, contacting the cell colony with a composition comprising CAR or a sequence encoding CAR, thereby generating a modified cell colony; (c) under conditions suitable for integrating the sequence encoding CAR, cultivating a modified cell colony; (d) amplifying and / or selecting at least one cell from a modified cell colony expressing CAR on the cell surface.

[0434] In some aspects, the cell colony can include leukocytes and / or CD4+ and CD8+ leukocytes. The cell colony can include CD4+ and CD8+ leukocytes at an optimized ratio. The optimized ratio of CD4+ to CD8+ leukocytes does not occur naturally in vivo. The cell colony can include tumor cells.

[0435] In some aspects, conditions sufficient to transfer the CAR or a sequence encoding the CAR, a transposon, or a vector across the cell membrane of at least one cell in the cell population include applying at least one of one or more electric pulses, a buffer, and one or more supplementary factors at a specific voltage. In some aspects, conditions suitable for integrating the sequence encoding the CAR include at least one of a buffer and one or more supplementary factors.

[0436] The buffer may comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplemental factors may comprise (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or a combination thereof; and (e) agents that modify or stabilize one or more nucleic acids. The recombinant human cytokines, chemokines, interleukins, or any combination thereof may comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-γ, IL-1α / IL-1F1, IL-1β / IL-1F2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 β, IL-32 γ, IL-33, LAP (TGF-β1), lymphotoxin-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSF11 / RANK L, or any combination thereof. The salts, minerals, metabolites, or any combination thereof can comprise HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacement, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof.The cell culture medium can comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or a combination thereof include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNApol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) inhibitors (e.g., TWS119), or any combination thereof. Examples of such inhibitors include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof. The agent that modifies or stabilizes one or more nucleic acids comprises a pH adjuster, a DNA binding protein, a lipid, a phospholipid, CaPO4, a net neutrally charged DNA binding peptide with or without an NLS sequence, a TREX1 enzyme, or any combination thereof.

[0437] The amplification and selection steps can occur concurrently or sequentially. Amplification can occur before selection. Amplification can occur after selection, and optionally, a further (i.e., second) selection can occur after amplification. Concurrent amplification and selection can be simultaneous. Amplification and / or selection steps can be performed over a period of 10 to 14 days, inclusive.

[0438] Amplification can include contacting at least one cell of the modified cell population with an antigen to stimulate at least one cell by CAR, thereby generating an amplified cell population. Antigens can be presented on the surface of a substrate. The substrate can have any form, including but not limited to a surface, a hole, a bead or multiple beads and a matrix. The substrate may further comprise a paramagnetic or magnetic component. Antigens can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic beads from the modified and amplified cell population. Antigens can be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells may include but are not limited to tumor cells and stem cells.

[0439] In some aspects where the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell in the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as having survived the selection and identifying cells that fail to express the selection gene as having failed to survive the selection step.

[0440] The present disclosure provides compositions comprising the modified, expanded, and selected cell populations of the methods described herein.

[0441] A more detailed description of methods for expressing CARs on the surface of cells is disclosed in PCT Publication Nos. WO 2019 / 049816 and PCT / US2019 / 049816.

[0442] The present disclosure provides cells or cell populations, wherein the cells constitute a composition comprising (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein after the constructs of (a) and (b) are integrated into the genomic sequence of the cell, the exogenous receptor is expressed, and wherein the exogenous receptor transduces an intracellular signal after binding a ligand or antigen, and the intracellular signal directly or indirectly targets the inducible promoter that regulates the expression of the inducible transgene (a) to modify gene expression.

[0443] The composition can modify gene expression by reducing gene expression. The composition can modify gene expression by transiently modifying gene expression (e.g., for the duration of binding of a ligand to an exogenous receptor). The composition can modify gene expression acutely (e.g., a ligand reversibly binds to an exogenous receptor). The composition can modify gene expression over a long period of time (e.g., a ligand irreversibly binds to an exogenous receptor).

[0444] An exogenous receptor may comprise an endogenous receptor relative to the genomic sequence of the cell.Exemplary receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G protein-coupled receptors.

[0445] Exogenous receptors may include non-natural receptors. Non-natural receptors may be synthetic, modified, recombinant, mutant or chimeric receptors. Non-natural receptors may include one or more sequences separated from or derived from a T cell receptor (TCR). Non-natural receptors may include one or more sequences separated from or derived from a scaffold protein. In some aspects, including those aspects in which the non-natural receptor does not include a transmembrane domain, the non-natural receptor interacts with a second transmembrane, membrane-bound and / or intracellular receptor, which, after contacting the non-natural receptor, transduces intracellular signals. Non-natural receptors may include a transmembrane domain. Non-natural receptors may interact with intracellular receptors that transduce intracellular signals. Non-natural receptors may include an intracellular signaling domain. Non-natural receptors may be chimeric ligand receptors (CLRs). CLRs may be chimeric antigen receptors (CARs).

[0446] Sequences encoding inducible promoters include sequences encoding NFĸB promoters, sequences encoding interferon (IFN) promoters, or sequences encoding interleukin-2 promoters. In some aspects, the IFN promoter is an IFNγ promoter. Inducible promoters can be isolated or derived from promoters of cytokines or chemokines. The cytokine or chemokine may comprise IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor β (TGFβ), colony stimulating factor 2 (GM-CSF), interferon γ (IFNγ), tumor necrosis factor α (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin 6 family cytokine (Lif).

[0447] Inducible promoters can be separated from or derived from promoters of genes comprising surface proteins that are involved in cell differentiation, activation, exhaustion, and function. In some aspects, genes comprise CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0448] Inducible promoters can be isolated or derived from promoters of genes involved in CD metabolism and differentiation. Inducible promoters can be isolated or derived from promoters of Nr4a1, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0449] In some aspects, the inducible transgenic construct comprises or drives the expression of signal transduction components, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes downstream of inhibitory checkpoint signals. Non-limiting examples of such proteins are disclosed in PCT Publication No. WO 2019 / 173636 and PCT Application No. PCT / US2019 / 049816.

[0450] Armored Cells

[0451] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to enhance their therapeutic potential. Alternatively or in addition, the modified cells can be further modified so as to render them less sensitive to immunological and / or metabolic checkpoints. This type of modification "arms" cells, after modification, which cells may be referred to herein as "armed" cells (e.g., armed T cells). Armed cells can be produced, for example, by blocking and / or diluting specific checkpoint signals (e.g., checkpoint inhibition) naturally delivered to cells within a tumor immunosuppressive microenvironment.

[0452] The armed cells of the present disclosure can be derived from any cell, for example, T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB) derived T cells (including T cells separated from or derived from peripheral blood mobilized by G-CSF) or umbilical cord blood (UCB) derived T cells. Armed cells (e.g., armed T cells) can include one or more of the following: chimeric ligand receptor (CLR comprising protein scaffold, antibody, ScFv or antibody mimics) / chimeric antigen receptor (CAR comprising protein scaffold, antibody, ScFv or antibody mimics), CARTyrin (CAR comprising Centyrin) and / or VCAR (CAR comprising camelid VHH or single domain VH). Armed cells (e.g., armed T cells) can include inducible pro-apoptotic polypeptides as disclosed herein. Armed cells (e.g., armed T cells) can include exogenous sequences. Exogenous sequences can include sequences encoding therapeutic proteins. Exemplary therapeutic proteins can be proteins that are nuclear, cytoplasmic, intracellular, transmembrane, cell surface bound or secreted. Exemplary therapeutic proteins expressed by armed cells (e.g., armed T cells) can modify the activity of the armed cells or can modify the activity of a second cell. The armed cells (e.g., armed T cells) can comprise a selection gene or selection marker. The armed cells (e.g., armed T cells) can comprise a synthetic gene expression cassette (also referred to herein as an inducible transgenic construct).

[0453] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding inhibitory checkpoint signal receptors to produce armed cells (e.g., armed CAR T cells). The receptors for inhibitory checkpoint signals are expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing the expression of genes encoding inhibitory checkpoint signal receptors results in the loss of protein expression of inhibitory checkpoint receptors on the surface of armed cells or in the cytoplasm. Therefore, armed cells with silenced or reduced expression of one or more genes encoding inhibitory checkpoint receptors are resistant, unacceptable or insensitive to checkpoint signals. In the presence of these inhibitory checkpoint signals, the resistance or sensitivity of armed cells to inhibitory checkpoint signals is reduced, which enhances the therapeutic potential of armed cells. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immune suppression) are disclosed in PCT Publication No. WO 2019 / 173636. Preferred examples of inhibitory checkpoint signals that can be silenced include, but are not limited to, PD-1 and TGFβRII.

[0454] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding intracellular proteins involved in checkpoint signaling, to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced by targeting any intracellular signaling protein involved in checkpoint signaling pathways, thereby achieving checkpoint inhibition or interference of one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in PCT Publication No. WO 2019 / 173636.

[0455] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding transcription factors that hinder the efficacy of therapy to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced or regulated by silencing or reducing the expression (or repressing function) of transcription factors that hinder the efficacy of therapy. Non-limiting examples of transcription factors that can be modified to silence or reduce expression or repress their function include, but are not limited to, the exemplary transcription factors disclosed in PCT Publication No. WO 2019 / 173636.

[0456] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding cell death or apoptosis receptors to produce armed cells (e.g., armed CAR T cells). The interaction of death receptors with their endogenous ligands leads to the onset of apoptosis. The destruction of the expression, activity or interaction of cell death and / or apoptosis receptors and / or ligands renders the modified cells less receptive to death signals, thereby making the armed cells more effective in the tumor environment. Non-limiting examples of cell death and / or apoptosis receptors and ligands are disclosed in PCT Publication No. WO 2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas (CD95).

[0457] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce the expression of one or more genes encoding metabolic sensor proteins to produce armed cells (e.g., armed CAR T cells). Metabolic sensing of the immune suppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose and other molecules) is destroyed by modified cells, resulting in prolonged retention of T cell function, and thus, each cell kills more tumor cells. Non-limiting examples of metabolic sensor genes and proteins are disclosed in PCT Publication No. WO 2019 / 173636. Preferred examples, HIF1a and VHL play a role in T cell function in an oxygen-deficient environment. Armed T cells may have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0458] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding proteins that confer sensitivity to cancer therapy including monoclonal antibodies to produce armed cells (e.g., armed CAR T cells). Therefore, in the presence of cancer therapy (e.g., chemotherapy, monoclonal antibody therapy or another anti-tumor treatment), armed cells can work and can demonstrate excellent function or efficacy. Non-limiting examples of proteins that confer sensitivity to cancer therapy are disclosed in PCT Publication No. WO2019 / 173636.

[0459] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding growth advantage factors to produce armed cells (e.g., armed CAR T cells). Silencing or reducing the expression of oncogenes can confer growth advantages on cells. For example, silencing or reducing the expression of the TET2 gene (e.g., destroying expression) during the CAR T cell manufacturing process results in the generation of armed CAR T cells with a significant ability to amplify and subsequently eradicate tumors when compared to unarmed CAR T cells lacking such amplification capacity. This strategy can be coupled with a safety switch (e.g., an iC9 safety switch as described herein) that allows targeted destruction of armed CAR T cells in the event of adverse reactions from the subject or uncontrolled growth of armed CAR T cells. Non-limiting examples of growth advantage factors are disclosed in PCT Publication No. WO 2019 / 173636.

[0460] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to express modified / chimeric checkpoint receptors to generate armed T cells of the present disclosure.

[0461] Modified / chimeric checkpoint receptors may comprise inactive receptors, decoy receptors, or dominant negative receptors. Inactive receptors, decoy receptors, or dominant negative receptors may be modified / chimeric receptors / proteins. Inactive receptors, decoy receptors, or dominant negative receptors may be truncated for expression of an intracellular signaling domain. Alternatively or in addition, inactive receptors, decoy receptors, or dominant negative receptors may be mutated at one or more amino acid positions within an intracellular signaling domain that are critical or essential for effective signal transduction. Truncation or mutation of inactive receptors, decoy receptors, or dominant negative receptors may result in the receptor losing the ability to transmit or transduce checkpoint signals to or within a cell.

[0462] For example, dilution or blocking of immune suppressive checkpoint signals from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing modified / chimeric PD-1 null receptors on the surface of armed cells (e.g., armed CAR T cells), wherein the null receptor effectively competes with the endogenous (unmodified) PD-1 receptors also expressed on the surface of armed cells to reduce or inhibit the transduction of immune suppressive checkpoint signals through the endogenous PD-1 receptors of armed cells. In this non-limiting example, competition between two different receptors for binding to PD-L1 expressed on tumor cells reduces or weakens the level of effective checkpoint signaling, thereby enhancing the therapeutic potential of armed cells expressing PD-1 null receptors.

[0463] Modification / chimeric checkpoint receptors can include invalid receptors, decoy receptors or dominant negative receptors, which are transmembrane receptors, membrane-associated or membrane-attached receptors / proteins or intracellular receptors / proteins. Exemplary invalid, decoy or dominant negative intracellular receptors / proteins include but are not limited to signal transduction components, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines and chemokine receptors are disclosed in PCT Publication No. WO 2019 / 173636.

[0464] Modification / chimeric checkpoint receptors can include switch receptors. Exemplary switch receptors include modified / chimeric receptors / proteins, in which natural or wild-type intracellular signaling domains are converted or replaced by different intracellular signaling domains, and the different intracellular signaling domains are non-natural and / or not wild-type domains for proteins. For example, replacing the inhibitory signaling domain with a stimulatory signaling domain converts the immune suppression signal into an immunostimulatory signal. Alternatively, replacing the inhibitory signaling domain with a different inhibitory domain can reduce or enhance the level of inhibitory signaling. By competing with endogenous wild-type checkpoint receptors (rather than switch receptors) for binding to homologous checkpoint receptors expressed in an immune suppressive tumor microenvironment, the expression or overexpression of the switch receptor can result in dilution and / or blocking of the homologous checkpoint signal. Armed cells (e.g., armed CAR T cells) can include sequences encoding switch receptors, resulting in the expression of one or more switch receptors, and thus changing the activity of armed cells. The armed cells (e.g., armed CAR T cells) can express switch receptors that target intracellularly expressed proteins downstream of checkpoint receptors, transcription factors, cytokine receptors, death receptors, metabolic sensing molecules, cancer therapies, oncogenes, and / or tumor suppressor proteins or genes.

[0465] Exemplary switch receptors may comprise or may be derived from proteins including, but not limited to, signaling components downstream of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapies, and oncogenes or tumor suppressor genes.

[0466] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to express CLR / CAR that mediates conditional gene expression to produce armed T cells. The combination of CLR / CAR and the conditional gene expression system in the nucleus of armed T cells constitutes a synthetic gene expression system that is conditionally activated when the cognate ligand is bound to CLR or the cognate antigen is bound to CAR. For example, the system can help 'arm' or enhance the therapeutic potential of modified T cells by reducing or limiting the expression of synthetic genes at the site where the ligand or antigen is bound, at the tumor environment, or within the tumor environment.

[0467] Gene editing compositions and methods

[0468] The modified cells are produced by introducing a transgene into the cells. The introducing step may comprise delivering the nucleic acid sequence, transgene and / or genome editing construct via a non-transposing delivery system.

[0469] Nucleic acid sequence, transgenic and / or genome editing construct are introduced into the cell in vitro, in vivo, in vitro or in situ, and can include one or more of local delivery, adsorption, absorption, electroporation, spin infection (spin-fection), co-cultivation, transfection, mechanical delivery, acoustic wave delivery, vibration delivery, magnetic transfection or delivery mediated by nanoparticles. Nucleic acid sequence, transgenic and / or genome editing construct are introduced into the cell in vitro, in vivo, in vitro or in situ, and can include liposome transfection, calcium phosphate transfection, fugene transfection and dendrimer-mediated transfection. Nucleic acid sequence, transgenic and / or genome editing construct are introduced into the cell in vitro, in vivo, in vitro or in situ by mechanical transfection, and can include cell extrusion, cell bombardment or gene gun technology. Nucleic acid sequence, transgenic and / or genome editing construct are introduced into the cell in vitro, in vivo, in vitro or in situ by nanoparticle-mediated transfection, and can include liposome delivery, micelle delivery and polymer vesicle (polymerosome) delivery.

[0470] The nucleic acid sequence, transgenic and / or genome editing construct is introduced into the cell in vitro, in vivo, in vitro or in situ and can include a non-viral vector. The non-viral vector can include nucleic acid. The non-viral vector can include plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmid, small circle DNA, single-stranded oligodeoxynucleotide (ssODN), DDNA oligonucleotide, single-stranded mRNA (ssRNA) and double-stranded mRNA (dsRNA). The non-viral vector can include a transposon as described herein.

[0471] The nucleic acid sequence, transgene and / or genome editing construct is introduced into the cell in vitro, in vivo, in vitro or in situ and can include a viral vector. The viral vector can be a non-integrated non-chromosomal vector. Non-limiting examples of non-integrated non-chromosomal vectors include adeno-associated virus (AAV), adenovirus and herpes virus. The viral vector can be an integrated chromosomal vector. Non-limiting examples of integrated chromosomal vectors include adeno-associated vectors (AAV), lentivirus and gamma retrovirus.

[0472] Introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can comprise a combination of vectors. Non-limiting examples of vector combinations include viral and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Non-limiting examples of vector combinations include a combination of DNA-derived vectors and RNA-derived vectors, a combination of RNA and reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0473] Genome modification can include introducing a nucleic acid sequence, a transgene, and / or a genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ to stably integrate the nucleic acid sequence, transiently integrate the nucleic acid sequence, produce site-specific integration of the nucleic acid sequence, or produce biased integration of the nucleic acid sequence. The nucleic acid sequence can be a transgene.

[0474] Genomic modification can include ex vivo, in vivo, in vitro or in situ introduction of nucleic acid sequences, transgenic and / or genome editing constructs into cells to stably integrate nucleic acid sequences. Stable chromosomal integration can be random integration, site-specific integration or biased integration. Site-specific integration can be non-auxiliary or auxiliary. Auxiliary site-specific integration is delivered together with a fixed-point nuclease. The fixed-point nuclease comprises a transgenic with 5' and 3' nucleotide sequence extensions, which contains the homology percentage with the upstream and downstream regions of the genomic integration site. The transgenic with homologous nucleotide extensions enables genomic integration by end connection or non-homologous end connection mediated by homologous recombination, microhomology. Site-specific integration can occur at safe harbor sites. Genomic safe harbor sites can accommodate the integration of new genetic material in a manner that ensures that newly inserted genetic elements work reliably (e.g., expressed at therapeutically effective expression levels) and do not cause harmful changes to the host genome that cause risks to the host organism. Non-limiting examples of potential genomic safe harbors include intronic sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of the AAV virus on chromosome 19, the site of the chemokine (CC motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0475] Site-specific transgenic integration can occur at the site of the expression of the target gene. By site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons and response elements, the destruction of target gene expression can occur. Non-limiting examples of the target gene targeted by site-specific integration include TRAC, TRAB, PDI, any immune suppression gene and the gene related to allogeneic rejection.

[0476] Site-specific transgenic integration can occur at sites that result in enhanced expression of the target gene. Enhancement of target gene expression can occur through site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements.

[0477] Enzyme can be used to produce chain breaks in the host genome to promote delivery or integration of transgenic. Enzyme can produce single-strand breaks or double-strand breaks. Non-limiting examples of break-inducing enzymes include transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™ and CPF1. Break-inducing enzymes can be delivered to cells with DNA encoding, with mRNA encoding, as proteins or as nucleoprotein complexes with guide RNA (gRNA).

[0478] Site-specific transgene integration can be controlled by vector-mediated integration site biasing, which can be controlled by the selected lentiviral vector or the selected gammaretroviral vector.

[0479] In one embodiment, the transgenic gene is inserted into the chromosome of the transgenic gene. The site-specific transgenic integration site can be an unstable chromosome insertion. The transgenic gene integrated can become silent, removed, excised or further modified. Genomic modification can be an unstable integration of transgenic. Unstable integration can be transient non-chromosomal integration, semi-stable non-chromosomal integration, semi-persistent non-chromosomal insertion or unstable chromosome insertion. Transient non-chromosomal insertion can be extrachromosomal or cytoplasmic. In one aspect, the transient non-chromosomal insertion of transgenic is not integrated into the chromosome, and the genetic material modified does not replicate during cell division.

[0480] The genome modification can be a semi-stable or permanent non-chromosomal integration of the transgene. The DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of non-viral vectors, allowing autonomous replication in the nucleus of dividing cells.

[0481] The genomic modification can be the unstable chromosomal integration of a transgene. The integrated transgene can be silenced, removed, excised, or further modified.

[0482] Modification of the genome by transgenic insertion can occur via host cell directed double strand break repair (homology-mediated repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or recombinase-mediated modification. Modification of the genome by transgenic insertion can occur via CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER™, and cpf1.

[0483] In gene editing systems involving the insertion of new or existing nucleotides / nucleic acids, in addition to a cutting enzyme (e.g., a nuclease, a recombinase, an integrase, or a transposase), an insertion tool (e.g., a DNA template vector, a transposable element (transposon or retrotransposon) must be delivered to the cell. Examples of such insertion tools for recombinases may include DNA vectors. Other gene editing systems require the delivery of an integrase along with an insertion vector, a transposase along with a transposon / retrotransposon, and the like. An example of a recombinase that can be used as a cutting enzyme is CRE recombinase. Non-limiting examples of integrases that can be used in the insertion tool include viral-based enzymes taken from any of a variety of viruses, including AAV, gamma retroviruses, and lentiviruses. Examples of transposons / retrotransposons that can be used in the insertion tool are described in more detail herein.

[0484] The cells with ex vivo, in vivo, in vitro or in situ genomic modifications can be germline cells or somatic cells. The modified cells can be human, non-human, mammalian, rat, mouse or dog cells. The modified cells can be differentiated, undifferentiated or immortalized. The modified undifferentiated cells can be stem cells. The modified undifferentiated cells can be induced pluripotent stem cells. The modified cells can be immune cells. The modified cells can be T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes or osteoclasts. The modified cells can be modified when the cells are dormant, in an activated state, static, in interphase, in early stage, in mid-stage, in late stage or in late stage. The modified cells can be fresh, cryopreserved, bulked, sorted into subpopulations, from whole blood, from leukapheresis or from immortalized cell lines. Detailed descriptions for separating cells from leukapheresis products or blood are disclosed in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0485] The present disclosure provides gene editing compositions and / or cells comprising gene editing compositions. The gene editing compositions may include sequences encoding DNA binding domains, and sequences encoding nuclease proteins or their nuclease domains. The sequences encoding nuclease proteins or sequences encoding their nuclease domains may include DNA sequences, RNA sequences, or a combination thereof. Nucleases or their nuclease domains may include one or more of CRISPR / Cas proteins, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases.

[0486] The nuclease or its nuclease domain may comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise a Clo051 nuclease or its nuclease domain. The gene editing composition may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or Clo051 nuclease domain. The gene editing composition may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0487] The present disclosure provides a composition comprising a small Cas9 (Cas9) operably connected to an effector. The present disclosure provides a fusion protein comprising a DNA positioning component and an effector molecule, consisting essentially of or consisting of, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 construct of the present disclosure can include an effector comprising an IIS type endonuclease. Staphylococcus aureus Cas9 with an active catalytic site comprises the amino acid sequence of SEQ ID NO: 79.

[0488] The present disclosure provides a composition comprising a small inactivated Cas9 (dSaCas9) operably linked to an effector. The present disclosure provides a fusion protein comprising a DNA positioning component and an effector molecule, consisting essentially of or consisting of a small inactivated Cas9 (dSaCas9), wherein the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease. dSaCas9 comprises the amino acid sequence of SEQ ID NO: 80, which includes D10A and N580A mutations to inactivate the catalytic site.

[0489] The present disclosure provides compositions comprising an inactivated Cas9 (dCas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, essentially consisting of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of the present disclosure can include an effector comprising a Type IIS endonuclease.

[0490] dCas9 can be isolated or derived from Streptococcus pyogenes ( Streptoccocus pyogenes ). dCas9 can include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A. dCas9 can include the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82.

[0491] An exemplary Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 83.

[0492] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 84. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 85. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0493] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 86. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 87. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0494] The cell comprising the gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is transiently expressed. The guide RNA can comprise a sequence complementary to the target sequence within the genomic DNA sequence. The target sequence within the genomic DNA sequence can be a target sequence within a safe harbor site of the genomic DNA sequence.

[0495] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024.

[0496] One or more micelles based on poly (histidine) can also be used to deliver gene editing tools to cells. Poly (histidine) (for example, poly (L-histidine)) is a pH-sensitive polymer, because the imidazole ring provides a lone electron pair on unsaturated nitrogen. That is, poly (histidine) has amphoteric properties by protonation-deprotonation. In particular, at certain pH, triblock copolymers containing poly (histidine) can be assembled into micelles with positively charged poly (histidine) units on the surface, so as to enable compounding with negatively charged gene editing molecules. These nanoparticles are used to combine and release proteins and / or nucleic acids in a pH-dependent manner, and effective and selective mechanisms can be provided to perform required genetic modification. In particular, this micelle-based delivery system provides a large amount of flexibility about charged materials and the targeted release of large payload capacity and nanoparticle payload. In one example, nuclease is delivered by using micelles based on poly (histidine) so that site-specific cutting of double-stranded DNA is possible. Without wishing to be bound by a particular theory, it is believed that in micelles formed from various triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidine) blocks on the ends to form one or more surrounding layers.

[0497] In one aspect, the present disclosure provides a triblock copolymer made of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block can be poly(ethylene oxide) (PEO), and the charged block can be poly(L-histidine). An example triblock copolymer that can be used is PEO-b-PLA-b-PHIS, where a variable number of repeat units in each block varies by design.

[0498] Diblock copolymers that can be used as intermediates for preparing triblock copolymers can have hydrophilic, biocompatible poly(ethylene oxide) (PEO) (chemically synonymous with PEG) coupled to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(orthoesters), poly(peptides), poly(phosphazenes), and poly(sugars), including but not limited to poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-coglycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC). Polymeric micelles composed of a 100% PEGylated surface have improved in vitro chemical stability, enhanced in vivo bioavailability, and prolonged blood circulation half-life.

[0499] Polysomes, polymersomes, and poly(histidine)-based micelles, including those comprising triblock copolymers, and methods for their preparation are further described in detail in U.S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO 2019 / 126589.

[0500] Transposon and vector compositions

[0501] The present disclosure provides compositions and methods for delivering antibodies (e.g., scFv) or CARs (e.g., comprising scFv) to cells or cell colonies. Non-limiting examples of compositions for delivering the compositions of the present disclosure to cells or cell colonies include transposons or vectors. Therefore, the present disclosure provides transposons comprising antibodies (e.g., scFv) or CARs (e.g., comprising scFv) or vectors comprising antibodies (e.g., scFv) or CARs (e.g., comprising scFv).

[0502] The transposon comprising the CAR of the present disclosure or the vector comprising the CAR of the present disclosure may further comprise a sequence encoding an inducible pro-apoptotic polypeptide. Alternatively or in addition, a transposon or a vector may comprise the CAR of the present disclosure, and a second transposon or a second vector may comprise a sequence encoding an inducible pro-apoptotic polypeptide of the present disclosure. Inducible pro-apoptotic polypeptides are described in more detail herein.

[0503] The transposon comprising the CAR of the present disclosure or the vector comprising the CAR of the present disclosure may further comprise a sequence encoding a chimeric stimulating receptor (CSR). Alternatively or in addition, a transposon or a vector may comprise a CAR of the present disclosure, and a second transposon or a second vector may comprise a sequence encoding a CSR of the present disclosure. Chimeric stimulating receptors are described in more detail herein.

[0504] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure may further comprise a sequence encoding a recombinant HLA-E polypeptide. Alternatively or additionally, one transposon or one vector may comprise a CAR of the present disclosure, while a second transposon or second vector may comprise a sequence encoding a recombinant HLA-E polypeptide. Recombinant HLA-E polypeptides are described in more detail herein.

[0505] The transposon comprising the CAR of the present disclosure or the vector comprising the CAR of the present disclosure may further comprise a selection gene. The selection gene may encode a gene product necessary for cell viability and survival. When challenged by selective cell culture conditions, the selection gene may encode a gene product necessary for cell viability and survival. The selective cell culture conditions may comprise a compound that is harmful to cell viability or survival, and wherein the gene product confers resistance to the compound. Non-limiting examples of selection genes include neo (confers resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase family 1, member A1), FRANCF, RAD51C (encoding RAD51 paralog C), GCS (encoding glucosylceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

[0506] In a preferred aspect, select gene encoding DHFR mutant protease.DHFR mutant protease comprises the aminoacid sequence of SEQ ID NO:88, is basically made up of it or is made up of it.DHFR mutant protease is by polynucleotide encoding, and described polynucleotide comprises the nucleotide sequence of SEQ ID NO:89, is basically made up of it or is made up of it.The aminoacid sequence of DHFR mutant protease can further be included in the sudden change of one or more positions in position 80,113 or 153.The aminoacid sequence of DHFR mutant protease can comprise following one or more: phenylalanine (F) or leucine (L) replace at position 80, leucine (L) or valine (V) replace at position 113 and valine (V) or aspartic acid (D) replace at position 153.

[0507] The transposon comprising the CAR of the present disclosure or the vector comprising the CAR of the present disclosure may further comprise at least one self-cleaving peptide. For example, the self-cleaving peptide may be located between the CAR (e.g., comprising an scFv) and an inducible pro-apoptotic polypeptide; or, the self-cleaving peptide may be located between the CAR (e.g., comprising an scFv) and a protein encoded by a selection gene.

[0508] The transposon comprising the CAR of the present disclosure or the vector comprising the CAR of the present disclosure may further comprise at least two self-cleaving peptides. For example, the first self-cleaving peptide is located upstream or immediately upstream of the CAR, and the second self-cleaving peptide is located downstream or immediately downstream of the CAR; or, the first self-cleaving peptide and the second self-cleaving peptide are flanked by the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of the inducible pro-apoptotic polypeptide, and the second self-cleaving peptide is located downstream or immediately downstream of the inducible pro-apoptotic polypeptide; or, the first self-cleaving peptide and the second self-cleaving peptide are flanked by the inducible pro-apoptotic polypeptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of the protein encoded by the selection gene, and the second self-cleaving peptide is located downstream or immediately downstream of the protein encoded by the selection gene; or, the first self-cleaving peptide and the second self-cleaving peptide are flanked by the protein encoded by the selection gene.

[0509] Non-limiting examples of self-cleaving peptides include T2A peptides, GSG-T2A peptides, E2A peptides, GSG-E2A peptides, F2A peptides, GSG-F2A peptides, P2A peptides, or GSG-P2A peptides. The T2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 90. The GSG-T2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 91. The GSG-T2A polypeptide is encoded by a polynucleotide comprising, or consisting of, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 92. The E2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 93. The GSG-E2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 94. The F2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 95. The GSG-F2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 96. The P2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 97.The GSG-P2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 98.

[0510] Transposition system

[0511] The present disclosure provides a transposon comprising a protein scaffold as disclosed herein, or the present disclosure provides a transposon comprising an antibody (e.g., scFv) or CAR (e.g., comprising scFv) as disclosed herein. In a preferred aspect, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding scFv or CAR (e.g., comprising scFv) as disclosed herein, flanked by two cis-regulatory insulator elements. The present disclosure also provides a composition comprising a transposon. In a preferred aspect, the composition comprising a transposon further comprises a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase can be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0512] The transposon of the present disclosure can be a piggyBac™ (PB) transposon. In some aspects, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. The sequence encoding the SPB transposase is an mRNA sequence.

[0513] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described in detail in: US Patent No. 6,218,182; US Patent No. 6,962,810; US Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296.

[0514] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) on the ends of the transposon and insert content between the ITRs at the sequence 5'-TTAT-3' within the chromosomal site (TTAT target sequence) or at the sequence 5'-TTAA-3' within the chromosomal site (TTAA target sequence). The target sequence of the PB or PBL transposon may comprise or consist of 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'-T ', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system has no payload restrictions for the gene of interest that can be included between the ITRs.

[0515] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; and U.S. Patent No. 8,399,643. In a preferred aspect, the PB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 99.

[0516] The PB or PBL transposase may comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or at each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 99. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of the sequence of SEQ ID NO: 99, wherein the amino acid substitution at position 30 may be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 may be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 may be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 may be a substitution of lysine (K) for asparagine (N). In a preferred aspect, the SPB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 100.

[0517] In certain aspects wherein the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591 of the sequence of SEQ ID NO: 99 or SEQ ID NO: 100, as described in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816 are described in more detail.

[0518] As described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816, the PB, PBL, or SPB transposase can be isolated or derived from an insect, a vertebrate, a crustacean, or a urochordate. In a preferred aspect, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni ( Trichoplusia ni ) (GenBank accession number AAA87375) or Bombyx mori ( Bombyx mori ) (GenBank accession number BAD11135).

[0519] A hyperactive PB or PBL transposase is a transposase that is more active than a naturally occurring variant from which it is derived. In a preferred aspect, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis ( Xenopus tropicalis Examples of highly active PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and WO 2019 / 173636. A list of highly active amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0520] In some aspects, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise its corresponding transposon but integrates the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and WO 2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0521] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636.

[0522] The transposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Patent No. 9,228,180) or a highly active Sleeping Beauty (SB100X) transposase. In a preferred aspect, the Sleeping Beauty transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 101. In a preferred aspect, the highly active Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 102.

[0523] The transposon of the present disclosure can be a Helaizer transposon. Exemplary Helaizer transposon includes Helibat1, which comprises a nucleotide sequence or consists of a nucleotide sequence that is identical to SEQ ID NO: 103 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage between the two). In some aspects, when the transposon is a Helaizer transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019 / 173636). In a preferred aspect, the Helitron transposase comprises an amino acid sequence or consists of an amino acid sequence that is identical to SEQ ID NO: 104 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage between the two).

[0524] The transposon of the present disclosure can be a Tol2 transposon. Exemplary Tol2 transposon, including inverted repeats, subterminal sequences and Tol2 transposase, comprises a nucleotide sequence or consists of a nucleotide sequence, and the nucleotide sequence is identical to SEQ ID NO: 105 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage between the two). In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019 / 173636). In a preferred aspect, the Tol2 transposase comprises an amino acid sequence or consists of an amino acid sequence, and the amino acid sequence is identical to SEQ ID NO: 106 at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage between the two).

[0525] The transposon of the present disclosure can be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a highly active TcBuster transposase (e.g., as disclosed in WO 2019 / 173636). The TcBuster transposase can comprise a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence, or consist of it. In a preferred aspect, the TcBuster transposase comprises an amino acid sequence or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage between the two) identical to SEQ ID NO: 107. The polynucleotide encoding the TcBuster transposase can comprise a naturally occurring nucleotide sequence or a non-naturally occurring nucleotide sequence, or consist of it. In a preferred aspect, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 108.

[0526] In some aspects, the mutant TcBuster transposase comprises one or more sequence variations when compared to the wild-type TcBuster transposase as described in more detail in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0527] The transposon can be a nanotransposon. The nanotransposon can comprise, be essentially composed of, or be composed of: (a) a sequence encoding a transposon insert comprising a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an ITR internal sequence; (b) a sequence encoding a backbone, wherein the sequence encoding the backbone comprises a sequence encoding a replication origin of 1 to 450 nucleotides (inclusive) and a sequence encoding a selectable marker of 1 to 200 nucleotides (inclusive), and (c) an ITR inter-sequence. In some aspects, the ITR inter-sequence of (c) comprises the sequence of (b). In some aspects, the ITR internal sequence of (a) comprises the sequence of (b).

[0528] The sequence encoding the backbone can comprise 1 to 600 nucleotides, inclusive. In some aspects, the sequence encoding the backbone consists of: 1 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, each range including the endpoints.

[0529] The inter-ITR sequence can comprise 1 to 1000 nucleotides, inclusive. In some aspects, the inter-ITR sequence consists of 1 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, 600 to 650 nucleotides, 650 to 700 nucleotides, 700 to 750 nucleotides, 750 to 800 nucleotides, 800 to 850 nucleotides, 850 to 900 nucleotides, 900 to 950 nucleotides, or 950 to 1000 nucleotides, each range inclusive.

[0530] The nanotransposon can be a short nanotransposon (SNT), wherein the inter-ITR sequence comprises 1 to 200 nucleotides, inclusive. The inter-ITR sequence can consist of 1 to 10 nucleotides, 10 to 20 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, or 90 to 100 nucleotides, each range inclusive.

[0531] The selectable marker having 1 to 200 nucleotides (inclusive) can comprise a sequence encoding a sucrose selectable marker. The sequence encoding a sucrose selectable marker can comprise a sequence encoding an RNA-OUT sequence. The RNA-OUT sequence encoding can comprise 137 base pairs (bp) or consist of 137 base pairs (bp). The selectable marker having 1 to 200 nucleotides (inclusive) can comprise a sequence encoding a fluorescent marker. The selectable marker having 1 to 200 nucleotides (inclusive) can comprise a sequence encoding a cell surface marker.

[0532] The sequence encoding the replication origin of 1 to 450 nucleotides (inclusive) may comprise a sequence encoding a mini-replication origin. In some aspects, the sequence encoding the replication origin of 1 to 450 nucleotides (inclusive) comprises a sequence encoding an R6K replication origin. The R6K replication origin may comprise a R6K gamma replication origin. The R6K replication origin may comprise a R6K mini-replication origin. The R6K replication origin may comprise a R6K gamma mini-replication origin. The R6K gamma mini-replication origin may comprise or consist of 281 base pairs (bp).

[0533] In some aspects of nano transposon, the sequence of coding backbone does not comprise recombination site, excision site, junction site or its combination. In some aspects, the sequence of nano transposon and coding backbone does not comprise the product of recombination site, excision site, junction site or its combination. In some aspects, the sequence of nano transposon and coding backbone is not derived from recombination site, excision site, junction site or its combination.

[0534] In some aspects of nano transposon, recombination site comprises the sequence resulting from a recombination event. In some aspects, recombination site comprises the sequence of the product that is a recombination event. In some aspects, recombination event comprises the activity of a recombinase (e.g., a recombinase site).

[0535] In some aspects of the nanotransposon, the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.

[0536] In some aspects of the nanotransposon, the sequence between the ITRs does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some aspects, the sequence between the ITRs does not comprise the product of a recombination event, an excision event, a ligation event, or a combination thereof. In some aspects, the sequence between the ITRs is not derived from a recombination event, an excision event, a ligation event, or a combination thereof. In some aspects, the sequence between the ITRs comprises a sequence encoding foreign DNA. In some aspects, the sequence within the ITRs comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell. The mammalian cell can be a human cell. In some aspects, the sequence within the ITRs comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, and a second sequence encoding an insulator. In some aspects, the sequence within the ITRs comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosine (poly-A) sequence, and a second sequence encoding an insulator. In some aspects, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (poly A) sequence, and a second sequence encoding an insulator.

[0537] Nanotransposons are described in more detail in PCT / US2019 / 067758.

[0538] Vector system

[0539] The carrier of the present disclosure can be a viral vector or a recombinant vector.The viral vector can include a sequence separated from or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof.The viral vector can include a sequence separated from or derived from an adeno-associated virus (AAV).The viral vector can include a recombinant AAV (rAAV).Exemplary adeno-associated virus and recombinant adeno-associated virus include two or more reverse terminal repeat (ITR) sequences located close to the sequence encoding the scFv or CAR of the present disclosure.Exemplary adeno-associated virus and recombinant adeno-associated virus include but are not limited to all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).Exemplary adeno-associated virus and recombinant adeno-associated virus include but are not limited to self-complementary AAV (scAAV) and AAV hybrids, which contain a genome of a serotype and a capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0540] The carrier of the present disclosure can be nanoparticle.The limiting examples of nanoparticle carrier comprises nucleic acid (for example, RNA, DNA, synthetic nucleotide, modified nucleotide or its any combination), amino acid (L-amino acid, D-amino acid, synthetic amino acid, modified amino acid or its any combination), polymer (for example, polymer vesicle (polymersome)), micelle, lipid (for example, liposome), organic molecule (for example, carbon atom, lamella, fiber, tube), inorganic molecule (for example, calcium phosphate or gold) or its any combination.Nanoparticle carrier can passively or actively transport across cell membrane.

[0541] Cell delivery compositions disclosed herein (e.g., transposon, vector) may include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0542] Inducible apoptosis-promoting peptide

[0543] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure are much less immunogenic. The inducible pro-apoptotic polypeptides are recombinant polypeptides and are therefore non-naturally occurring. Further, the sequences are recombined to produce inducible pro-apoptotic polypeptides that do not contain non-human sequences that the host human immune system can recognize as "non-self" and thereby induce an immune response in a subject who receives an inducible pro-apoptotic polypeptide, a cell comprising an inducible pro-apoptotic polypeptide, or a composition comprising an inducible pro-apoptotic polypeptide or a cell containing an inducible pro-apoptotic polypeptide.

[0544] 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 aspects, the non-human sequence comprises a restriction site. In certain aspects, the ligand binding region can be a multimeric ligand binding region. In certain aspects, the pro-apoptotic peptide is a caspase polypeptide. Non-limiting examples of caspase polypeptides include 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. Preferably, the caspase polypeptide is a caspase 9 polypeptide. The caspase 9 polypeptide can be a truncated caspase 9 polypeptide. The inducible pro-apoptotic polypeptide can be non-naturally occurring. When the caspase is caspase 9 or a truncated caspase 9, the inducible pro-apoptotic polypeptide may also be referred to as an "iC9 safety switch."

[0545] The inducible caspase polypeptide may comprise (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 aspects, the inducible caspase polypeptide comprises (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.

[0546] The ligand-binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. 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). The FKBP12 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 109. The FKBP12 polypeptide may be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 110.

[0547] The linker region can comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 111, or the linker region can be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 112. In some aspects, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0548] The truncated caspase 9 polypeptide may comprise an amino acid sequence that does not include arginine (R) at position 87 of the sequence. Alternatively or additionally, the truncated caspase 9 polypeptide may comprise an amino acid sequence that does not include alanine (A) at position 282 of the sequence. The truncated caspase 9 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 113, or the truncated caspase 9 polypeptide may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 114.

[0549] In certain aspects, when the polypeptide comprises a truncated caspase 9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to SEQ ID NO: 115, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to SEQ ID NO: 116.

[0550] The inducible pro-apoptotic polypeptide can be expressed in a cell under the transcriptional control of any promoter known in the art, which is capable of initiating and / or regulating the expression of the inducible pro-apoptotic polypeptide in the cell.

[0551] Activation of inducible pro-apoptotic polypeptides can be accomplished, for example, by chemically induced dimerization (CID) mediated by an inducing agent to produce conditionally controlled proteins or polypeptides. Not only are pro-apoptotic polypeptides inducible, but the induction of these polypeptides is also reversible due to degradation of unstable dimerization agents or administration of monomeric competitive inhibitors.

[0552] In certain aspects, when 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-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1 (R*),2R*[S*[S*[1 (R*),2R*]]]]]-(9Cl) CAS Registry Number: 195514-63-7; Molecular Formula: C78H98N4O20; Molecular Weight: 1411.65); AP20187 (CAS Registry Number: 195514-80-8 and Molecular Formula: C82H107N5O20) or an AP20187 analog, such as AP1510. As used herein, the inducers AP20187, AP1903, and AP1510 can be used interchangeably.

[0553] Inducible pro-apoptotic peptides and methods of inducing these peptides are described in detail in U.S. Patent Publication No. WO 2019 / 0225667 and PCT Publication No. WO 2018 / 068022.

[0554] Formulation, dosage and mode of administration

[0555] The present disclosure provides formulations, dosages, and methods for administering the compositions described herein.

[0556] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable adjuvants, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods for preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, ed., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. As is well known in the art or as described herein, a pharmaceutically acceptable carrier may be conventionally selected that is suitable for the mode of administration, solubility, and / or stability of the protein scaffold, fragment, or variant composition.

[0557] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, constituting 1-99.99% by weight or volume alone or in combination. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / protein components that may also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. A preferred amino acid is glycine.

[0558] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), inositol, and the like. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0559] The composition may also include a buffer or pH adjuster; typically, a buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate.

[0560] Additionally, the disclosed compositions can include polymeric excipients / additives such as polyvinylpyrrolidone, sucrose (polymeric sugars), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0561] Many known and developed modes can be used to administer a therapeutically effective amount of a composition or pharmaceutical composition disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal means.

[0562] The compositions of the present disclosure can be prepared for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, particularly in the form of liquid solutions or suspensions; for vaginal or rectal administration, particularly in the form of semisolid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as, but not limited to, tablets or capsules; or intranasal administration, such as, but not limited to, powders, nasal drops or aerosols or certain medicaments; or transdermal administration, such as, but not limited to, gels, ointments, lotions, suspensions or patch delivery systems with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or increase drug concentration in transdermal patches (Junginger et al., in "Drug Permeation Enhancement;" Hsieh, DS, ed., pp. 59-90 (Marcel Dekker, Inc. New York 1994), or with oxidizing agents that enable application of formulations containing proteins and peptides to the skin (WO 98 / 53847), or applying an electric field to create a transient transport pathway, such as electroporation, or increasing the mobility of charged drugs through the skin, such as iontophoresis, or applying ultrasound, such as sonophoresis (U.S. Patent Nos. 4,309,989 and 4,767,402) (the above disclosures and patents are incorporated herein by reference in their entirety).

[0563] For parenteral administration, any of the compositions disclosed herein can be formulated as solutions, suspensions, emulsions, granules, powders, or lyophilized powders, either in combination with or separately from a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, hydronaphthalenes, and the like as common excipients. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Medicaments for injection can be non-toxic, parenteral diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. As acceptable vehicles or solvents, water, Ringer's solution, isotonic saline, and the like are permitted; as common solvents or suspending solvents, sterile fixed oils can be used. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semisynthetic fatty oils or fatty acids; natural, synthetic, or semisynthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, a pneumatic needle-free injection device such as described in US Pat. No. 5,851,198, and a laser perforation device such as described in US Pat. No. 5,839,446.

[0564] Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and aprotinin (trasylol)) to inhibit enzymatic degradation. Formulations for delivering hydrophilic agents including proteins and protein scaffolds and combinations of at least two surfactants are described in U.S. Patent No. 6,309,663, which are intended for oral, buccal, mucosal, nasal, pulmonary, vaginal, transmembrane or rectal administration. The active ingredient compound of a solid dosage form for oral administration may be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, arginine, chitin, chitosan, pectin, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, α-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, perfuming agents, and the like.

[0565] Tablets and pills can be further processed into enteric coating preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions and solution preparations that can allow medical use. These preparations can contain inert diluents, such as water, that are generally used in the field. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Patent number 4,239,754). Recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used for drug delivery (U.S. Patent number 4,925,673). In addition, U.S. Patent number 5,879,681 and U.S. Patent number 5,871,753 describe and are known in the art for carrier compounds for oral delivery of bioactive agents.

[0566] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size that effectively reaches the lower airways or sinuses of the lungs. The compositions or pharmaceutical compositions can be delivered by any of various inhalation or nasal devices known in the art, which are used for administering therapeutic agents by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of the patient include metered dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, sprayers, etc. All such devices can use formulations suitable for administration for distributing the compositions or pharmaceutical compositions described herein as aerosols. Such aerosols can be made up of solutions (aqueous and non-aqueous) or solid particles. In addition, a spray comprising the compositions or pharmaceutical compositions described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), a propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a tank as a mixture comprising a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in a size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulations, and related devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0567] For absorption through mucosal surfaces, compositions include emulsions comprising a plurality of submicron particles, mucoadhesive macromolecules, bioactive peptides, and an aqueous continuous phase that promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Patent No. 5,514,670). Mucus surfaces suitable for application of the emulsions of the present disclosure may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain, as excipients, such as polyalkylene glycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration may be solid and contain, as excipients, such as lactose, or may be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulation is disclosed in PCT Publication No. WO 2019 / 049816.

[0568] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as a liposome or polymer nanoparticles, microparticles, microcapsules or microspheres (collectively referred to as microparticles unless otherwise indicated). Many suitable devices are known, including microparticles made from synthetic polymers and natural polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides and polyphosphazenes, such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, preparations and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0569] It can be expected that the disclosed compounds will be delivered to the subject over an extended period of time, for example, a period of one week to one year from the start of a single administration. Various slow release, depot or implant dosage forms can be utilized. For example, the dosage form can contain a pharmaceutically acceptable non-toxic salt of a compound having low solubility in body fluids, for example, (a) an acid addition salt with a polyacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemonosulfonic acid or naphthalenedisulfonic acid, polygalacturonic acid, or the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or the like, or a salt with an organic cation such as N,N'-dibenzylethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), for example, zinc tannate. In addition, the disclosed compounds or preferably relatively insoluble salts, such as those just described, can be formulated in a gel suitable for injection, such as an aluminum monostearate gel with, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate, pamoate, and the like. Another type of slow-release depot formulation for injection will contain a compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as the polylactic acid / polyglycolic acid polymers described in U.S. Patent No. 3,773,919. The compounds or preferably relatively insoluble salts, such as those described above, can also be formulated in a cholesterol matrix silicone rubber pellet, particularly for use in animals. Additional slow-release, depot, or implant formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," edited by J.R. Robinson, Marcel Dekker, Inc., NY, 1978).

[0570] Suitable dosages are well known in the art. See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd ed., Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ. Preferred dosages may optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or to achieve a serum concentration of about 0.1-5000 μg / ml / single or multiple administrations, or any range, value, or fraction thereof. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are about 1 mg / kg, up to about 3, about 6, or about 12 mg / kg of the subject's body weight.

[0571] Alternatively, the dosage administered may vary depending on known factors such as the pharmacodynamic properties of a particular agent, as well as its mode and route of administration; the age, health, and weight of the recipient; the nature and extent of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Generally, the dosage of the active ingredient may be about 0.1 to 100 mg / kg body weight. Typically, 0.1 to 50, and preferably 0.1 to 10 mg / kg / administration or in a sustained-release form is effective to achieve the desired result.

[0572] As a non-limiting example, treatment of humans or animals can be provided using single, infusion, or repeated doses, at least one day out of days 1-40, or alternatively or additionally, at least one week out of weeks 1-52, or alternatively or additionally, at least one year out of years 1-20, or any combination thereof, as a one-time or periodic dose of about 0.1 to 100 mg / kg, or any range, value, or fraction thereof, per day of a composition or pharmaceutical composition disclosed herein.

[0573] Dosage forms suitable for internal administration generally contain from about 0.001 mg to about 500 mg of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is generally present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.

[0574] An effective amount can comprise an amount of about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple, or continuous administration to achieve a serum concentration of 0.01-5000 μg / ml per single, multiple, or continuous administration, or any effective range or value therein, as accomplished and determined using known methods, as described herein or known in the relevant art.

[0575] In aspects where the composition to be administered to a subject in need thereof is a modified cell as disclosed herein, about 1 x 10 3 Up to 1x10 15 cells; approximately 1x10 4 Up to 1x10 12 cells; approximately 1x10 5 Up to 1x10 10 cells; approximately 1x10 6 Up to 1x10 9 cells; approximately 1x10 6 Up to 1x10 8 cells; approximately 1x10 6 Up to 1x10 7 cells; or approximately 1x10 6 Up to 25x10 6 In one aspect, administration of about 5x10 6 Up to 25x10 6 cells of cells.

[0576] A more detailed description of the disclosed compositions and pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 049816.

[0577] Methods of using the compositions of the present disclosure

[0578] The present disclosure provides uses of the disclosed compositions or pharmaceutical compositions for treating a disease or condition in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting a cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0579] The present disclosure provides methods for regulating or treating at least one malignant disease or condition in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or conditions include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, B cell, T cell, or FAB. ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc.

[0580] In preferred aspects, the treatment of malignant diseases or illnesses includes adoptive cell therapy.For example, in one aspect, present disclosure provides cells expressing at least one disclosed antibody (eg, scFv) and / or the modification of the CAR comprising an antibody (eg, scFv), which has been selected and / or expanded for application to a subject in need. The modified cells can be formulated for storage at any temperature including room temperature and body temperature. The modified cells can be formulated for frozen storage and subsequent thawing. The modified cells can be formulated in a pharmaceutically acceptable carrier for direct application to a subject from a sterile package. The modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and / or CAR expression level to ensure the minimum level of cell function and CAR expression. The modified cells can be formulated in a pharmaceutically acceptable carrier with a specified density using one or more reagents to suppress further expansion and / or prevent cell death.

[0581] Any method may include administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods may optionally further include co-administration or combination therapy for treating such diseases or conditions, wherein administration of any composition or pharmaceutical composition disclosed herein further includes prior, simultaneous, and / or subsequent administration of at least one chemotherapeutic agent (e.g., an alkylating agent, a mitotic inhibitor, a radiopharmaceutical).

[0582] In some aspects, the subject does not develop graft-versus-host (GvH) and / or host-versus-graft (HvG) following administration. In one aspect, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or intravenous infusion. In one aspect, administration is local. Topical administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral injection or infusion, intraspinal injection or infusion, intraventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0583] In some aspects, the therapeutically effective dose is a single dose. 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 therebetween, produced simultaneously. In some aspects, when the composition is autologous cells or allogeneic cells, the dose is an amount sufficient to allow the cells to implant and / or for a sufficient time to treat the disease or condition.

[0584] In one example, present disclosure provides a method for treating a cancer in a subject in need thereof, comprising administering a composition to the subject, the composition comprising an antibody (e.g., scFv) or a CAR comprising an antibody (e.g., scFv), the antibody or CAR specifically binding to an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell colony, the cell or cell colony can be autologous or allogeneic.

[0585] In some aspects of the methods of treatment described herein, treatment can be modified or terminated. Specifically, in aspects where the compositions for treatment include inducible pro-apoptotic polypeptides, apoptosis can be selectively induced in cells by contacting cells with inducers. Treatment can be modified or terminated in response to signs such as signs of recovery or signs of reduced disease severity / progress, signs of disease remission / stopping, and / or adverse events. In some aspects, the method includes administering an inhibitor of an inducer to suppress the step of modification of cell therapy, thereby restoring the function and / or efficacy of cell therapy (for example, when signs or symptoms of disease reappear, or severity increases and / or adverse events are resolved).

[0586] Antibody / scFv production, screening and purification

[0587] At least one antibody of the present disclosure (e.g., a monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single chain variable fragment (scFv), an antigen binding fragment (Fab), or a Fab fragment) can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, 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).

[0588] Amino acids from a scFv can 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 property, as known in the art.

[0589] Optionally, scFv can be transformed, with the retention of high affinity for antigen and other favorable biological properties. In order to achieve this goal, scaffold protein can be optionally prepared by using the three-dimensional model of the parental sequence and the transformed sequence, analyzing the process of the parental sequence and various conceptual transformation products. The three-dimensional model is generally available and is familiar to those skilled in the art. Computer programs are available, which illustrate and display the possible three-dimensional conformational structure of selected candidate sequences, and can measure possible immunogenicity (for example, the Immunofilter program of Xencor, Inc., Monrovia, Calif.). The inspection of these displays allows analysis of the possible effects of residues in the function of candidate sequences, that is, analysis affects the residues of candidate scFv in conjunction with the ability of its antigen. In this way, residues can be selected and combined from parental sequence and reference sequence to achieve desired characteristics, for example, affinity for target antigens. Alternatively or in addition to the above-mentioned program, other suitable transformation methods can be used.

[0590] Nucleotide (DNA or RNA display) or peptide display libraries can be used, for example, in in vitro display, to easily realize the screening of scFv that specifically binds to similar proteins or fragments. This method involves screening large peptide collections for each member with the desired function or structure. The length of the nucleotide or peptide sequence displayed can be 3 to 5000 or more nucleotides or amino acids, frequently 5-100 amino acids long, and often about 8 to 25 amino acids long. In addition to the direct chemical synthesis method for generating peptide libraries, several recombinant DNA methods have also been described. One type relates to the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a specific display peptide sequence. Such methods are described in PCT Patent Publications WO 91 / 17271, WO 91 / 18980, WO 91 / 19818 and WO 93 / 08278.

[0591] Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis and recombinant methods. See PCT Patent Publication Nos. WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif.) and Cambridge Antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Patent Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5856456, all assigned to Enzon; ​​5,223,409, 5,403,484, 5,571,698, 5,837,500, all assigned to Dyax; 5,427,908, 5,580,717, all assigned to Affymax; and 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5856456, all assigned to Enzon; Technologies, 5,885,793; Genentech, 5,750,373; Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra.

[0592] The scFvs of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred aspect, at least one scFv of the present disclosure can optionally bind to a target protein with high affinity, e.g., with a KD equal to or less than about 10 −7 M, for example but not limited to, 0.1-9.9 (or any range or value therein) x 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.

[0593] The affinity or avidity of an scFv for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and methods described herein). The affinity of a particular scFv-antigen interaction measured may be different if measured under different conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using standardized solutions of the protein scaffold and antigen, and standardized buffers such as those described herein.

[0594] Competitive assays can be performed with the scFv of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the scFv of the present disclosure for binding to a target protein and / or shared epitope regions. These assays, as readily known to those of ordinary skill in the art, assess competition between antagonists or ligands for a limited number of binding sites on a protein. Proteins and / or antibodies are fixed or insoluble before or after competition, and samples bound to the target protein are separated from unbound samples, for example, by decantation (wherein the protein / antibody is pre-insoluble) or by centrifugation (wherein the protein / antibody is precipitated after the competitive reaction). Additionally, competitive binding can be determined by whether function is altered by binding or lack of binding of the scFv to the target protein, for example, whether the scFv molecule inhibits or enhances, for example, the enzymatic activity of a marker. ELISA and other functional assays can be used, as well known in the art.

[0595] Nucleic acid molecules

[0596] The nucleic acid molecules encoding scFv of the present disclosure can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained or synthesized by cloning, or any combination thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be a coding strand, also referred to as a sense strand, or it can be a non-coding strand, also referred to as an antisense strand.

[0597] The isolated nucleic acid molecules of the present disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, such as, but not limited to, at least one specified portion of at least one scFv; nucleic acid molecules comprising a coding sequence for a protein scaffold or loop region that binds to a target protein; and nucleic acid molecules comprising a nucleotide sequence that is substantially different from those described above, but still encodes a protein scaffold as described herein and / or as known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it will be routine for those skilled in the art to generate such degenerate nucleic acid variants encoding a specific scFv of the present disclosure. See, for example, Ausubel et al., supra, and such nucleic acid variants are included in the present disclosure.

[0598] As indicated herein, nucleic acid molecules of the present disclosure comprising nucleic acids encoding scFvs can include, but are not limited to, those encoding the amino acid sequence of the scFv fragment itself; the coding sequence for the entire protein scaffold or a portion thereof; the coding sequence for the scFv, fragment, or portion thereof, and additional sequences, such as the coding sequence for at least one signal leader or fusion peptide, with or without such additional coding sequences, such as at least one intron, together with additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, such as transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and mRNA stability); additional coding sequences encoding additional amino acids, such as those that provide additional functionality. Thus, the sequence encoding the protein scaffold can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fusion protein scaffold comprising the protein scaffold fragment or portion.

[0599] Polynucleotides that selectively hybridize to a polynucleotide as described herein

[0600] The present disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Therefore, the polynucleotides can be used to separate, detect and / or quantitatively comprise nucleic acids of this type of polynucleotide. For example, the polynucleotides of the present disclosure can be used to identify, separate or amplify partial or full-length clones in a preserved library. The polynucleotides can be genomic or cDNA sequences isolated from a human or mammalian nucleic acid library, or otherwise complementary to a cDNA from a human or mammalian nucleic acid library or a cDNA sequence.

[0601] In some embodiments, the cDNA library comprises at least 80% full-length sequence, preferably at least 85% or 90% full-length sequence, and more preferably at least 95% full-length sequence. The cDNA library can be standardized to increase the representation of rare sequences. Usually but not exclusively, low or medium stringency hybridization conditions are adopted for the sequence with the sequence identity that reduces with respect to the complementary sequence. Medium and high stringency conditions can be optionally adopted for the sequence with higher identity. Low stringency conditions allow the selective hybridization of a sequence with about 70% sequence identity, and can be used to identify straight homology or paralogous sequence.

[0602] Optionally, the polynucleotide will encode at least a portion of a protein scaffold encoded by a polynucleotide described herein. The polynucleotide includes a nucleic acid sequence that can be used to selectively hybridize with a polynucleotide encoding a protein scaffold of the present disclosure. See, e.g., Ausubel, supra; Colligan, supra, each of which is incorporated herein by reference in its entirety.

[0603] Nucleic acid construction

[0604] The isolated nucleic acids of the present disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques and / or (d) combinations thereof, as is well known in the art.

[0605] The nucleic acid may conveniently comprise nucleotide sequences other than the polynucleotides of the present disclosure. For example, a multiple cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotides. Additionally, a translatable sequence may be inserted to aid in the isolation of the translated polynucleotides of the present disclosure. For example, a hexahistidine tag sequence provides a convenient means for purifying the proteins of the present disclosure. The nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotides of the present disclosure.

[0606] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0607] Recombinant methods for constructing nucleic acids

[0608] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some aspects, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library under stringent conditions. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, for example, Ausubel, supra; or Sambrook, supra).

[0609] Nucleic acid screening and isolation methods

[0610] Probes based on the polynucleotide sequences of the present disclosure can be used to screen cDNA or genomic libraries. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will appreciate that various degrees of hybridization stringency can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, a greater degree of complementarity must exist between the probe and target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization can be conveniently varied by manipulating the formamide concentration, for example, within a range of 0% to 50%, thereby altering the polarity of the reactant solution. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0611] Methods of amplification of RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation based on the teachings and guidance presented herein.

[0612] Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al.; 5,142,033 to Innis; 5,122,464 to Wilson et al.; 5,122,464 to Innis et al. 5,091,310 to Gyllensten et al.; 5,066,584 to Gyllensten et al.; 4,889,818 to Gellensten et al.; 4,994,370 to Silver et al.; 4,766,067 to Biswas; 4,656,134 to Ringold), and RNA-mediated amplification using antisense RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al., under the trade name NASBA), the entire contents of which are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.)

[0613] For example, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences encoding proteins to be expressed, prepare nucleic acids to be used as probes for detecting the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide technicians in completing in vitro amplification methods are found in: Berger, supra, Sambrook, supra, and Ausubel, supra, and Mullis et al., U.S. Patent No. 4,683,202 (1987); and Innis et al., PCR Protocols A Guide to Methods and Applications, ed., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0614] Synthetic methods for constructing nucleic acids

[0615] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis via known methods (see, e.g., Ausubel et al., supra). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that although chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligating shorter sequences.

[0616] Recombinant expression cassette

[0617] The present disclosure further provides a recombinant expression cassette comprising a nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure, for example, a cDNA or genomic sequence encoding a protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically comprises a polynucleotide of the present disclosure that is operably connected to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of a nucleic acid of the present disclosure.

[0618] In some aspects, an isolated nucleic acid acting as a promoter, enhancer, or other element can be introduced into an appropriate position (upstream, downstream, or intron) of a non-heterologous form of a polynucleotide of the present disclosure to up-regulate or down-regulate the expression of the polynucleotide of the present disclosure. For example, an endogenous promoter can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0619] Expression vectors and host cells

[0620] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically modified with the recombinant vectors, and production of at least one protein scaffold by recombinant techniques, as are well known in the art. See, for example, Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0621] The polynucleotide can optionally be linked to a vector containing a selectable marker for propagation in a host. Typically, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

[0622] The DNA insert should be operably connected with a suitable promotor. The expression construct will further contain sites for transcription initiation, termination, and in the transcription region, for the ribosome bind site of translation. The coding portion of the mature transcript expressed by the construct will preferably be included in the translation initiation at the beginning and the terminator codon (for example UAA, UGA or UAG) suitably placed at the end of mRNA to be translated, wherein UAA and UAG are preferably used in mammals or eukaryotic cell expression.

[0623] The expression vector will preferably but optionally include at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, zeocin ( Sh bla gene), puromycin ( pac gene), hygromycin B ( hygB gene), G418 / Geneticin ( neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin ( bsd gene) resistance genes, and ampicillin, zeocin ( Sh bla gene), puromycin ( pac gene), hygromycin B ( hygB gene), G418 / Geneticin ( neo Genes, such as kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes (the aforementioned patents are hereby incorporated by reference in their entirety). Appropriate culture media and conditions for the above-mentioned host cells are known in the art. Suitable vectors will be apparent to those skilled in the art. Introduction of the vector construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, for example, in Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0624] The expression vector will preferably but optionally include at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or protein groups that distinguish cells or cell subsets from another limited cell subset. Preferably, the selectable cell surface markers distinguish those cells modified by the compositions or methods of the present disclosure from those cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, "designated clusters" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8): 1277-87).

[0625] The expression vector will preferably but optionally include at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0626] At least one protein scaffold of the present disclosure can be expressed in a modified form, such as a fusion protein, and can include not only a secretion signal, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and durability in the host cell, during purification, or during subsequent processing and storage. In addition, peptide moieties can be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to the final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17, and 18.

[0627] Those of ordinary skill in the art are aware of numerous expression systems that can be used to express nucleic acids encoding proteins of the present disclosure. Alternatively, nucleic acids of the present disclosure can be expressed in host cells by opening (by manipulation) in a host cell containing endogenous DNA encoding the protein scaffold of the present disclosure. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

[0628] The example of the cell culture that can be used for producing protein scaffold, its specified part or variant is bacterium, yeast and mammalian cell as known in the art.Mammalian cell system is often the form of monolayer cell, although mammalian cell suspension or bioreactor can also be used.Many suitable host cell lines that can express complete glycosylated protein have been developed in this area, and include COS-1 (for example ATCC CRL 1650), COS-7 (for example ATCC CRL-1651), HEK293, BHK21 (for example ATCC CRL-10), CHO (for example ATCC CRL 1610) and BSC-1 (for example ATCC CRL-26) cell line, Cos-7 cell, CHO cell, hep G2 cell, P3X63Ag8.653, SP2 / 0-Ag14, 293 cell, HeLa cell etc., it can be from for example American Type Culture Collection (American Type Culture Collection), Manassas, Va. (www.atcc.org) easily obtain. Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0629] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., a late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer and / or processing information sites, such as a ribosome binding site, an RNA splice site, a polyadenylation site (e.g., an SV40 large T Ag poly A addition site), and a transcription terminator sequence. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells that can be used to produce the nucleic acids or proteins of the disclosure are known and / or are available, for example, from the American Type Culture Collection's catalog of cell lines and hybridomas (www.atcc.org) or other known or commercial sources.

[0630] When using eukaryotic host cells, a polyadenylation sequence or transcription terminator sequence is typically introduced into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of transcripts may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). In addition, as is known in the art, gene sequences that control replication in the host cell may be introduced into the vector.

[0631] scFv purification

[0632] scFv can be recovered and purified from recombinant cell culture by well-known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

[0633] The scFvs of the present disclosure include purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, E. coli, yeast, higher plants, insects, and mammalian cells. Depending on the host employed in the recombinant production procedure, the protein scaffolds of the present disclosure may be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18, and 20, Colligan, Protein Science, supra, chapters 12-14, all of which are incorporated herein by reference in their entirety.

[0634] Amino acid code

[0635] The amino acids constituting the protein scaffolds of the present disclosure are often abbreviated. Amino acid nomenclature can be indicated by specifying amino acids via their single-letter codes, their three-letter codes, names, or trinucleotide codons, as is well known in the art (see Alberts, B. et al., Molecular Biology of The Cell, 3rd edition, Garland Publishing, Inc., New York, 1994). The protein scaffolds of the present disclosure may include one or more amino acid substitutions, deletions, or additions from spontaneous or mutational and / or artificial manipulation, as specified herein. The amino acids essential for function in the protein scaffolds of the present disclosure can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecule is then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).

[0636] As will be appreciated by those skilled in the art, the present disclosure includes at least one bioactive protein scaffold of the present disclosure. The bioactive protein scaffold has a specific activity that is at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or more of the specific activity of a natural (non-synthetic), endogenous, or related and known protein scaffold. Methods for determining and quantifying enzymatic activity and substrate specificity are well known to those skilled in the art.

[0637] In another aspect, the disclosure relates to protein scaffolds and fragments as described herein, which are modified by the covalent attachment of an organic moiety. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In particular aspects, the hydrophilic polymer group can have a molecular weight of approximately 800 to approximately 120,000 daltons, and can be a polyalkane diol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group can comprise approximately eight to approximately 40 carbon atoms.

[0638] The modified protein scaffolds and fragments of the present disclosure may comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety bonded to the protein scaffold or fragment of the present disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic acids and dicarboxylic acids. As the term is used herein, a "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, protein scaffolds modified by covalent attachment of polylysine are encompassed by the present disclosure. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure can be linear or branched and include, for example, polyalkylene glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers that modify the protein scaffolds of the present disclosure have a molecular weight of about 800 to about 150,000 daltons as separate molecular entities. For example, PEG5000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by using suitable methods. For example, a polymer containing amine groups can be coupled to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate (eg, activated with N,N-carbonyldiimidazole) on the fatty acid or fatty acid ester can be coupled to a hydroxyl group on the polymer.

[0639] Suitable fatty acids and fatty acid esters for modifying the protein scaffolds of the present disclosure may be saturated or may contain one or more unsaturated units. Suitable fatty acids for modifying the protein scaffolds of the present disclosure include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), n-triacontanoic acid (C30), n-tetracontanoic acid (C40), cis-Δ9-octadecanoic acid (C18, oleic acid), all cis Δ5,8,11,14-eicosatetraenoic acid (C20, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosandioic acid, and the like. Suitable fatty acid esters include dicarboxylic acid monoesters containing straight or branched lower alkyl groups. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

[0640] Suitable methods can be used, for example, by reacting with one or more properties-modifying agents, to prepare protein scaffolds and fragments of modification. As used in this article, " properties-modifying agent" refers to a ...

Claims

1. A non-naturally occurring chimeric stimulatory receptor (CSR) comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

39.

2. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

43.

3. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

47.

4. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

51.

5. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

55.

6. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

59.

7. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

63.

8. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

67.

9. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

71.

10. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

37.

11. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

41.

12. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

45.

13. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

49.

14. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

53.

15. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

57.

16. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

61.

17. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

65.

18. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

69.

19. A CSR comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR is SEQ ID NO:

73.

20. A nucleic acid sequence encoding the CSR of any one of claims 1-19.

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