Allogeneic cell compositions and methods of use

Abstract

Disclosed are chimeric stimulatory receptors (CSRs), cellular compositions containing CSRs, methods for making them, and methods for using them to treat a disease or disorder in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 896,495, filed September 5, 2019, and U.S. Provisional Patent Application No. 62 / 976,536, filed February 14, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Field of Disclosure FIELD OF THE DISCLOSURE The present disclosure relates to molecular biology, and more particularly to chimeric receptors, allogeneic cell compositions, and methods of making and using the same.

[0003] Incorporation by reference of sequence listing The contents of the file named "POTH-055_001WO_SequenceLissting_ST25.txt", created on August 21, 2020, and having a size of 291KB, are incorporated herein by reference in their entirety. [Background technology]

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

[0005] The present disclosure provides a non-natural chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation moiety, wherein the signal peptide comprises a CD2 signal peptide, and the activation moiety comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and 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 the signaling domain comprises a CD3ζ protein or a portion thereof, wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0006] In some embodiments, the CD2 signal peptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 5. In one embodiment, the CD2 signal peptide comprises the amino acid sequence of SEQ ID NO: 5.

[0007] The present disclosure also provides a non-natural chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation moiety, wherein the signal peptide comprises a CD8α signal peptide, and the activation moiety comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and 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 the signaling domain comprises a CD3ζ protein or a portion thereof, wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0008] In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 7. In preferred embodiments, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 7.

[0009] The present disclosure also provides non-natural chimeric stimulating receptors (CSRs) in which the activating moiety comprises a modification. In some embodiments, the modification comprises a mutation or truncation of the amino acid sequence of the agonist-binding CD2 extracellular domain or portion thereof compared to the wild-type sequence of the CD2 extracellular domain or portion thereof. In some embodiments, a non-natural CSR comprising a mutation or truncation of the agonist-binding CD2 extracellular domain or portion thereof does not bind to CD58. In some embodiments, the CD2 extracellular domain or portion thereof comprising the mutation or truncation comprises an amino acid sequence 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 embodiment, the CD2 extracellular domain or portion thereof comprising the mutation or truncation comprises the amino acid sequence of SEQ ID NO:3.

[0010] In some embodiments, 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 preferred embodiments, the CD2 transmembrane domain or a portion thereof comprises the amino acid sequence of SEQ ID NO: 9.

[0011] In some embodiments, the CD2 intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 13. In a preferred embodiment, the CD2 intracellular domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CD28 intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 15. In a preferred embodiment, the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 17. In a preferred embodiment, the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the IL17RA intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 19. In a preferred embodiment, the IL17RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the IL15RA intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 21. In a preferred embodiment, the IL15RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL21R intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: XX. In a preferred embodiment, the IL21R intracellular domain comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ICOS intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 25. In a preferred embodiment, the ICOS intracellular domain comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CD27 intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:27.In a preferred embodiment, the CD27 intracellular domain comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the OX40 intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 29. In a preferred embodiment, the OX40 intracellular domain comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the GITR intracellular domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 31. In a preferred embodiment, the GITR intracellular domain comprises the amino acid sequence of SEQ ID NO: 31.

[0012] In some embodiments, the signaling domain comprising a CD3 zeta protein or a portion thereof comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11. In preferred embodiments, the signaling domain comprising a CD3 zeta protein or a portion thereof comprises the amino acid sequence of SEQ ID NO: 11.

[0013] In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 39. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the non-naturally occurring CSR is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 43. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 47. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 51. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 55. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 59. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 63. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 67. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 67.In some embodiments, 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 preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 71.

[0014] In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 37. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 41. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 45. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 49. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 53. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 57. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 61. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 65. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 65.In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 69. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 73. In preferred embodiments, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 73.

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

[0016] The present disclosure provides a cell comprising any of the CSRs disclosed herein.The present disclosure provides a cell comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0017] The present disclosure also provides modified T lymphocytes (T cells) comprising: (a) a modification in 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 or autologous cells. In some preferred embodiments, the modified cells are allogeneic cells. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0018] The present disclosure provides a composition comprising any of the CSRs disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein, or a composition comprising a plurality of modified cells disclosed herein.

[0019] 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 stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain 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.

[0020] The modified T cells can further comprise an inducible pro-apoptotic polypeptide. The modified T cells can further comprise a modification of an endogenous sequence encoding beta-2-microglobulin (B2M), where the modification reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0021] The modified T cells can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is disposed between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising HLA-E can further comprise a first linker disposed between the B2M signal peptide and the peptide, and a second linker disposed between the B2M polypeptide and the peptide encoding HLA-E.

[0022] The modified T cells can further comprise a non-native antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-native antigen receptor can comprise a chimeric antigen receptor (CAR).

[0023] The CSR may be transiently expressed in the modified T cells. The CSR may be stably expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be transiently expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be stably expressed in the modified T cells. An inducible apoptosis-promoting polypeptide may be transiently expressed in the modified T cells. An inducible apoptosis-promoting polypeptide may be stably expressed in the modified T cells. A sequence encoding a non-natural antigen receptor or therapeutic protein may be transiently expressed in the modified T cells. A sequence encoding a non-natural antigen receptor or therapeutic protein may be stably expressed in the modified T cells.

[0024] 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 (TCM ), or stem cell-like T cells.

[0025] The present disclosure provides compositions comprising any of the modified T cells disclosed herein. The present disclosure also provides compositions comprising a population of modified T lymphocytes (T cells), wherein a plurality of the modified T cells of the population comprise a CSR disclosed herein. The present disclosure also provides compositions comprising a population of T lymphocytes (T cells), wherein a plurality of the T cells of the population comprise a modified T cell disclosed herein.

[0026] The present disclosure provides methods of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of any of the compositions disclosed herein or compositions for use in treating the disease or disorder. In one embodiment, the composition is a modified T cell or population of modified T cells disclosed herein. The present disclosure also includes methods of treating a disease or disorder, comprising administering to a subject in need thereof a composition disclosed herein and a therapeutically effective amount of at least one non-naturally occurring molecule that binds to CSR.

[0027] The present disclosure provides methods for producing a population of modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells. The present disclosure provides compositions comprising the population of modified T cells produced by this method. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR are stem memory T cells (T SCM ) or T SCMIn some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CM The modified T cells express one or more cell surface markers of CD45RO-like cells, and wherein the one or more cell surface markers include CD45RO and CD62L. The composition can be used to treat a disease or disorder. The present disclosure also provides uses of the compositions produced by the methods for treating a disease or disorder. The disclosure further provides a method of treating a disease or disorder, comprising administering a therapeutically effective amount of a composition produced by the methods to a subject in need thereof. The treatment method can further comprise administering an activator composition to the subject to activate the population of modified T cells in vivo, induce cell division of the population of modified T cells in vivo, or a combination thereof.

[0028] The present disclosure provides methods for producing a population of modified T cells, comprising, consisting essentially of, or consisting of introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of primary human T cells under conditions that transiently express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells. The present disclosure provides compositions comprising the population of modified T cells produced by this method. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR are stem memory T cells (T SCM ) or T SCM In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CMThe modified T cells express one or more cell surface markers of CD45-like cells, and wherein the one or more cell surface markers include CD45RO and CD62L. The composition can be used to treat a disease or disorder. The present disclosure also provides uses of the compositions produced by the methods for treating a disease or disorder. The present disclosure further provides methods of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition produced by the method. In some embodiments, the modified T cells within the population of modified T cells administered to the subject no longer express CSR.

[0029] The present disclosure provides methods for expanding a population of modified T cells, comprising introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of primary human T cells under conditions that stably express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not stably express the CSR under the same conditions. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR are stem memory T cells (T SCM ) or T SCMIn some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CM The modified T cells express one or more cell surface markers of CD45RO-like cells, wherein the one or more cell surface markers include CD45RO and CD62L. The present disclosure provides a composition comprising a population of modified T cells expanded by this method. The composition can be used to treat a disease or disorder. The present disclosure also provides a use of a composition expanded by the method for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering a therapeutically effective amount of a composition expanded by the method to a subject in need thereof. The treatment method can further comprise administering to the subject an activator composition to activate the population of modified T cells in vivo, inducing cell division of the population of modified T cells in vivo, or a combination thereof.

[0030] The present disclosure provides a method of expanding a population of modified T cells, comprising introducing a composition comprising a CSR of the present disclosure or a sequence encoding the same into a plurality of primary human T cells under conditions that transiently express the CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, and contacting the cells with an activator composition to produce a plurality of activated modified T cells, wherein the expansion of the plurality of modified T cells is at least two-fold greater than the expansion of a plurality of wild-type T cells that do not transiently express the CSR under the same conditions. The present disclosure provides a composition comprising the population of modified T cells expanded by the method. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising CSRs are stem memory T cells (T SCM ) or T SCM In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CMThe composition expresses one or more cell surface markers of CD45-like cells, wherein the one or more cell surface markers include CD45RO and CD62L. This composition can be used to treat a disease or disorder. The present disclosure also provides uses of the composition expanded by the method for treating a disease or disorder. The present disclosure further provides a method of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition expanded by the method. In some embodiments, the modified T cells within the population of modified T cells administered to the subject no longer express CSR.

[0031] Any of the above aspects can be combined with any other aspect.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular includes the plural unless the context clearly dictates otherwise. By way of example, the terms "a," "an," and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. By way of example, an "element" means one or more elements. Throughout this specification, the word "comprising," or variations such as "comprises" or "comprising," are understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, or steps, or group of elements, integers, or steps. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0033] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims.

[0034] The patent or application file contains at least one color drawing. 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. [Brief explanation of the drawings]

[0035] [Figure 1] Figure 1 is a schematic diagram showing an example of a CSR CD2z-D111H mutant for enhancing allogeneic or autologous CAR-T manufacturing. CSR CD2z-D111H mutants 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 encoded in mRNA or stably encoded in transposons.

[0036] [Figure 2]Figure 2 is a schematic diagram showing an example of a CSR CD2z-D111H mutant with a CD8a signal peptide to enhance allogeneic or autologous CAR-T manufacturing. 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 by mRNA or stably as encoded by transposons.

[0037] [Figure 3] Figure 3 is a graph showing that delivery of CSR enhances CAR T cell expansion during production. PanT cells isolated from normal donor blood were genetically modified using the piggyBac® DNA modification system in combination with the Cas-CLOVER™ gene editing system. Cells were electroporated in a single reaction with at least a transposon encoding the CAR and a selection gene, mRNA encoding the CSR, mRNA encoding the super-piggyBac® transposase enzyme, mRNA encoding Cas-CLOVER™, and multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out the TCR and MHCI (double knockout; DKO). Cells were then stimulated with agonist monoclonal antibodies anti-CD2, anti-CD3, and anti-CD28 and subsequently selected for gene modification over a 14-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of gene-modified cells. Significantly more DKO cell expansion was observed in samples expressing CSR.

[0038] [Figure 4]Figure 4 is a schematic diagram illustrating the experimental protocol for evaluating in vivo tumor control by CAR T cells generated using different CSRs. Allogeneic CAR T cells were generated using different boosting agents, as described in Figures 1 and 3 and Tables 1 and 2. A mouse xenograft model of multiple myeloma was utilized to evaluate the in vivo antitumor efficacy of allogeneic CAR T cells generated using 10 different boosting agents. Specifically, RPMI-8226 cell lines were injected subcutaneously (SC) into female NSG mice at a dose of 1 × 10 cells (day -7). Then, on day 0, when tumors were established (75–125 mm3 by caliper measurement [target average, approximately 100 mm3]), allogeneic CAR T cells were injected intravenously (IV) at a "stress" dose (5 × 106). The "stress" dose was used for higher resolution in detecting potential functional differences in efficacy between CAR T cells generated with different boosting molecules.

[0039] [Figure 5] Figure 5 is a graph showing tumor volume over time after alloCAR T cell treatment. In vivo tumor control was assessed using "stress" doses of CAR T cells produced according to the protocol shown in Figure 4 and using different CSRs. Tumor volume assessment by caliper measurement for all animals is displayed as group means with error bars and SEM (standard error of the mean).

[0040] [Figure 6] Figure 6 is a graph showing total T cells in the blood over time after alloCAR T cell treatment. In vivo tumor control was assessed using a "stress" dose of CAR T cells produced according to the protocol shown in Figure 4 and using different CSRs. Total T cells in the blood were measured by TruCount staining of human CD45+ cells per μl (hCD45+ / μL) for all animals and are displayed as group means with error bars SEM.

[0041] [Figure 7]Figure 7 is a graph showing peak T cells in the blood (T cell Cmax). In vivo tumor control was assessed using a "stress" dose of CAR T cells produced according to the protocol shown in Figure 4 and using different CSRs. For all animals, peak T cell levels in the blood, measured by TruCount staining of human CD45+ (hCD45+) cells, are displayed as group means with error bars and SEM.

[0042] [Figure 8] Figure 8 is a graph showing the area under the curve for blood T cells (hCD45+). In vivo tumor control was assessed using a "stress" dose of CAR T cells produced according to the protocol shown in Figure 4 and using different CSRs. For all animals, blood T cell AUC calculated from TruCount staining of human CD45+ cells is displayed as the group mean with error bars as SEM.

[0043] [Figure 9] Figure 9 is a series of graphs showing the phenotype of CD8+ T cells in the blood. In vivo tumor control was assessed using a "stress" dose of CAR T cells generated according to the protocol shown in Figure 4 and using different CSRs. For all animals, the phenotype of CD8+ T cells in the blood, measured by FACS staining, is displayed as the group mean with error bars as SEM at 14 and 35 days after CAR-T treatment. Cells were stained for surface CD45RA, CD45RO, and CD62L expression to define TSCM, TCM, TEM, and TEFF cells: TSCM (CD45RA+CD45RO-CD62L+; blue), TCM (CD45RA-CD45RO+CD62L+; red), TEM (CD45RA-CD45RO+CD62L-; green), and TEFF (CD45RA+CD45RO-CD62L-; purple).

[0044] All documents cited herein, including cross-referenced or related patents or patent applications, are incorporated herein by reference in their entirety for all purposes, unless expressly excluded or otherwise limited. The citation of a document is not an admission that it is prior art with respect to the disclosed or claimed invention, or that it alone or in combination with any other reference teaches, suggests, or discloses the invention herein. Furthermore, if the meaning or definition of a term in that document conflicts with the meaning or definition of the same term in this specification incorporated by reference, the meaning or definition assigned to that term in this specification shall control. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description of the Invention The present disclosure provides allogeneic cell compositions, methods of making and using these compositions, which contain non-naturally occurring structural modifications to restore responsiveness of allogeneic cells to environmental stimuli and reduce or prevent rejection due to natural killer cell-mediated cytotoxicity.

[0046] Chimeric stimulating receptor (CSR) and recombinant HLA-E polypeptides

[0047] An adoptive cell composition that is "universally" safe for administration to any patient requires significant reduction or elimination of alloreactivity. To this end, the cells (e.g., allogeneic cells) of the present disclosure can be modified to disrupt the expression or function of T cell receptors (TCRs) and / or major histocompatibility complex (MHC) classes. TCRs mediate graft-versus-host (GvH) reactions, while MHCs mediate host-versus-graft (HvG) reactions. In preferred embodiments, TCR expression and / or function is eliminated to prevent T cell-mediated GvH, which can potentially cause death in the subject. Thus, in preferred embodiments, the present disclosure provides pure TCR-negative allogeneic T cell compositions (e.g., each cell of the composition expresses TCRs at such low levels that they are undetectable or nonexistent).

[0048] To prevent HvG and thus improve cell engraftment in a subject, expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated. Improved engraftment results in longer cell persistence and thus a larger therapeutic window for the subject. Specifically, expression and / or function of beta-2-microglobulin (B2M), a structural component of MHC-I, is reduced or eliminated.

[0049] The above strategies pose additional challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), a part of the TCR complex. The loss of CD3ζ in TCR-KO T cells dramatically reduces the ability of these cells to be optimally activated and expanded using standard stimulating / activating reagents, including, but not limited to, agonistic anti-CD3 mAbs. Disruption of the expression or function of any component of the TCR complex results in the loss of all components of the complex, including TCR-alpha (TCRα), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ). Both CD3ε and CD3ζ are required for T cell activation and expansion. Agonistic anti-CD3 mAbs typically recognize CD3ε and possibly another protein within the complex, which then transmits signals to CD3ζ. CD3ζ provides the primary stimulus for T cell activation (together with a secondary costimulatory signal) for optimal activation and expansion. Under normal conditions, full T cell activation depends on TCR engagement in combination with a second signal mediated by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL) that enhances the immune response. However, in the absence of the TCR, stimulation with standard activation / stimulation reagents containing agonist anti-CD3 mAbs significantly reduces T cell expansion. In fact, T cell expansion is reduced to only 20–40% of normal expansion levels when stimulated with standard activation / stimulation reagents containing agonist anti-CD3 mAbs.

[0050] Thus, the present disclosure provides a non-natural chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component; (b) a transmembrane domain; and (c) an endodomain comprising a cytoplasmic domain and a signaling domain, wherein the signal peptide and the cytoplasmic domain are not derived from the same protein.

[0051] The activating component can include one or more portions of a T cell receptor (TCR), a TCR complex, a TCR co-receptor, a TCR co-stimulatory protein, a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activating component binds. The activating component can include the CD2 extracellular domain or a portion thereof to which an agonist binds.

[0052] The signaling domain can include one or more of a human signaling domain component, a T cell receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signaling domain can include a CD3 protein or a portion thereof. The CD3 protein can include a CD3 zeta protein or a portion thereof.

[0053] 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 protein and the second protein can be the same. The first protein and the third protein can be the same. The first protein and the fourth protein cannot be the same. The first protein and the fifth protein cannot be the same. The second protein and the third protein can be the same. The second protein and the fourth protein cannot be the same. The second protein and the fifth protein cannot be the same. The third protein and the fourth protein cannot be the same. The third protein and the fifth protein cannot be the same. The fourth protein and the fifth protein cannot be the same.

[0054] In some embodiments, the activating component does not bind to a naturally occurring molecule. In some embodiments, the activating component binds to a naturally occurring molecule, but CSR does not transduce a signal when the activating component binds to a naturally occurring molecule. In some embodiments, the activating component binds to a non-natural molecule. In some embodiments, the activating component does not bind to a naturally occurring molecule, but does bind to a non-natural molecule. CSR can selectively transduce a signal when the activating component binds to a non-natural molecule.

[0055] The present disclosure provides a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0056] The present disclosure provides a cell comprising any of the CSRs disclosed herein.The present disclosure provides a cell comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0057] The modified cells disclosed herein can be allogeneic or autologous cells. In some preferred embodiments, the modified cells are allogeneic cells. In some embodiments, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0058] The present disclosure provides a composition comprising any of the CSRs disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein, or a composition comprising a plurality of modified cells disclosed herein.

[0059] 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 stimulating receptor (CSR) comprising: (i) an ectodomain comprising an activating component isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signaling domain isolated from or derived from a second protein, wherein the first protein and the second protein are not identical.

[0060] The modified T cells can further comprise an inducible pro-apoptotic polypeptide. The modified T cells can further comprise a modification of an endogenous sequence encoding beta-2-microglobulin (B2M), wherein the modification reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0061] The modified T cells can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is disposed between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising HLA-E can further comprise a first linker disposed between the B2M signal peptide and the peptide, and a second linker disposed between the B2M polypeptide and the peptide encoding HLA-E.

[0062] The modified T cells can further comprise a non-native antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-native antigen receptor can comprise a chimeric antigen receptor (CAR).

[0063] The CSR may be transiently expressed in the modified T cells. The CSR may be stably expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be transiently expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be stably expressed in the modified T cells. An inducible apoptosis-promoting polypeptide may be transiently expressed in the modified T cells. An inducible apoptosis-promoting polypeptide may be stably expressed in the modified T cells. A sequence encoding a non-natural antigen receptor or therapeutic protein may be transiently expressed in the modified T cells. A sequence encoding a non-natural antigen receptor or therapeutic protein may be stably expressed in the modified T cells.

[0064] As described in detail herein, gene editing compositions, including, but not limited to, RNA-guided fusion proteins, including dCas9-Clo051, can be used to target and reduce or eliminate the expression of endogenous T cell receptors. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (such as a promoter) of a gene encoding an endogenous T cell receptor. Non-limiting examples of primers (including T7 promoters, genomic target sequences, and gRNA scaffolds) for generating guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α), TCR-beta (TCR-β), and beta-2-microglobulin (β2M) are disclosed in PCT Application No. PCT / US2019 / 049816.

[0065] Gene editing compositions containing RNA-guided fusion proteins, including, but not limited to, dCas9-Clo051, can be used to target and reduce or eliminate the expression of endogenous MHC1, MHCII, or MHC activators. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (such as a promoter) of a gene encoding one or more components of endogenous MHC1, MHCII, 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, the entire contents of which are incorporated herein by reference.

[0066] A detailed description of non-naturally occurring polypeptides, including genetic modifications of endogenous sequences encoding non-naturally occurring chimeric stimulating receptors, TCR-alpha (TCR-α), TCR-beta (TCR-β), and / or beta-2-microglobulin (β2M), and HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptides, is disclosed in PCT Application No. PCT / US2019 / 049816, which is incorporated herein by reference in its entirety.

[0067] Chimeric stimulating receptors of the present disclosure

[0068] The present disclosure provides a chimeric stimulating receptor (CSR) comprising an activating component comprising, consisting essentially of, or consisting of an agonist-binding CD2 extracellular domain or portion thereof. The agonist-binding CD2 extracellular domain or portion thereof is [ka] In a preferred embodiment, the CD2 extracellular domain or portion thereof to which the agonist binds comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:1 that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:1.

[0069] In some embodiments, the CD2 extracellular domain or portion thereof to which the agonist binds is [ka] In a preferred embodiment, the CD2 extracellular domain or portion thereof to which the agonist binds is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:2.

[0070] The present disclosure provides chimeric stimulating receptors (CSRs) comprising an ectodomain comprising an activating component comprising, consisting essentially of, or consisting of a non-native CD2 ectodomain. In some embodiments, the ectodomain of a CSR of the present disclosure can comprise a modification. The modification can comprise a mutation or truncation in the amino acid sequence of the activating component compared to the wild-type amino acid sequence of the activating component. The mutation or truncation in the amino acid sequence of the activating component can comprise a mutation or truncation in the CD2 ectodomain or a portion thereof to which an agonist binds. The mutated or truncated CD2 ectodomain binds to an anti-CD2 activating agonist and an anti-CD2 activating molecule but does not bind to native CD58. In some embodiments, the mutation present in the CD2 ectodomain that binds to an anti-CD2 activating agonist but does not bind to CD58 is a D111H mutation. A CD2 ectodomain with a D111H mutation is [ka] In a preferred embodiment, the extracellular domain of CD2 having the D111H mutation comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:3 that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:3.

[0071] In some embodiments, the CD2 extracellular domain having the D111H mutation is [ka] In a preferred embodiment, the CD2 extracellular domain having the D111H mutation is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:4.

[0072] The internal domain of the CSR of the present disclosure can further comprise, consist essentially of, or consist of a signal peptide. In some embodiments, the signal peptide can comprise, consist essentially of, or consist of the CD2 signal peptide or a portion thereof. In some embodiments, the signal peptide can comprise, consist essentially of, or consist of the CD8a signal peptide or a portion thereof.

[0073] In some embodiments, the CD2 signal peptide comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percent therebetween) identical to MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 5). In preferred embodiments, the CD2 signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5.

[0074] In some embodiments, the CD2 signal peptide is [ka] In a preferred embodiment, the CD2 signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:6.

[0075] In some embodiments, the CD8a signal peptide comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percent therebetween) identical to MALPVTALLLPLALLLHAARP (SEQ ID NO: 7). In preferred embodiments, the CD8a signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7.

[0076] In some embodiments, the CD8a signal peptide is [ka] In a preferred embodiment, the CD8a signal peptide is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:8.

[0077] The present disclosure provides CSRs comprising a transmembrane domain. In some embodiments, the transmembrane domain can comprise, consist essentially of, or consist of a CD2 transmembrane domain or a portion thereof. In some embodiments, the CD2 transmembrane domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 9). In preferred embodiments, the CD2 transmembrane domain or a portion thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9.

[0078] In some embodiments, the CD2 transmembrane domain or portion thereof comprises: [ka] In a preferred embodiment, the CD2 transmembrane domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO: 10.

[0079] The present disclosure provides CSRs comprising an endodomain comprising at least one signaling domain. In some embodiments, the signaling domain can comprise, consist essentially of, or consist of the CD3ζ intracellular domain or a portion thereof. In some embodiments, the CD3ζ intracellular domain or a portion thereof is [ka] In a preferred embodiment, the CD3 zeta intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO:11.

[0080] In some embodiments, the CD3 zeta intracellular domain, or a portion thereof, [ka] In a preferred embodiment, 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 at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence of SEQ ID NO: 12.

[0081] The endodomain of the CSR of the present disclosure can further comprise, consist essentially of, or consist of a cytoplasmic domain. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the CD2 intracellular domain (ICD) or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the CD28 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the 4-1BB intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the IL17RA intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the IL15RA intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the IL21R intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the ICOS intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the CD27 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the OX40 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain can comprise, consist essentially of, or consist of the GITR intracellular domain or a portion thereof.

[0082] In some embodiments, the CD2 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD2 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO: 13.

[0083] In some embodiments, the CD2 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD2 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO: 14.

[0084] In some embodiments, the CD28 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD28 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO: 15.

[0085] In some embodiments, the CD28 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD28 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO: 16.

[0086] In some embodiments, the 4-1BB intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the 4-1BB intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO: 17.

[0087] In some embodiments, the 4-1BB intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, 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 at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO: 18.

[0088] In some embodiments, the IL17RA intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL17RA intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO: 19.

[0089] In some embodiments, the IL17RA intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL17RA intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:20.

[0090] In some embodiments, the IL15RA intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL15RA intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO:21.

[0091] In some embodiments, the IL15RA intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL15RA intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:22.

[0092] In some embodiments, the IL21R intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL21R intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO:23.

[0093] In some embodiments, the IL21R intracellular domain, or a portion thereof, [ka] In a preferred embodiment, the IL21R intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:24.

[0094] In some embodiments, the ICOS intracellular domain or portion thereof comprises: [ka] In a preferred embodiment, the ICOS intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO:25.

[0095] In some embodiments, the ICOS intracellular domain or portion thereof comprises: [ka] In a preferred embodiment, the ICOS intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence of SEQ ID NO:26.

[0096] In some embodiments, the CD27 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD27 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO:27.

[0097] In some embodiments, the CD27 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the CD27 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:28.

[0098] In some embodiments, the OX40 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the OX40 intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the amino acid sequence of SEQ ID NO:29.

[0099] In some embodiments, the OX40 intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the OX40 intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent therebetween) to the nucleic acid sequence of SEQ ID NO:30.

[0100] In some embodiments, the GITR intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the GITR intracellular domain or a portion thereof comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the amino acid sequence of SEQ ID NO: 31.

[0101] In some embodiments, the GITR intracellular domain or a portion thereof comprises: [ka] In a preferred embodiment, the GITR intracellular domain or a portion thereof is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence of SEQ ID NO: 32.

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[0144] The compositions of the present disclosure (eg, CSR) bind to anti-CD2 activating agonists and anti-CD2 activating molecules, but do not bind to native CD58.

[0145] Compositions comprising the CSR of the present disclosure can be incorporated into a cell delivery composition (e.g., a transposon or vector) as described in detail herein, and optionally, can be incorporated into a cell.

[0146] Cells and modified cells of the present disclosure

[0147] The cells and modified cells of the present disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure can be immune cells. Immune cells of the present disclosure include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem and memory T cells (T SCM cells), central memory T cells (T CM ), stem cell-like T cells, B lymphocytes (B cells), 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.

[0148] Immune progenitor cells can include any cells that can differentiate into one or more types of immune cells. Immune progenitor cells can include pluripotent stem cells that can self-renew and develop into immune cells. Immune progenitor cells can include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells can include progenitor cells that can develop into immune cells. Immune progenitor cells can include hematopoietic progenitor cells (HPCs).

[0149] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs are found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0150] HSCs can be isolated or derived from primary or cultured stem cells. HSCs can be isolated or derived from embryonic stem cells, multipotent stem cells, pluripotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0151] Immune progenitor cells can include HSCs or HSC progeny, non-limiting examples of HSC progeny 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.

[0152] HSCs generated by the disclosed methods can retain the characteristics of "primitive" stem cells, which are isolated or derived from adult stem cells and share the characteristics of embryonic stem cells while committed to a single lineage. For example, "primitive" HSCs generated by the disclosed methods retain their "stemness" and do not differentiate after division. As a result, as adoptive cell therapy, "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs generated by the disclosed methods can be therapeutically effective when administered as a single dose.

[0153] Primitive HSCs can be CD34+. Primitive HSCs can be CD34+ and CD38-. Primitive HSCs can be CD34+, CD38-, and CD90+. Primitive HSCs can be CD34+, CD38-, CD90+, and CD45RA-. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0154] Primitive HSCs, HSCs, and / or HSC progeny can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). The modified primitive HSCs, modified HSCs, and / or modified HSC progeny can be forward differentiated to produce modified immune cells, including, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells.

[0155] The modified immune cells or immune progenitor cells can be NK cells. NK cells can be cytotoxic lymphocytes differentiated from lymphocyte progenitor cells. The modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some embodiments, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0156] The modified immune cells or immune precursor cells can be B cells. B cells are a type of lymphocyte that express B cell receptors on their cell surface. B cell receptors bind to specific antigens. The modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0157] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapeutic agents, the disclosed modified T cells have the ability to rapidly replicate upon antigen recognition, thus potentially eliminating the need for repeated treatments. To achieve this, in some embodiments, the modified T cells not only promote an initial response but also persist in the patient as a stable population of viable memory T cells to prevent potential relapse. Alternatively, in some embodiments, the modified T cells do not persist in the patient if this is not desired.

[0158] Development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, and early memory T cells, especially stem cell memory (T SCM Intensive efforts have been made to develop modified T cell products comprising central memory (T) or stem cell-like T cells. The stem cell-like modified T cells of the present disclosure exhibit the greatest potential for self-renewal and pluripotency, and are thought to be capable of functioning as central memory (T CM ) T cells or T CM -like cells, effector memory (T EM ) and effector T cells (T E ), thereby achieving better tumor eradication and long-term modified T cell engraftment. A linear pathway of differentiation leads to the differentiation of these cells (naive T cells (T N )>T SCM >>T EM >T E >T TE ), where T N is T SCM These are the parent progenitor cells that directly give rise to T CM The T cell compositions of the present disclosure can include one or more of each parental T cell subset, SCM Cells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0159] Immune cell progenitor cells include early memory T cells, stem cell-like T cells, and naïve T cells (T N), T SCM , TCM, T EM , T E , or T TE The immune cell precursors can be primitive HSCs, HSCs, or HSC progeny cells of the present disclosure. The immune cells can be early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE It could be.

[0160] The disclosed methods can modify and / or produce 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 one or more cell surface markers of early memory T cells. The population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. The population of modified early memory T cells can comprise a plurality of modified T cells. SCM The population of modified early memory T cells includes multiple modified T CM Contains cells.

[0161] The disclosed methods can modify and / or produce 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 within the population express one or more cell surface markers of stem cell-like T cells. A population of modified stem cell-like T cells can include a plurality of modified T cells. SCM The population of modified stem cell-like T cells comprises multiple modified T CM Contains cells.

[0162] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage therebetween, of the plurality of modified T cells in the population are stem memory T cells (T SCM ) or T SCM and wherein the one or more cell surface markers include CD45RA and CD62L. The cell surface markers can include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. The cell surface markers can include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0163] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage therebetween, of the plurality of modified T cells in the population are central memory T cells (T CM ) or T CM and wherein the one or more cell surface markers include CD45RO and CD62L. The cell surface markers can include one or more of CD45RO, IL-2Rβ, CCR7, and CD62L.

[0164] The methods of the disclosure can modify and / or produce 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 the plurality of modified T cells in the population are naive T cells (T N ) The cell surface markers can include one or more of CD45RA, CCR7, and CD62L.

[0165] The disclosed methods can modify and / or produce 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 the plurality of modified T cells in the population are effector T cells (T EFF ) The cell surface markers can include one or more of CD45RA, CD95, and IL-2Rβ.

[0166] The methods of the disclosure can modify and / or produce 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 the plurality of modified T cells in the population are stem cell-like T cells, stem memory T cells (T SCM ), or central memory T cells (T CM ) express one or more cell surface markers.

[0167] A plurality of the modified cells of 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 cells of the population comprise a transgene or a sequence encoding a transgene, wherein at least 70%, at least 75%, at least 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% express one or more cell surface markers including CD34, or wherein at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% of the population of modified cells express one or more cell surface markers including CD34 (e.g., including the cell surface marker phenotype CD34+).

[0168] A plurality of the modified cells of 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 of the population comprise 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 population of modified cells. %, 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 population of modified cells express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38, or wherein at least about 45% to about 90%, about 50% to about 80%, or about 65% to about 75% of the population of modified cells express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38 (e.g., cell surface marker phenotypes CD34+ and CD38-).

[0169] A plurality of the modified cells of 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 cells of the population comprise 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% are CD3 and CD90, and do not express one or more cell surface markers including 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 population of modified cells express one or more cell surface markers including CD34 and CD90, and do not express one or more cell surface markers including CD38 (e.g., cell surface marker phenotypes CD34+, CD38-, and CD90+).

[0170] a plurality of modified cells of the population comprise 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 of the population comprise 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 cells comprise one or more of CD34 and CD90. and do not express one or more cell surface markers including CD34 and CD90, 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 population of modified cells express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including CD38 and CD45RA (e.g., cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA-).

[0171] A plurality of the modified cells of the population comprise 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 cells of the population comprise 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% express one or more cell surface markers including CD34, CD90, and CD49f, and do not express one or more cell surface markers including CD38 and CD45RA, or wherein at least about 0.02% to about 30%, 0.02% to about 2%, about 0.04% to about 2%, or about 0.Between 0.4% and approximately 1% express one or more cell surface markers including CD34, CD90, and CD49f, and do not express one or more cell surface markers including one or more cell surface markers including CD38 and CD45RA (e.g., cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA-, and CD49f+).

[0172] A plurality of the modified cells of 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 cells of the population comprise a transgene or a sequence encoding a transgene, wherein at least 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 at least 99.9%, or 100%, of the population of modified cells express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including 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 population of modified cells express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including CD45RA (e.g., cell surface marker phenotypes CD34+, CD90+, and CD45RA-).

[0173] Compositions and methods for producing and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells), and buffers for maintaining or enhancing cell viability and / or the level of stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells), are disclosed elsewhere herein and in more detail in U.S. Pat. No. 10,329,543 and PCT Publication No. WO2019 / 173636.

[0174] 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 or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0175] Methods for expressing chimeric antigen receptors

[0176] The present disclosure provides a method for expressing a CAR on the surface of a cell, the method comprising: (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding a CAR under conditions sufficient to transport the CAR across the cell membrane of at least one cell in the cell population, thereby producing a modified cell population; (c) culturing the modified cell population under conditions suitable for incorporation of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on its cell surface.

[0177] In some embodiments, the cell population can comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio. The optimal ratio of CD4+ and CD8+ leukocytes does not naturally occur in vivo. The cell population can comprise tumor cells.

[0178] In some embodiments, the conditions sufficient to transfer the CAR, or a sequence encoding the CAR, transposon, or vector, across the cell membrane of at least one cell in the cell population comprise application of at least one of one or more electrical pulses of a particular voltage, a buffer, and one or more cofactors. In some embodiments, the conditions suitable for integration of the sequence encoding the CAR comprise at least one buffer and one or more cofactors.

[0179] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplemental factors can include (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, metabolic, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof; and (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins, or any combination thereof, include 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-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12p70, IL-12 / IL-35p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANKL, or any combination thereof.The salts, minerals, metabolites, or any combination thereof can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacers, antibiotics, pH adjusters, Earl's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl, NaHPO, NAHPO, sodium lactobionate, mannitol, sodium succinate, sodium chloride, ClNa, glucose, Ca(NO), Tris / HCl, KHPO, KHPO, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium can include PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XFT Cell Expansion Medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof, include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (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 reagent that modifies or stabilizes one or more nucleic acids can include a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net-neutral-charge DNA-binding peptide with or without an NLS sequence, a TREX1 enzyme, or a combination thereof.

[0180] The expansion and selection steps can be performed simultaneously or sequentially. Expansion can occur before selection. Expansion can occur after selection, and optionally, expansion can be followed by a further (i.e., second) selection. Joint expansion and selection can occur simultaneously. The expansion and / or selection steps can continue for 10 to 14 days, inclusive.

[0181] Expansion can include contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell via the CAR, thereby generating an expanded cell population. The antigen can be presented on the surface of a substrate. The substrate can be in any configuration, including but not limited to, a surface, a well, a bead, or a combination thereof, and a matrix. The substrate can further include a paramagnetic or magnetic component. The antigen can be presented on the surface of a substrate, where the substrate is a magnetic bead, and a magnet can be used to remove or separate the magnetic bead from the modified and expanded cell population. The antigen can be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells can include, but are not limited to, tumor cells and stem cells.

[0182] In some embodiments, where the transposon or vector comprises a selection gene, the selection step involves contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thus identifying cells that express the selection gene as those that survive the selection and cells that fail to express the selection gene as those that do not survive the selection step.

[0183] The present disclosure provides compositions comprising modified, expanded, and selected cell populations of the methods described herein.

[0184] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication Nos. WO2019 / 049816 and PCT / US2019 / 049816.

[0185] The present disclosure provides a cell or population of cells, wherein the cells comprise 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 the exogenous receptor is expressed upon integration of the construct of (a) and the construct of (b) into the genomic sequence of the cell, wherein the exogenous receptor, upon binding to a ligand or antigen, transduces an intracellular signal that directly or indirectly targets the inducible promoter and regulates expression of the inducible transgene (a) to modify gene expression.

[0186] The composition can modify gene expression by decreasing gene expression. The composition can modify gene expression by modifying gene expression transiently (e.g., while a ligand is bound 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 chronically (e.g., a ligand irreversibly binds to an exogenous receptor).

[0187] Exogenous receptors can include receptors that are endogenous with respect to the genomic sequence of a 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.

[0188] The exogenous receptor can include a non-natural receptor. The non-natural receptor can be synthetic, modified, recombinant, mutated, or chimeric. The non-natural receptor can include one or more sequences isolated or derived from a T cell receptor (TCR). The non-natural receptor can include one or more sequences isolated or derived from a scaffold protein. In some embodiments, including those 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 transmits an intracellular signal after contact with the non-natural receptor. The non-natural receptor can include a transmembrane domain. The non-natural receptor can interact with an intracellular receptor that transmits an intracellular signal. The non-natural receptor can include an intracellular signaling domain. The non-natural receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR).

[0189] The sequence encoding the inducible promoter includes a sequence encoding an NFκB promoter, a sequence encoding an interferon (IFN) promoter, or a sequence encoding an interleukin-2 promoter. In some embodiments, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated or derived from a cytokine or chemokine promoter. The cytokine or chemokine can include IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony-stimulating factor 2 (GM-CSF), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), CC-C motif chemokine ligand 5 (CCL5), CC-C motif chemokine ligand 4 (Ccl4), CC-C motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin 6 family cytokine (Lif).

[0190] Inducible promoters can be isolated from or derived from promoters of genes including surface proteins involved in cell differentiation, activation, depletion, and function. In some embodiments, the genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0191] The inducible promoter can be isolated from or derived from the promoters of genes involved in CD4+ metabolism and differentiation, such as Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp3612, Gadd45b, Dusp5, Dusp6, and Neto2.

[0192] In some embodiments, the inducible transgene construct comprises or drives expression of 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 therapy, and oncogenes or tumor suppressor genes, non-limiting examples of which are disclosed in PCT Publication No. WO2019 / 173636 and PCT Application No. PCT / US2019 / 049816.

[0193] armored cells

[0194] 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 to make them less sensitive to immunological and / or metabolic checkpoints. This type of modification "armores" the cells, and the modified cells are referred to herein as "armored" cells (e.g., armored T cells). Armored cells can be generated by blocking and / or diluting (e.g., checkpoint inhibition) certain checkpoint signals that are naturally delivered to cells, for example, within the tumor immunosuppressive microenvironment.

[0195] The armored cells of the present disclosure can be derived from any cell, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells (including T cells isolated or derived from G-CSF-mobilized peripheral blood), or umbilical cord blood (UCB)-derived T cells. The armored cells (e.g., armored T cells) can comprise one or more of chimeric ligand receptors (CLRs comprising a protein scaffold, antibody, ScFv, or antibody mimetic) / chimeric antigen receptors (CARs comprising a protein scaffold, antibody, ScFv, or antibody mimetic), CARTyrin (CARs comprising centrin), and / or VCARs (CARs comprising camelid VHHs or single-domain VHs). The armored cells (e.g., armored T cells) can comprise an inducible apoptosis-promoting polypeptide as disclosed herein. The armored cells (e.g., armored T cells) can comprise an exogenous sequence. The exogenous sequence can include a sequence encoding a therapeutic protein. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell surface-associated, or secreted proteins. Exemplary therapeutic proteins expressed by armored cells (e.g., armored T cells) may modify the activity of the armored cells or may modify the activity of a second cell. The armored cells (e.g., armored T cells) may comprise a selection gene or selection marker. The armored cells (e.g., armored T cells) may comprise a synthetic gene expression cassette (also called an inducible transgene construct).

[0196] 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 receptors for inhibitory checkpoint signals to generate armored cells (e.g., armored CAR T cells). 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 receptors for inhibitory checkpoint signals results in a loss of protein expression of the inhibitory checkpoint receptor on the surface or in the cytoplasm of the armored cells. Thus, armored cells in which the expression of one or more genes encoding inhibitory checkpoint receptors has been silenced or reduced are resistant, non-receptive, or insensitive to checkpoint signals. The resistance or reduced sensitivity of armored cells to inhibitory checkpoint signals enhances the therapeutic potential of the armored cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in PCT Publication No. WO2019 / 173636. Suitable examples of inhibitory checkpoint signals that can be silenced include, but are not limited to, PD-1 and TGFβRII.

[0197] 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 intracellular proteins involved in checkpoint signaling to produce armored cells (e.g., armored CAR T cells). The activity of the modified cells can be enhanced by targeting any intracellular signaling protein involved in a checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference with one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in PCT Publication No. WO2019 / 173636.

[0198] 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 transcription factors that interfere with the effectiveness of the treatment to produce armored cells (e.g., armored CAR T cells). The activity of the modified cells can be enhanced or modulated by silencing or reducing the expression (or inhibiting the function) of transcription factors that interfere with the effectiveness of the treatment. Non-limiting examples of transcription factors that can be modified to silence or reduce their expression or inhibit their function include, but are not limited to, the exemplary transcription factors disclosed in PCT Publication No. WO2019 / 173636.

[0199] 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 cell death or cell apoptosis receptors to produce armored cells (e.g., armored CAR T cells). The interaction of a death receptor with its endogenous ligand results in the initiation of apoptosis. Disruption of the expression, activity, or interaction of a cell death and / or cell apoptosis receptor and / or ligand reduces the sensitivity of the modified cells to death signals, thereby making the armored cells more effective in the tumor environment. Non-limiting examples of cell death and / or cell apoptosis receptors and ligands are disclosed in PCT Publication No. WO2019 / 173636. A suitable example of a cell death receptor that can be modified is Fas (CD95).

[0200] 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 sensing proteins to produce armored cells (e.g., armored CAR T cells). Disruption of metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) by the modified cells results in prolonged retention of T cell function, resulting in more tumor cell deaths per cell. Non-limiting examples of metabolic sensing genes and proteins are disclosed in PCT Publication No. WO2019 / 173636. Suitable examples, HIF1a and VHL, are involved in T cell function in hypoxic environments. Armored T cells may have suppressed or reduced expression of one or more genes encoding HIF1a or VHL.

[0201] 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 proteins that confer sensitivity to cancer treatments, including monoclonal antibodies, to produce armored cells (e.g., armored CAR T cells). The armored cells are thus capable of functioning and may exhibit superior function or efficacy in the presence of a cancer treatment (e.g., chemotherapy, monoclonal antibody therapy, or another anti-tumor treatment). Non-limiting examples of proteins involved in conferring sensitivity to cancer treatments are disclosed in PCT Publication No. WO2019 / 173636.

[0202] 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 growth advantage factors to produce armored cells (e.g., armored CAR T cells). Silencing or reducing the expression of an oncogene can confer a growth advantage to the cells. For example, silencing or reducing (e.g., disrupting expression of) the TET2 gene during the CAR T cell manufacturing process results in the generation of armored CAR T cells with a significant ability to expand and subsequently eradicate tumors when compared to non-armored CAR T cells that lack expansion capacity. This strategy can be combined with a safety switch (e.g., the iC9 safety switch described herein), which allows for targeted destruction of armored CAR T cells upon adverse reactions from the subject or uncontrollable growth of the armored CAR T. Non-limiting examples of growth advantage factors are disclosed in PCT Publication No. WO2019 / 173636.

[0203] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to express modified / chimeric checkpoint receptors to produce the armored T cells of the present disclosure.

[0204] Modified / chimeric checkpoint receptors can include null receptors, decoy receptors, or dominant-negative receptors. The null receptors, decoy receptors, or dominant-negative receptors can be modified / chimeric receptors / proteins. The null receptors, decoy receptors, or dominant-negative receptors can be truncated to express the intracellular signaling domain. Alternatively or additionally, the null receptors, decoy receptors, or dominant-negative receptors can be mutated at one or more amino acid positions within the intracellular signaling domain that are critical or necessary for effective signal transduction. Truncation or mutation of the null receptors, decoy receptors, or dominant-negative receptors can result in the loss of the receptor's ability to send or transmit a checkpoint signal to or within a cell.

[0205] For example, dilution or blocking of immunosuppressive checkpoint signals from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing a modified / chimeric PD-1 null receptor on the surface of armored cells (e.g., armored CAR T cells), which effectively competes with the endogenous (unmodified) PD-1 receptor also expressed on the surface of the armored cells, reducing or inhibiting the transmission of immunosuppressive checkpoint signals through the armored cells' endogenous PD-1 receptor. In this non-limiting example, competition between the two different receptors for binding to PD-L1 expressed on tumor cells reduces or diminishes the level of effective checkpoint signaling, thus enhancing the therapeutic potential of armored cells expressing PD-1 null receptors.

[0206] Modified / chimeric checkpoint receptors can include null, decoy, or dominant-negative receptors that are transmembrane receptors, membrane-bound or membrane-linked receptors / proteins, or intracellular receptors / proteins. Exemplary null, decoy, or dominant-negative intracellular receptors / proteins include, but are not limited to, downstream signaling components 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 treatments, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication No. WO2019 / 173636.

[0207] Modified / chimeric checkpoint receptors can include switch receptors. Exemplary switch receptors include modified / chimeric receptors / proteins in which a native or wild-type intracellular signaling domain is switched or replaced with a different intracellular signaling domain that is not native to the protein and / or is not the wild-type domain. For example, replacing an inhibitory signaling domain with a stimulatory signaling domain switches an immunoinhibitory signal to an immunostimulatory signal. Alternatively, replacing an inhibitory signaling domain with another inhibitory domain can reduce or enhance the level of inhibitory signaling. Expression or overexpression of a switch receptor can result in dilution and / or blocking of a cognate checkpoint signal through competition with endogenous wild-type checkpoint receptors (not the switch receptor) for binding to the cognate checkpoint receptor expressed in the immunosuppressive tumor microenvironment. Armor cells (e.g., armored CAR T cells) can include a sequence encoding a switch receptor, resulting in the expression of one or more switch receptors, thereby altering the activity of the armored cell. The armored cells (e.g., armored 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 therapeutics, oncogenes, and / or tumor suppressor proteins or genes.

[0208] Exemplary switch receptors can include or be derived from proteins, including, but not limited to, downstream signaling components 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 therapy, and oncogenes or tumor suppressor genes.

[0209] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to express a CLR / CAR that mediates conditional gene expression to generate armored T cells. The combination of the CLR / CAR and conditional gene expression system in the nucleus of the armored T cell constitutes a synthetic gene expression system that is conditionally activated upon binding of the cognate ligand to the CLR or the cognate antigen to the CAR. This system may be useful for "armoring" or enhancing the therapeutic potential of modified T cells, for example, by reducing or limiting synthetic gene expression at the site of ligand or antigen binding or within the tumor environment.

[0210] Gene editing compositions and methods

[0211] The modified cells are generated by introducing a transgene into the cells. The introducing step can include delivery of the nucleic acid sequence, transgene, and / or genome editing construct via a non-transposition delivery system.

[0212] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include one or more of local delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, sonic delivery, vibration delivery, magnetofection, or nanoparticle-mediated delivery. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include liposome transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ by mechanical transfection can include cell squeezing, cell bombardment, or gene gun techniques. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ by nanoparticle-mediated transfection can include liposome delivery, micelle delivery, and polymerosome delivery.

[0213] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include non-viral vectors. Non-viral vectors can include nucleic acids. Non-viral vectors can include plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircular DNA, single-stranded oligodeoxynucleotides (ssODN), dDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). Non-viral vectors can include transposons as described herein.

[0214] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include viral vectors. The viral vector can be a non-integrating, non-chromosomal vector. Non-limiting examples of non-integrating, non-chromosomal vectors can include adeno-associated virus (AAV), adenovirus, and herpesvirus. The viral vector can be an integrating, chromosomal vector. Non-limiting examples of integrating, chromosomal vectors can include adeno-associated vector (AAV), lentivirus, and gammaretrovirus.

[0215] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include a combination of vectors. Non-limiting examples of vector combinations can include a viral vector and a non-viral vector, multiple non-viral vectors, or multiple viral vectors. Non-limiting examples of vector combinations can include a combination of a DNA-based vector and an RNA-based vector, 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.

[0216] Genome modifications can be made by introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably integrate nucleic acid sequences, transiently integrate nucleic acid sequences, generate site-specific integration of nucleic acid sequences, or generate biased integration of nucleic acid sequences. The nucleic acid sequence can be a transgene.

[0217] Genome modification can involve introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably integrate nucleic acid sequences. Stable chromosomal integration can be random, site-specific, or biased. Site-specific integration can be unassisted or assisted. Assisted site-specific integration is co-delivered with a site-specific nuclease. The site-specific nuclease includes a transgene with 5' and 3' nucleotide sequence extensions that contain percent homology to regions upstream and downstream of the genomic integration site. A transgene with homologous nucleotide extensions allows for genome integration via homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration can occur at a safe harbor site. A genomic safe harbor site can accommodate the integration of new genetic material to ensure that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective expression levels) and does not cause deleterious changes to the host genome that pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intron sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), the natural 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.

[0218] Site-specific transgene integration can occur at a site that disrupts expression of the target gene. Disruption of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. Non-limiting examples of target genes that can be targeted for site-specific integration include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allogeneic rejection.

[0219] Site-specific transgene integration can occur at sites that result in enhanced expression of the target gene. Enhancement of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0220] Enzymes can be used to create strand breaks in the host genome to facilitate transgene delivery or integration. The enzymes can create single-strand or double-strand breaks. Non-limiting examples of cleavage-inducing enzymes include transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™, and CPF1. Cleavage-inducing enzymes can be delivered to cells encoded by DNA, encoded by mRNA, as proteins, or as nucleoprotein complexes with guide RNAs (gRNAs).

[0221] Site-specific transgene integration can be controlled by vector-mediated integration site bias, which can be controlled by the selected lentiviral or gammaretroviral vector.

[0222] The site-specific transgene integration site can be an unstable chromosomal insertion. The integrated transgene can be silenced, removed, excised, or further modified. The genome modification can be an unstable integration of the transgene. The unstable integration can be a transient non-chromosomal integration, a semi-stable non-chromosomal integration, a semi-persistent non-chromosomal insertion, or an unstable chromosomal insertion. The transient non-chromosomal insertion can be epichromosomal or cytoplasmic. In one embodiment, the transient non-chromosomal insertion of the transgene is not integrated into a chromosome, and the modified genetic material is not replicated during cell division.

[0223] The genome modification can be semi-stable or persistent 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 nuclei of dividing cells.

[0224] The genome modification can be an unstable chromosomal integration of the transgene. The integrated transgene can be silenced, removed, excised, or further modified.

[0225] Modification of the genome by inserting a transgene can occur via host cell-directed double-strand break repair (homology-directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinase enzyme-mediated modification. Modification of the genome by inserting a transgene can occur via CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER™, and cpfl.

[0226] In gene editing systems involving the insertion of new or existing nucleotides / nucleic acids, an insertion tool (e.g., a DNA template vector, a transposable element (transposon or retrotransposon)) must be delivered to the cell in addition to a cutting enzyme (e.g., a nuclease, recombinase, integrase, or transposase). Examples of such insertion tools for recombinases include DNA vectors. Other gene editing systems require the delivery of an integrase together with an insertion vector, a transposon together with a transposon / retrotransposon, etc. 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 insertion tools include viral-based enzymes from any of a number of viruses, such as AAV, gammaretroviruses, and lentiviruses. Examples of transposons / retrotransposons that can be used in insertion tools are described in further detail herein.

[0227] Cells with ex vivo, in vivo, in vitro, or in situ genomic modifications can be germline or somatic cells. The modified cells can be human, non-human, mammalian, rat, mouse, or canine 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 while the cells are in a quiescent, activated, resting, interphase, prophase, metaphase, anaphase, or telophase state. The modified cells can be fresh, cryopreserved, bulk, or sorted into subpopulations from whole blood, leukapheresis, or from immortalized cell lines. Detailed descriptions for isolating cells from leukapheresis products or blood are disclosed in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0228] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition may include a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding the nuclease protein or the sequence encoding the nuclease domain may include a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may include one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0229] The nuclease or nuclease domain thereof can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0230] The present disclosure provides compositions comprising a small Cas9 (Cas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 constructs of the present disclosure can include an effector comprising a type IIS endonuclease. A Staphylococcus aureus Cas9 having an active catalytic site comprises the amino acid sequence of SEQ ID NO:79.

[0231] The present disclosure provides compositions comprising an inactivated small Cas9 (dSaCas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, 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. The dSaCas9 comprises the amino acid sequence of SEQ ID NO: 80, which includes D10A and N580A mutations to inactivate the catalytic site.

[0232] The present disclosure provides compositions comprising an inactivated Cas9 (dCas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially 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.

[0233] dCas9 can be isolated or derived from Streptococcus pyogenes. dCas9 can include dCas9 with substitutions at amino acid positions 10 and 840, which inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A. dCas9 can include the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:82.

[0234] An exemplary Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:83.

[0235] 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.

[0236] 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.

[0237] The cell containing 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 in the genomic DNA sequence. The target sequence in the genomic DNA sequence can be a target sequence in a safe harbor site of the genomic DNA sequence.

[0238] 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.

[0239] Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles. Poly(histidine) (e.g., poly(L-histidine)) is a pH-sensitive polymer due to the imidazole ring that provides a lone electron pair on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties due to protonation-deprotonation. In particular, at a specific pH, poly(histidine)-containing triblock copolymers assemble into micelles with positively charged poly(histidine) units on their surfaces, thus enabling complexation with negatively charged gene editing molecules. Using these nanoparticles to bind and release proteins and / or nucleic acids in a pH-dependent manner can provide an efficient and selective mechanism for achieving desired genetic modifications. In particular, this micelle-based delivery system offers substantial flexibility in terms of charged materials, as well as large payload capacity and targeted release of nanoparticle payloads. In one example, site-specific cleavage of double-stranded DNA is enabled by the delivery of nucleases using poly(histidine)-based micelles. Without wishing to be bound by any particular theory, it is believed that in micelles formed by various triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic and poly(histidine) blocks at the ends to form one or more surrounding layers.

[0240] In one embodiment, the present disclosure provides a triblock copolymer consisting of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block can be poly(ethylene oxide) (PEO), and the charged block can be poly(L-histidine). An example of a triblock copolymer that can be used is PEO-b-PLA-b-PHIS, where the number of repeating units in each block varies by design.

[0241] Diblock copolymers, which can be used as intermediates for creating triblock copolymers, can have hydrophilic, biocompatible poly(ethylene oxide) (PEO) (chemically synonymous with PEG) conjugated to a variety of hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(orthoesters), poly(peptides), poly(phosphazenes), and poly(saccharides) [including, but not limited to, poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC)]. Polymeric micelles composed of 100% PEGylated surfaces have improved in vitro chemical stability, increased in vivo bioavailability, and prolonged blood circulation half-lives.

[0242] Polymeric vesicles, polymersomes, and poly(histidine)-based micelles, including those containing triblock copolymers, and methods for making the same are described in more detail in U.S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Patent Application Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO2019 / 126589.

[0243] Transposon and vector composition

[0244] The present disclosure provides compositions and methods for delivering an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv) to a cell or population of cells. Non-limiting examples of compositions for delivering the compositions of the present disclosure to a cell or population of cells include a transposon or a vector. Thus, the present disclosure provides a transposon comprising an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv), or a vector comprising an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv).

[0245] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise a sequence encoding an inducible apoptosis-promoting polypeptide. Alternatively, or in addition, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding an inducible apoptosis-promoting polypeptide of the present disclosure. Inducible apoptosis-promoting polypeptides are described in more detail herein.

[0246] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise a sequence encoding a chimeric stimulating receptor (CSR). Alternatively or additionally, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding a CSR of the present disclosure. Chimeric stimulating receptors are described in more detail herein.

[0247] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise a sequence encoding a recombinant HLA-E polypeptide. Alternatively or additionally, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding a recombinant HLA-E polypeptide. Recombinant HLA-E polypeptides are described in more detail herein.

[0248] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise a selection gene. The selection gene can encode a gene product essential for cell viability and survival. The selection gene can encode a gene product essential for cell viability and survival when stimulated by selective cell culture conditions. The selective cell culture conditions can include compounds that are deleterious to cell viability or survival, where the gene product confers resistance to such compounds. Non-limiting examples of selection genes include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0249] In a preferred embodiment, the selection gene encodes a DHFR mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 88. The DHFR mutein enzyme is encoded by a polynucleotide comprising, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 88. The amino acid sequence of the DHFR mutein enzyme can further comprise one or more mutations at positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme can comprise one or more of a phenylalanine (F) or leucine (L) substitution at position 80, a leucine (L) or valine (V) substitution at position 113, and a valine (V) or aspartic acid (D) substitution at position 153.

[0250] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise at least one self-cleaving peptide. For example, the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the inducible apoptosis-promoting polypeptide. Alternatively, the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the protein encoded by the selection gene.

[0251] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can 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. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of the inducible apoptosis-promoting polypeptide, and the second self-cleaving peptide is located downstream or immediately downstream of the inducible apoptosis-promoting polypeptide. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the inducible apoptosis-promoting 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. Alternatively, the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the protein encoded by the selection gene.

[0252] Non-limiting examples of self-cleaving peptides include a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 90. A GSG-T2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 91. The GSG-T2A polypeptide is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 92. The E2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 93. The GSG-E2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 94. The F2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 95. The GSG-F2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 96.The P2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 97. The GSG-P2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 98.

[0253] Transposition System

[0254] 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., an scFv) or a CAR (e.g., comprising an scFv) as disclosed herein. In a preferred embodiment, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding an scFv or a CAR (e.g., comprising an scFv) as disclosed herein, flanked by two cis-regulatory insulator elements. The present disclosure also provides a composition comprising the transposon. In a preferred embodiment, the composition comprising the 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.

[0255] The transposon of the present disclosure can be a PiggyBac™ (PB) transposon. In some embodiments, 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.

[0256] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643, and PCT Publication No. WO2010 / 099296.

[0257] The PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of the transposon and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) within the chromosomal site or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of PB or PBL transposon are 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'TGAA-3', 5' The PB or PBL transposon system may comprise or consist of the following sequences: 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system does not limit the payload of the gene of interest that can be included between the ITRs.

[0258] 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 preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:99.

[0259] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 99. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 99, wherein the amino acid substitution at position 30 can be a substitution of isoleucine (I) with valine (V), the amino acid substitution at position 165 can be a substitution of glycine (G) with serine (S), the amino acid substitution at position 282 can be a substitution of methionine (M) with valine (V), and the amino acid substitution at position 538 can be a substitution of asparagine (N) with lysine (K). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 100.

[0260] In certain embodiments, where the transposase comprises an above-described mutation at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases can 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 SEQ ID NO:99 or SEQ ID NO:100, and which are described in further detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0261] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or the silkworm Bombyx mori (GenBank Accession No. BAD11135).

[0262] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from the silkworm, Bombyx mori, or Xenopus tropicalis. Examples of hyperactive 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 WO2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0263] In some embodiments, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise the 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 WO2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0264] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to nuclear localization signals 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 WO2019 / 173636.

[0265] The transposon of the present disclosure can be a "Sleeping Beauty" transposon. In some embodiments, when the transposon is a "Sleeping Beauty" transposon, the transposase is a "Sleeping Beauty" transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180) or a hyperactive "Sleeping Beauty" (SB100X) transposase. In preferred embodiments, the "Sleeping Beauty" transposase comprises or consists of an amino acid sequence 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 embodiment, the hyperactive "Sleeping Beauty" (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 102.

[0266] The transposon of the present disclosure may be a Hellaiser transposon. Exemplary Hellaiser transposons include Helibat1, which comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 103. In some embodiments, when the transposon is a Hellaiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO2020 / 173636). In preferred embodiments, the Helitron transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 104.

[0267] The transposon of the present disclosure may be a Tol2 transposon. Exemplary Tol2 transposons, including an inverted repeat sequence, a subterminal sequence, and a Tol2 transposase, comprise or consist of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 105. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO2019 / 173636). In preferred embodiments, the Tol2 transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage) identical to SEQ ID NO: 106.

[0268] The transposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in WO2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. In preferred embodiments, the TcBuster transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 107. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence. In a preferred embodiment, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:108.

[0269] In some embodiments, the mutant TcBuster transposase comprises one or more sequence changes when compared to the wild-type TcBuster transposase, as described in detail in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0270] The transposon can be a nanotransposon. The nanotransposon comprises, consists essentially of, or consists of: (a) a transposon insert-encoding sequence, including a first inverted terminal repeat (ITR)-encoding sequence, a second inverted terminal repeat (ITR)-encoding sequence, and an intra-ITR sequence; (b) a backbone-encoding sequence, the backbone-encoding sequence including a sequence encoding an origin of replication having 1 to 450 nucleotides (including the endpoints) and a sequence encoding a selectable marker having 1 to 200 nucleotides (including the endpoints); and (c) inter-ITR sequences. In some embodiments, the inter-ITR sequences of (c) comprise the sequence of (b). In some embodiments, the inter-ITR sequences of (a) comprise the sequence of (b).

[0271] The sequence encoding the backbone can comprise 1 to 600 nucleotides (including the endpoints). In some embodiments, 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, or 550 to 600 nucleotides (each including the endpoints).

[0272] The inter-ITR sequence can comprise 1 to 1,000 nucleotides (including the endpoints). In some embodiments, 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 1,000 nucleotides (each range includes the endpoints).

[0273] The nanotransposon may be a short nanotransposon (SNT), where the inter-ITR sequence comprises 1-200 nucleotides (including the endpoints). The inter-ITR sequence can consist of 1-10 nucleotides, 10-20 nucleotides, 20-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides (each range including the endpoints).

[0274] A selectable marker having 1 to 200 nucleotides (including the endpoint) can comprise a sequence encoding a sucrose selectable marker. The sequence encoding the sucrose selectable marker can comprise a sequence encoding an RNA-OUT sequence. The sequence encoding the RNA-OUT sequence can comprise or consist of 137 base pairs (bp). A selectable marker having 1 to 200 nucleotides (including the endpoint) can comprise a sequence encoding a fluorescent marker. A selectable marker having 1 to 200 nucleotides (including the endpoint) can comprise a sequence encoding a cell surface marker.

[0275] The sequence encoding an origin of replication having 1 to 450 nucleotides (including the endpoints) can comprise a sequence encoding a mini-origin of replication. In some embodiments, the sequence encoding an origin of replication having 1 to 450 nucleotides (including the endpoints) comprises a sequence encoding an R6K origin of replication. The R6K origin of replication can comprise an R6K gamma origin of replication. The R6K origin of replication can comprise an R6K mini-origin of replication. The R6K origin of replication can comprise an R6K gamma mini-origin of replication. The R6K gamma mini-origin of replication can comprise or consist of 281 base pairs (bp).

[0276] In some embodiments of the nanotransposon, the backbone-encoding sequence does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, neither the nanotransposon nor the backbone-encoding sequence comprises the product of a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, neither the nanotransposon nor the backbone-encoding sequence is derived from a recombination site, an excision site, a ligation site, or a combination thereof.

[0277] In some embodiments of the nanotransposon, the recombination sites comprise sequences that result from a recombination event. In some embodiments, the recombination sites comprise sequences that are the product of a recombination event. In some embodiments, the recombination event comprises recombinase activity (e.g., a recombinase site).

[0278] In some embodiments of the nanotransposon, the backbone-encoding sequence does not further comprise a sequence encoding foreign DNA.

[0279] In some embodiments of the nanotransposon, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, the inter-ITR sequence does not comprise the product of a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence does not result from a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence comprises a sequence encoding foreign DNA. In some embodiments, the inter-ITR sequence 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 embodiments, the inter-ITR sequence 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 embodiments, the inter-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator. In some embodiments, the inter-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator.

[0280] Nanotransposons are described in further detail in PCT / US2019 / 067758.

[0281] Vector System

[0282] The vectors of the present disclosure may be viral vectors or recombinant vectors. Viral vectors may include sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may include sequences isolated or derived from adeno-associated viruses (AAV). Viral vectors may include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include two or more inverted terminal repeat (ITR) sequences located in cis next to the scFv or CAR encoding sequence of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids comprising a genome of one 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.

[0283] The vector of the present disclosure can be a nanoparticle. Non-limiting examples of nanoparticle vectors include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold), or any combination thereof. Nanoparticle vectors can be passively or actively transported across cell membranes.

[0284] The cell delivery compositions (e.g., transposons, vectors) disclosed herein can include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO2019 / 173636 and PCT / US2019 / 049816.

[0285] Inducible Pro-Apoptotic Polypeptides

[0286] The inducible apoptosis-promoting polypeptides disclosed herein are superior to existing inducible apoptosis-promoting polypeptides because they are much less immunogenic. The inducible apoptosis-promoting polypeptides are recombinant polypeptides and therefore do not occur in nature. Furthermore, they are recombined to produce inducible apoptosis-promoting polypeptides that are free of non-human sequences that can be recognized as "non-self" by the host's human immune system, thereby inducing an immune response in a subject receiving the inducible apoptosis-promoting polypeptide, cells comprising the inducible apoptosis-promoting polypeptide, or compositions comprising the inducible apoptosis-promoting polypeptide, or cells comprising the inducible apoptosis-promoting polypeptide.

[0287] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand-binding region can be a multimeric ligand-binding region. In certain embodiments, 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 naturally occurring. When the caspase is caspase 9 or a truncated caspase 9, the inducible pro-apoptotic polypeptide can also be referred to as an "iC9 safety switch."

[0288] The inducible caspase polypeptide can comprise (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences. In certain embodiments, 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 non-human sequences.

[0289] The ligand-binding region can 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 can comprise a modification at position 36 of the sequence. The modification can be a substitution of phenylalanine (F) with valine (V) at position 36 (F36V). The FKBP12 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 109. The FKBP12 polypeptide can comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 110. It can be encoded by a polynucleotide.

[0290] The linker region can comprise, consist essentially of, or consist of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the amino acid sequence of SEQ ID NO: 111, or the linker region can be encoded by a polynucleotide that comprises or consists of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the nucleic acid sequence of SEQ ID NO: 112. In some embodiments, the nucleic acid sequence encoding the linker does not contain any restriction sites.

[0291] A truncated caspase-9 polypeptide can comprise an amino acid sequence that does not include an arginine (R) at position 87 of the sequence. Alternatively, or in addition, a truncated caspase-9 polypeptide can comprise an amino acid sequence that does not include an alanine (A) at position 282 of the sequence. A truncated caspase-9 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 113, or can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 1144.

[0292] In certain embodiments, wherein the polypeptide comprises a truncated caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:115, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:116.

[0293] The inducible pro-apoptotic polypeptide may be expressed in a cell under the transcriptional control of any promoter known in the art that is capable of initiating and / or regulating expression of the inducible pro-apoptotic polypeptide in that cell.

[0294] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, by chemically induced dimerization (CID) mediated by an inducer to generate a conditionally regulated protein or polypeptide. Not only are the pro-apoptotic polypeptides inducible, but the induction of these polypeptides is also reversible upon degradation of the labile dimerizer or administration of a monomeric competitive inhibitor.

[0295] In certain embodiments, when the ligand binding region comprises an FKBP12 polypeptide having a substitution of phenylalanine (F) with valine (V) at position 36 (F36V), the inducer is the synthetic drug AP1903 (CAS index name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1(R * ), 2R * [S * [S * [1(R * ), 2R * ]]]]]-(9Cl) CAS Registry Number: 195514-63-7; molecular formula: C78H98N4O20; molecular weight: 1411.65); 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.

[0296] Inducible pro-apoptotic peptides and methods for inducing these peptides are described in detail in U.S. Patent Publication No. WO2019 / 0225667 and PCT Publication No. WO2018 / 068022.

[0297] Formulations, Dosages, and Methods of Administration

[0298] The present disclosure provides formulations, dosages, and methods of administration of the compositions described herein.

[0299] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary substance, including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, and auxiliary substances. Pharmaceutically acceptable auxiliary substances are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, including, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers can be routinely selected to suit the administration method, solubility, and / or stability of the protein scaffold, fragment, or variant composition, as well known in the art or as described herein.

[0300] 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, e.g., alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which can be present alone or in combination and comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / protein components that can also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0301] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0302] The composition may also contain a buffer or a pH adjuster. Typically, the buffer is a salt prepared from an organic acid or an organic base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts such as citrate.

[0303] Additionally, the disclosed compositions can include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweetening agents, 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).

[0304] A therapeutically effective amount of the compositions disclosed herein or the disclosed pharmaceutical compositions can be administered using many known and developed modes. Non-limiting examples of modes of administration include bolus, buccal, injection, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavity, intraperitoneal, 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.

[0305] The compositions of the present disclosure may be administered parenterally (subcutaneously, intramuscularly, or intravenously) or any other way, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams or suppositories; for buccal or sublingual administration, such as, but not limited to, tablets or capsules; or intranasally, such as, but not limited to, powders, nasal drops, or aerosols, or in the form of specific medications; or transdermally, such as, but not limited to, gels, lotions, suspensions, or with chemical enhancers such as dimethyl sulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, DS, Eds., pp. 59-90, (Marcel Dekker, Inc. New York 1994)), or with oxidizing agents to allow application of protein or peptide containing formulations to the skin (WO 98 / 53847), or with the application of electric fields to create temporary transport pathways, e.g., electroporation, or with iontophoresis to enhance the mobility of charged drugs across the skin, or with the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) for use in patch delivery systems (the above publications and patents are incorporated herein by reference in their entireties).

[0306] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granule, powder, or lyophilized powder, either in combination with a pharmaceutically acceptable parenteral vehicle or provided separately. Parenteral preparations may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalene, 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. Injectable preparations may be in the form of non-toxic, non-oral diluents, such as aqueous solutions, sterile injection solutions, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, and isotonic saline. Sterile, fixed oils can be used as common solvents or suspending agents. For these purposes, any type of fixed oil and fatty acid, such as natural, synthetic, or semisynthetic fatty oils or fatty acids, natural, synthetic, or semisynthetic monoglycerides, diglycerides, or triglycerides, can be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas-pressure needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446.

[0307] Formulations for oral administration rely on the co-administration of auxiliary substances (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 enzymatic inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. Formulations for delivering hydrophilic drugs, including proteins and protein scaffolds, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, transvaginal, or rectal administration are described in U.S. Pat. No. 6,309,663. The active ingredient compound in a solid dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms can also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, α-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, fragrances, etc.

[0308] Tablets and pills can be further processed into enteric-coated formulations. Liquid formulations for oral administration include emulsions, syrups, elixirs, suspensions, and medically acceptable solution formulations. These preparations can contain inert diluents commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Additionally, carrier compounds used for orally delivering biologically active agents are known in the art, as described in U.S. Pat. Nos. 5,879,681 and 5,871,753.

[0309] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, atomizers, and the like. All such devices can employ formulations suitable for administration for dispensing the compositions or pharmaceutical compositions described herein in aerosols. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers (MDIs), a propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles preferably in the 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. WO2019 / 049816.

[0310] For absorption through mucosal surfaces, the composition comprises an emulsion containing a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase that promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure may include the 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 excipients such as polyalkylene glycols, petrolatum, and cocoa butter. Formulations for intranasal administration are solid and may contain excipients such as lactose, or may be aqueous or oily solutions for nasal sprays. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO2019 / 049816.

[0311] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as a liposome or polymer nanoparticle, a microparticle, a microcapsule, or a microsphere (collectively referred to as a microparticle unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids, e.g., polylactic acid, polyglycolic acid, and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers 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, formulations, and suitable devices is disclosed in PCT Publication No. WO2019 / 049816.

[0312] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, e.g., over a period of one week to one year from a single administration. A variety of sustained-release, depot, or implant dosage forms can be utilized. For example, dosage forms can include pharmaceutically acceptable, non-toxic salts of compounds that are poorly soluble in body fluids, such as (a) acid addition salts with polybasic acids, e.g., phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, and the like; (b) salts with polyvalent metal cations, e.g., zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like, or salts with organic cations formed, e.g., from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), e.g., zinc tannate salts. Furthermore, the disclosed compounds, or preferably relatively insoluble salts such as those described above, can be formulated in gels, such as aluminum monostearate gels containing sesame oil suitable for injection. Particularly preferred salts include zinc salts, zinc tannate salts, and pamoate salts. Another type of sustained-release depot formulation for injection would include the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as a polylactic acid / polyglycolic acid polymer, as described, for example, 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 cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0313] Appropriate dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, 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 Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. A preferred dose can optionally include about 0.1-99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or can include a serum concentration of about 0.1-5000 μg / ml per single or multiple dose, or any range, value, or fraction thereof. A preferred dose range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg / kg up to about 3, about 6, or about 12 mg / kg of subject body weight.

[0314] Alternatively, the administered dose may vary depending on known factors, such as the pharmacodynamic properties of the particular drug and its mode and route of administration; the age, health, and weight of the recipient; the nature and severity of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Typically, the dose of the active ingredient is about 0.1 to 100 mg per kilogram of body weight. Typically, a single dose of 0.1 to 50 mg, preferably 0.1 to 10 mg, per kilogram of body weight per administration or in sustained-release form is effective to achieve the desired results.

[0315] As non-limiting examples, human or animal treatment can be provided as a single or periodic dose of a composition or pharmaceutical composition disclosed herein of about 0.1-100 mg / kg per day, or any range, value, or fraction thereof, for at least one day for 1-40 days, or alternatively or additionally for at least one week for 1-52 weeks, or alternatively or additionally for at least one year for 1-20 years, or any combination thereof, in a single infusion or multiple doses.

[0316] 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 usually present in an amount of from about 0.5 to 99.999% by weight based on the total weight of the composition.

[0317] An effective amount can include an amount of from about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple, or continuous dose to achieve a serum concentration of 0.01 to 5000 μg / ml in single, multiple, or continuous administrations, as performed and measured using known methods, such as those described herein or known in the relevant art.

[0318] In embodiments in which the composition administered to a subject in need thereof is modified cells as disclosed herein, about 1 x 10 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells, approximately 1 x 10 5 ~1×10 10 cells, approximately 1 x 10 6 ~1×10 9 cells, approximately 1 x 10 6 ~1×10 8 cells, approximately 1 x 10 6 ~1×10 7 cells, or approximately 1 x 10 6 ~25×10 6 In one embodiment, cells may be administered in an amount of about 5×10 6 ~25×10 6 Cells are administered.

[0319] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO2019 / 049816.

[0320] Methods of Using the Compositions of the Present Disclosure

[0321] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one embodiment, the subject is a mammal. Preferably, the subject is human. The terms "subject" and "patient" are used interchangeably.

[0322] The present disclosure provides methods for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB. These include ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myelocytic 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 carcinoma, malignant histiocytosis, paraneoplastic syndrome / malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis carcinoma, 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, and cancer-related bone pain.

[0323] In a preferred embodiment, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in one embodiment, the present disclosure provides modified cells expressing at least one disclosed antibody (e.g., scFv) and / or a CAR comprising the antibody (e.g., scFv), selected and / or expanded for administration to a subject in need thereof. The modified cells can be prepared for storage at any temperature, including room temperature and body temperature. The modified cells can be prepared for cryopreservation and subsequent thawing. The modified cells can be prepared in a pharmaceutically acceptable carrier for administration to a subject directly from the sterile packaging. The modified cells can be prepared in a pharmaceutically acceptable carrier with an indicator of cell viability and / or CAR expression level to ensure cell function and a minimum level of CAR expression. The modified cells can be prepared in a pharmaceutically acceptable carrier at a predetermined density with one or more reagents to inhibit further expansion and / or prevent cell death.

[0324] Any of the methods can include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally further include co-administration or combination therapy for treating such a disease or disorder, including administering any of the compositions or pharmaceutical compositions disclosed herein before, concurrently with, and / or after at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical).

[0325] In some embodiments, the subject does not develop graft-versus-host disease (GvH) and / or host-versus-graft disease (HvG) after administration. In one embodiment, 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 infusion. In one embodiment, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0326] In some embodiments, a therapeutically effective dose is a single dose. In some embodiments, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number in between, doses produced simultaneously. In some embodiments where the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or last for a sufficient time to treat the disease or disorder.

[0327] In one example, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an antibody (e.g., an scFv) or a CAR comprising an antibody (e.g., an scFv), wherein the antibody or CAR specifically binds to an antigen on a tumor cell. In embodiments where the composition comprises modified cells or cell populations, the cells or cell populations can be autologous or allogeneic.

[0328] In some embodiments of the therapeutic methods described herein, treatment can be modified or terminated. Specifically, in embodiments in which the composition used for treatment comprises an inducible apoptosis-promoting polypeptide, apoptosis can be selectively induced in cells by contacting the cells with an inducer. Treatment can be modified or terminated, for example, in response to signs of recovery or signs of a decrease in disease severity / progression, signs of disease remission / halt, and / or the occurrence of adverse events. In some embodiments, the method includes administering an inhibitor of the inducer to inhibit modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., if signs or symptoms of disease recur, or if increased severity and / or adverse events are resolved).

[0329] Antibody / scFv production, screening, and purification

[0330] 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) may 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. For example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

[0331] Amino acids from the scFv can be altered, added, and / or deleted, as known in the art, to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility, or other suitable characteristics.

[0332] Optionally, scFvs can be engineered to retain high affinity for the antigen and other favorable biological properties. To achieve this goal, scaffold proteins can optionally be prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate sequences and can measure their potential immunogenicity (e.g., the Immunofilter program from Xencor, Inc. of Monrovia). Inspection of these displays allows for analysis of the possible role of residues in the function of the candidate sequence, i.e., analysis of residues that affect the ability of the candidate scFv to bind to its antigen. In this way, residues from the parental and reference sequences can be selected and combined to achieve desired properties, such as affinity for the target antigen. Alternatively, or in addition to the above procedures, other suitable engineering methods can be used.

[0333] Screening scFvs for specific binding to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. This method involves screening large collections of peptides for individual members with the desired function or structure. The displayed nucleotide or peptide sequences can be 3 to 5,000 or more nucleotides or amino acids in length, frequently 5 to 100 amino acids in length, and often about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves displaying peptide sequences on the surface of bacteriophage or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in PCT Patent Publication Nos. WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0334] Other systems for generating libraries of peptides include 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 suppliers such 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, and 5,856,456 assigned to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500 assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717 assigned to Affymax; and Cambridge Antibody See U.S. Patent No. 5,885,793 assigned to Genentech; U.S. Patent No. 5,750,373 assigned to Genentech; U.S. Patent Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, and 5,698,417 assigned to Xoma; Colligan, supra; Ausubel, supra; and Sambrook, supra.

[0335] The scFvs of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one scFv of the present disclosure optionally binds to a target protein with high affinity, e.g., at least about 10 as measured by surface plasmon resonance or Kinexa methods, as practiced by those skilled in the art. -7 KD less than or equal to M, for example, but not limited to, 0.1 to 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.

[0336] The affinity or avidity of an scFv for an antigen can be determined experimentally using any suitable method (e.g., 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 measured affinity of a particular scFv-antigen interaction may vary if measured under different conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, K on, K off) are preferably performed using standardized solutions of the protein scaffold and antigen, and standardized buffers, such as those described herein.

[0337] Competitive assays can be performed using the scFvs of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the scFvs of the present disclosure for binding to a target protein and / or share epitope regions. These assays, readily known to those skilled in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and / or antibody are immobilized or insolubilized before or after competition, and the target protein-bound sample is isolated from the unbound sample by, for example, decantation (where the protein / antibody was pre-insolubilized) or centrifugation (where the protein / antibody was precipitated after the competitive reaction). Competitive binding can also be determined by whether binding or lack of binding of the scFv to the target protein alters function, e.g., whether the scFv molecule inhibits or enhances, for example, the enzymatic activity of a label. ELISAs and other functional assays can be used, as are well known in the art.

[0338] nucleic acid molecule

[0339] The nucleic acid molecule of the present disclosure encoding an scFv 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 synthetically by cloning, or any combination thereof. The DNA can be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand.

[0340] Isolated nucleic acid molecules of the present disclosure can include nucleic acid molecules containing an open reading frame (ORF), optionally containing one or more introns, such as, but not limited to, at least one specific portion of at least one scFv; nucleic acid molecules containing coding sequences for protein scaffolds or loop regions that bind to a target protein; and nucleic acid molecules containing nucleotide sequences that are substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode protein scaffolds described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one of ordinary skill in the art to generate such degenerate nucleic acid variants encoding specific scFvs of the present disclosure. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are encompassed by the present disclosure.

[0341] As provided herein, nucleic acid molecules of the present disclosure, including nucleic acids encoding scFvs, include, but are not limited to, those that themselves encode the amino acid sequence of an scFv fragment; coding sequences for an entire protein scaffold or a portion thereof; coding sequences for an scFv, fragment, or portion, as well as additional sequences, such as coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, for example, at least one intron along with additional non-coding sequences, including, but not limited to, non-coding 5' and 3' sequences, such as transcribed, untranslated sequences that play a role in mRNA processing (e.g., ribosome binding and mRNA stability), including transcription, splicing, and polyadenylation signals; and additional coding sequences that encode additional amino acids that provide additional functional groups. Thus, the protein scaffold-encoding sequence 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.

[0342] Polynucleotides that selectively hybridize to the polynucleotides described herein

[0343] The present disclosure provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. That is, the polynucleotides can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present disclosure can be used to identify, isolate, or amplify partial-length or full-length clones in a deposited library. The polynucleotides can be genomic or cDNA sequences isolated from a human or mammalian nucleic acid library, or complementary to cDNAs from such libraries.

[0344] Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, more preferably at least 95% full-length sequences. The cDNA library can be normalized to improve the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, used with sequences that have reduced sequence identity compared to the complementary sequence. Moderate and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0345] Optionally, the polynucleotide encodes at least a portion of a protein scaffold encoded by a polynucleotide described herein. The polynucleotide comprises a nucleic acid sequence that can be used for selective hybridization to 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.

[0346] Nucleic acid construction

[0347] The isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0348] A nucleic acid can conveniently contain nucleotide sequences in addition to the polynucleotide of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the present disclosure. A nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of the polynucleotide of the present disclosure.

[0349] 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 the polynucleotide, or to improve the introduction of the polynucleotide 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).

[0350] Recombinant methods for constructing nucleic acids

[0351] 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 of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. RNA isolation and the construction of cDNA and genomic libraries are well known to those of skill in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

[0352] Nucleic Acid Screening and Isolation Methods

[0353] Probes based on the sequences of the polynucleotides disclosed herein 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 understand that various degrees of hybridization stringency can be used in assays; either the hybridization or the wash medium can be stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and target must be for duplex formation to occur. The degree of stringency can be controlled by temperature, ionic strength, pH, and the presence of a partially denaturing solvent, such as formamide. For example, hybridization stringency can be conveniently varied by manipulating the concentration of formamide, e.g., within the range of 0% to 50%, and by changing the polarity of the reaction solution. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or wash medium. The degree of complementarity will optimally be 100%, or between 70-100%, or any range or value therebetween, however, it will be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or washing medium.

[0354] Methods for amplifying 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 provided herein.

[0355] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 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.; 5,091,310 to Innis; 5,066 to Gyllensten et al.). Nos. 4,889,818 to Gelfand et al.; 4,994,370 to Silver et al.; 4,766,067 to Biswas; and 4,656,134 to Ringold), and RNA-mediated amplification (U.S. Pat. No. 5,130,238 to Maled et al., under the trade name NASBA), which uses antisense RNA against a target sequence as a template for double-stranded DNA synthesis, the entire contents of these references are incorporated herein by reference (see, e.g., Ausubel, supra; or Sambrook, supra).

[0356] For example, polymerase chain reaction (PCR) techniques can be used to amplify the sequences of the 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 expressed proteins, to generate nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a technician in in vitro amplification methods can be found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, the yield of long PCR products can be improved using, for example, T4 gene 32 protein (Boehringer Mannheim).

[0357] Synthetic methods for constructing nucleic acids

[0358] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, 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 using a DNA polymerase with the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA may be limited to sequences of about 100 bases or more, but longer sequences can be obtained by linking shorter sequences.

[0359] Recombinant Expression Cassette

[0360] The present disclosure further provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. A nucleic acid sequence of the present disclosure, e.g., a cDNA or genomic sequence encoding a protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette, which can then be introduced into at least one desired host cell. The recombinant expression cassette will typically comprise a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the host cell of interest. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present disclosure.

[0361] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a non-heterologous form of a polynucleotide of the disclosure at an appropriate location (upstream, downstream, or within an intron) to up-regulate or down-regulate expression of the polynucleotide of the disclosure. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0362] Expression vectors and host cells

[0363] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each incorporated herein by reference in its entirety).

[0364] The polynucleotide can optionally be linked to a vector containing a selectable marker for propagation in a host.Generally, the plasmid vector is introduced into the precipitate, such as calcium phosphate precipitate, or in a complex with charged lipid.If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line, and then introduced into host cells.

[0365] The DNA insert must be operably linked to a suitable promoter. Expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiation codon at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0366] Expression vectors preferably, but optionally, include at least one selectable marker, such as, but 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 methotrexate resistance), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), eukaryotic cell culture resistance genes, as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring methotrexate resistance), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), and eukaryotic cell culture resistance genes. Examples of suitable host cells include genes for resistance to erythromycin (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline for culturing in E. coli and other bacterial or prokaryotic cells (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of the vector construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described, for example, in Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0367] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for isolation of cells modified by the disclosed compositions and methods. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or a subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Examples of such cell surface markers include, but are not limited to, "cluster of designation" or "classification determinant" proteins (often abbreviated as "CD"), such as CD19, CD271, CD34, CD22, CD20, CD33, CD52 truncated or full-length forms, 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).

[0368] The expression vector will preferably, but optionally, include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the present disclosure. Selectable drug resistance markers of the present disclosure can include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0369] 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 persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in standard laboratory manuals, e.g., Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17, and 18.

[0370] Those skilled in the art are familiar with the numerous expression systems available for expressing nucleic acids encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by turning on (by engineering) endogenous DNA encoding the protein scaffolds of the present disclosure in the host cell. Such methods are well known in the art and are described, for example, 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 entireties.

[0371] Examples of cell cultures useful for the production of protein scaffolds, specific portions or variants thereof are bacterial, yeast, and mammalian cells known in the art. Mammalian cell lines are often in the form of monolayers of cells, although mammalian cell suspensions and bioreactors can also be used. Many suitable host cell lines capable of expressing natively glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). 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 embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0372] Expression vectors for these cells can include one or more of the following expression control sequences, including, 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 site, such as a ribosome binding site, an RNA splice site, a polyadenylation site (e.g., an SV40 large T Ag polyA addition site), and a transcription terminator sequence. See, e.g., Ausubel, et al., supra; Sambrook, et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and available, for example, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org), or from other known or commercial sources.

[0373] When eukaryotic host cells are used, a polyadenylation or transcription terminator sequence is usually incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. A sequence for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Furthermore, as is known in the art, gene sequences for regulating replication in host cells can be incorporated into the vector.

[0374] scFv purification

[0375] scFvs can be recovered and purified from recombinant cell cultures 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, and 10 (each of which is incorporated herein by reference in its entirety).

[0376] The scFvs of the present disclosure include purified products, products of chemical synthetic procedures, and products produced by recombinant technology from prokaryotic or eukaryotic hosts, including, for example, E. coli, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production method, the protein scaffolds of the present disclosure can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, e.g., 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 entireties.

[0377] Amino acid code

[0378] The amino acids comprising the protein scaffolds of the present disclosure are often abbreviated. Amino acids are designated by their single-letter code, their three-letter code, their name, or their three-nucleotide codon, as is well known in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). Protein scaffolds of the present disclosure can include one or more amino acid substitutions, deletions, or additions, either natural or due to mutation and / or human manipulation, as specified herein. Amino acids in the protein scaffolds of the present disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, particularly, but not limited to, at least one neutralizing activity. Sites important for binding of the protein scaffold may 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)).

[0379] As will be appreciated by those skilled in the art, the present disclosure includes at least one biologically active protein scaffold of the present disclosure. A biologically active protein scaffold has a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or more of the specific activity of a native (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those skilled in the art.

[0380] In another aspect, the present disclosure relates to protein scaffolds and fragments described herein that are modified by the covalent attachment of an organic moiety. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties (e.g., extended in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain aspects, the hydrophilic polymer group can have a molecular weight of about 800 to about 120,000 daltons and can be a polyalkane glycol (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 contain about 8 to about 40 carbon atoms.

[0381] The modified protein scaffolds and fragments of the present disclosure can include one or more organic moieties covalently attached directly or indirectly to an antibody. Each organic moiety attached to a protein scaffold or fragment of the present disclosure can 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 and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Thus, protein scaffolds modified by the covalent attachment of polylysine are included in the present disclosure. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure can be linear or branched, and can include, for example, polyalkane 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 modifying 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 one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared using appropriate methods. For example, a polymer containing an amine group can be attached to the carboxylate of a fatty acid or fatty acid ester, or an activated carboxylate on a fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be attached to a hydroxyl group on the polymer.

[0382] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the present disclosure can be saturated or can contain one or more unsaturated units. Fatty acids suitable 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, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight-chain or branched lower alkyl group, which can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0383] Modified protein scaffolds and fragments can be prepared using suitable methods, such as reaction with one or more modifying agents. As used herein, the term "modifying agent" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be attached to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or via linker moieties, such as divalent C1-C12 groups (where one or more carbon atoms can be replaced by heteroatoms such as oxygen, nitrogen, or sulfur). Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. A modifying agent containing a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or the resulting product can be cyclized by reaction with maleic anhydride to generate an activated maleimide derivative of the fatty acid (see, e.g., Thompson, et al., WO 92 / 16221, the entire teachings of which are incorporated herein by reference).

[0384] The modified protein scaffolds of the present disclosure can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be attached to the protein scaffold in a non-site-specific manner using amine-reactive modifiers, such as NHS esters of PEG. Modified protein scaffolds and fragments comprising organic moieties attached to specific sites on the protein scaffolds of the present disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)).

[0385] definition

[0386] As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes a plurality of such methods, reference to a "dosage" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0387] The terms "about" or "approximately" refer to an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When particular values ​​are described in patent applications and claims, the term "about" meaning within an acceptable range of error for the particular value should be inferred unless otherwise specified.

[0388] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein-encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, an isolated polynucleotide can comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure or a biologically active portion thereof is produced by recombinant methods, an optimal culture medium exhibits less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or undesired proteins.

[0389] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. The term "fragment," as used throughout this disclosure, refers to a portion of a DNA sequence or an amino acid sequence, and thus a portion of the protein encoded thereby. Fragments of DNA sequences, including coding sequences, retain the biological activity of the native protein and may therefore encode protein fragments that retain DNA recognition or binding activity to target DNA sequences as described herein. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or promoter activity. Thus, fragments of DNA sequences can range from at least about 20, 50, or 100 nucleotides, up to the full-length polynucleotides of the present disclosure.

[0390] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach, which involves pre-assembling monomeric and / or repeating units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the present disclosure can include repeating monomers of the present disclosure and can be constructed by a modular approach, which involves pre-assembling repeating units in a target vector, which can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by this method, as well as nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells comprising nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0391] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope of the present specification to use natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to as "analogs") of the antibodies defined herein. Thus, in one embodiment herein, the term "antibody of the present specification" in its broadest sense also encompasses such analogs. Generally, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to the antibodies defined herein.

[0392] "Antibody fragment," and all grammatical variations thereof, as used herein, is defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, where the portion does not contain the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of a single uninterrupted sequence of contiguous amino acid residues (referred to as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) a single-chain Fv (scFv) molecule; (2) a single-chain polypeptide containing only a light-chain variable domain or a fragment thereof containing the three CDRs of a light-chain variable domain without the associated heavy-chain portion; and (3) a single-chain polypeptide containing only a heavy-chain variable domain or a fragment thereof containing the three CDRs of a heavy-chain variable domain without the associated light-chain portion; and multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chains can include any constant domain sequence found in the non-Fc region of native antibodies (e.g., the CHI of an IgG isotype), and / or can include a hinge region sequence found in native antibodies, and / or can include a leucine zipper sequence fused to or positioned within the hinge region sequence or constant domain sequence of the heavy chain. The term also includes single domain antibodies ("sdAB"), which generally refer to antibody fragments having a single monomeric variable antibody domain (e.g., from camelids). Such fragment antibody types will be readily understood by those skilled in the art.

[0393] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0394] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements of any essential importance to the combination, when used for its intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. "Consisting of" means excluding all but trace elements of other ingredients or substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0395] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope can include three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more usually, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

[0396] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0397] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, editing, and proteins modified by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, glycosylation, and the like.

[0398] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0399] The term "operatively linked" or its equivalents (e.g., "operably linked") means that two or more molecules are positioned relative to one another so that they can interact to affect the function attributable to one or both molecules or a combination thereof.

[0400] Non-covalently linked components and methods of making and using non-covalently linked components are disclosed. The various components can take a variety of different forms as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for transient interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only or primarily under circumstances where such association is required for the desired activity. The association can be of sufficient duration to allow for the desired effect.

[0401] Disclosed is a method for targeting a protein to a specific locus in the genome of an organism, which can include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule can be operably associated via a non-covalent linkage.

[0402] The term "scFv" refers to a single-chain variable fragment. An scFv is a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL), connected by a linker peptide. The linker peptide can be about 5 to 40 amino acids, or about 10 to 30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable fragments lack the Fc region found in intact antibody molecules and therefore lack consensus binding sites (e.g., protein G) used in antibody purification. The term also includes scFvs, which are intrabodies, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0403] The term "single-domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single-domain antibodies are peptide chains, typically about 110 amino acids long, containing one variable domain (VH) of a heavy-chain antibody or common IgG, which generally have similar affinity for antigen as the whole antibody but are more heat-resistant and stable to detergents and high concentrations of urea. Examples include those derived from camel or fish antibodies. Alternatively, single-domain antibodies can be generated from common mouse or human IgG, which has four chains.

[0404] As used herein, the terms "specifically bind" and "specific binding" refer to the ability of an antibody, antibody fragment, or nanobody to preferentially bind to a particular antigen present in a homogeneous mixture of different antigens. In some embodiments, a specific binding interaction discriminates between desired and undesired antigens in a sample. In some embodiments, by about 10-fold to 100-fold or more (e.g., about 1000-fold or 10,000-fold or more). "Specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigenic target over a different antigenic target, and does not necessarily imply high affinity.

[0405] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0406] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of a described single strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0407] The probes of the present disclosure can comprise single-stranded nucleic acids that can hybridize to a target sequence under stringent hybridization conditions. Thus, the nucleic acids of the present disclosure can refer to probes that hybridize under stringent hybridization conditions.

[0408] The nucleic acids of the present disclosure can be single-stranded or double-stranded. The nucleic acids of the present disclosure can contain double-stranded sequences even when the majority of the molecule is single-stranded. The nucleic acids of the present disclosure can contain single-stranded sequences even when the majority of the molecule is double-stranded. The nucleic acids of the present disclosure can include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure can include combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure can include combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure can be synthesized to include unnatural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis or recombinant methods.

[0409] The nucleic acids of the present disclosure may be non-naturally occurring, either in their entirety or any portion thereof. The nucleic acids of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, rendering the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, resulting in a sequence that is non-naturally occurring, rendering the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic nucleotides, rendering the entire nucleic acid sequence non-naturally occurring.

[0410] Given the redundancy of the genetic code, multiple nucleotide sequences may encode a particular protein, and all such nucleotide sequences are contemplated herein.

[0411] The term "operably linked" as used throughout this disclosure refers to the expression of a gene under the control of a promoter to which it is spatially linked. A promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0412] The term "promoter," as used throughout this disclosure, refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located up to several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an EF-1 alpha promoter, a CAG promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0413] The term "substantially complementary," as used throughout this disclosure, refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0414] The term "substantially identical," as used throughout this disclosure, refers to whether a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or, with respect to nucleic acids, whether the first sequence is substantially complementary to the complement of the second sequence.

[0415] As used throughout this disclosure, the term "variant" when used to describe a nucleic acid refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0416] The term "vector" as used throughout this disclosure refers to a nucleic acid sequence that includes an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain a combination of amino acids and DNA sequences, RNA sequences, or both DNA and RNA sequences.

[0417] As used throughout this disclosure, the term "variant" when used to describe a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. Variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0418] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted while maintaining protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 (incorporated herein by reference in its entirety).

[0419] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, compatible amino acid substitutions in biological function depend on the relative similarity of amino acids, particularly the side chains of those amino acids, as evidenced by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0420] As used herein, "conservative" amino acid substitutions can be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modification of a polynucleotide encoding a polypeptide of the present disclosure. Amino acids can be classified according to their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are shown in Table A.

[0421] [Table 1]

[0422] Alternatively, conservative amino acids can be grouped as described in Table B by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0423] [Table 2]

[0424] Alternatively, exemplary conservative substitutions are shown in Table C.

[0425] [Table 3]

[0426] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure can include one or more conservative substitutions.

[0427] As used throughout this disclosure, the term "two or more" of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the listed amino acid substitutions. The term "two or more" can refer to 2, 3, 4, or 5 of the listed amino acid substitutions.

[0428] The polypeptides and proteins of the present disclosure may not occur in nature in their entirety or in any part thereof. The polypeptides and proteins of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, which cause the entire amino acid sequence to not occur in nature. The polypeptides and proteins of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, which result in a sequence that does not occur in nature, which causes the entire amino acid sequence to not occur in nature. The polypeptides and proteins of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic amino acids, which cause the entire amino acid sequence to not occur in nature.

[0429] As used throughout this disclosure, "sequence identity" can be determined by using a standalone executable BLAST engine program to blast two sequences (bl2seq), which can be retrieved using default parameters from the National Center for Biotechnology Information (NCBI) ftp site (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). The term "identical" or "identity," as used in the context of two or more nucleic acid or polypeptide sequences, refers to a certain percentage of residues that are identical over a specific region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specific region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specific region, and multiplying the result by 100 to obtain the percent sequence identity. If the two sequences are of different lengths, or if the alignment produces one or more offset ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using computer alignment algorithms such as BLAST and BLAST 2.0.

[0430] The term "endogenous" as used throughout this disclosure refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0431] The term "exogenous" as used throughout this disclosure refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, and includes non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., a DNA sequence, or a naturally occurring nucleic acid sequence present in a non-naturally occurring genomic location.

[0432] The present disclosure provides methods for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing," it is intended to mean presenting the polynucleotide construct to the cell so that the construct has access to the interior of the host cell. The methods of the present disclosure do not depend on a particular method for introducing the polynucleotide construct into the host cell, but only on the polynucleotide construct gaining access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation, transient transformation, and virus-mediated methods.

[0433] Example

[0434] Example 1 - Construction of Chimeric Stimulatory Receptors (CSRs)

[0435] Stimulation is enhanced by expression of chimeric stimulating receptors (CSRs) in the presence or absence of TCRs. Enhanced primary and secondary costimulatory signals are delivered when T cells are treated with reagents displaying agonist mAbs in the presence of transiently or stably expressed surface-expressed CSR / s. In one embodiment, this schematic represents allogeneic cells. More complete T cell activation is achieved by stimulatory signals via CSRs, thereby promoting T cell activation and expansion.

[0436] A chimeric stimulating receptor (CSR) was designed to contain an antigen recognition region that encompasses the CD2 extracellular domain. A panel of CSR mutants was engineered within the extracellular domain of CD2. The goal of this panel was to identify mutants that no longer bind to CD58 but retain receptivity to binding by anti-CD2 activator reagents. This may be desirable for two main reasons: 1) CD58 expression by activated T cells may interact with wild-type (WT) CSR, preventing optimal performance of CSR, and 2) WT CSR may function as a natural ligand, CAR, such that T cells expressing CSR may mediate cytotoxic activity against CD58-expressing cells, including activated T cells. Therefore, mutant CSRs that cannot interact with CD58 but retain the ability to bind activating anti-CD2 reagents for optimal cell expansion are desirable.

[0437] The D111H mutation in the CD2 extracellular domain ("CD2ECD(D111H)") retains the ability to bind activating CD2 reagents for optimal cell expansion and does not interact with CD58. Schematic diagrams of the CSRs of the present disclosure are shown in Figures 1 and 2. Figure 1 shows a schematic diagram of the CSR with the CD2 signal peptide. Figure 2 shows a schematic diagram of the CSR with the CD8 signal peptide. These CSRs can be used to enhance the 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 encoded in mRNA or stably encoded in transposons.

[0438] Example 2 - Functional characterization of chimeric stimulatory receptors

[0439] To test the effect of CSR on CAR-T cell expansion, PanT cells isolated from normal donor blood were genetically modified using the PiggyBac® DNA modification system combined with the Cas-CLOVER™ gene editing system. Cells were electroporated in a single reaction with a transposon encoding at least the CAR and a selection gene, mRNA encoding the CSR, mRNA encoding the super PiggyBac® transposase enzyme, mRNA encoding the Cas-CLOVER™ gene, and multi-guide RNAs (gRNAs) targeting TCRb and b2M to knock out the TCR and MHCI (double knockout; DKO). Cells were then stimulated with agonist mAbs anti-CD2, anti-CD3, and anti-CD28, followed by selection for gene modification over a 14-day culture period. At the end of the initial culture period, all T cells expressed the CAR, indicating successful selection of gene-modified cells. Larger LLCs were observed in DKO cells expressing CSR compared to cells that did not express CSR (no booster) (Figure 3). Thus, expression of CSR enhances the expansion of CAR-T cells during production.

[0440] We examined the effect of CSR expression on the memory phenotype of DKO CAR-T cells. DKO CAR-T cells generated with or without CSR (no booster) were stained for surface CD45RA, CD45RO, and CD62L expression to define Tscm, Tcm, Tem, and Teff cells: Tscm (CD45RA+CD45RO-CD62L+), Tcm (CD45RA-CD45RO+CD62L+), Tem (CD45RA-CD45RO+CD62L-), and Teff (CD45RA+CD45RO-CD62L-). The ratios of Teff, Tscm, Tcm, and Tem for DKO CAR-T cells with and without CSR are shown in Tables 1 and 2. Regardless of whether CSR was present, DKO CAR-T cells were primarily composed of very high levels of favorable Tscm and Tcm cells. Therefore, the memory phenotype of DKO CAR-T cells was not significantly affected by CSR coexpression.

[0441] [Table 4]

[0442] [Table 5]

[0443] Example 3 - In vivo efficacy of CAR-T cells expressing chimeric stimulating receptors (CSR)

[0444] To test the antitumor effects of allogeneic CAR-T cells expressing the different CSRs described in Example 2, an in vivo experiment was conducted using a mouse xenograft model of multiple myeloma. The experimental procedure is outlined in Figure 4, and 10 different allogeneic CAR-T cells were generated using 10 different CSRs. Specifically, RPMI-8226 cell lines were cultured at 1 × 10 7 Female NSG mice were subcutaneously injected with 100 μg of cells (day -7) and then incubated for 1 h at 4°C ... 3 [Target average approximately 100mm 3 ]), and then administered allogeneic CAR-T cells at a "stress" dose (5 × 10 6 ) by intravenous (IV) injection. The "stress" dose was used to obtain a higher resolution in detecting possible functional differences in efficacy between CAR-T cells generated with different CSRs. Treatment with PBS was used as a negative control. Allogeneic CAR-T cells expressing CD2.DHz were used as a positive control.

[0445] The results of this experiment are shown in Figures 5-9. Treatment of animals with CAR-T cells expressing various CSRs of the present disclosure resulted in a reduction in tumor volume compared to the PBS control (Figure 5). Tumor size 56 days after treatment with allogeneic CAR-T cells expressing CSR was comparable to that of the positive control. Total T cells in the blood were quantified after treatment with allogeneic CAR-T cells expressing CSR (Figure 6). Peak levels of T cells in the blood were also quantified after treatment with CAR-T cells expressing CSR and displayed as the group average across all animals (Figure 7). All animals showed an increase in peak levels of T cells compared to the PBS negative control. CAR-T cells expressing CD2.DH28z, CD2.DH.BBz, CD2.DH.15z, CD2.DH.Oxz, and CD2.DH.Gz showed an increase in peak levels of T cells in the blood compared to the CD2.DH.CD2z (also known as CD2.DH.z) control. The T cell area under the curve (T cell AUC) was calculated after treatment with each allogeneic CAR-T cell (Figure 8 and Table 3). All animals showed an increase in T cell AUC compared to the PBS negative control. CAR-T cells expressing CD2.DH28z, CD2.DH.BBz, CD2.DH.15z, CD2.DH.Oxz, and CD2.DH.Gz showed increased peak levels of blood T cells compared to the CD2.DH.CD2z (also known as CD2.DH.z) control. The effects on the proportions of Teff, Tem, Tcm, and Tscm cells after treatment with each allogeneic CAR-T cell expressing various CSRs were comparable to treatment with the positive control (CAR T cells expressing CD2.DH.z CSR).

[0446] [Table 6] Some aspects of the invention are described below. 1. (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises the CD2 signal peptide or the CD8α signal peptide, and the activation component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and 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 the signaling domain comprises a CD3ζ protein or a portion thereof; A non-natural chimeric stimulating receptor (CSR) comprising: The receptor, wherein the signal peptide and the cytoplasmic domain are not derived from the same protein. 2. The CSR of item 1, wherein the signal peptide comprises a CD2 signal peptide. 3. The CSR of item 1, wherein the CD2 signal peptide comprises the amino acid sequence of SEQ ID NO: 5. 4. The CSR of item 1, wherein the signal peptide comprises a CD8α signal peptide. 5. The CSR of item 1, wherein the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO: 7. 6. A CSR according to any of items 1 to 5, wherein the activating component comprises a modification. 7. The CSR according to any of items 1 to 6, wherein the modification comprises a mutation or truncation of the amino acid sequence of the CD2 extracellular domain or part thereof to which the agonist binds, compared to the wild-type sequence of the CD2 extracellular domain or part thereof. 8. The CSR according to item 7, wherein the CSR containing a mutation or truncation of the CD2 extracellular domain or part thereof to which the agonist binds does not bind to CD58. 9. The CSR of item 7, wherein the CD2 extracellular domain or part thereof containing the mutation or truncation comprises the amino acid sequence of SEQ ID NO: 3. 10. The CSR according to any one of items 1 to 9, wherein the CD2 transmembrane domain or a part thereof comprises the amino acid sequence of SEQ ID NO: 9. 11. The CSR according to any one of items 1 to 10, wherein the CD2 intracellular domain comprises the amino acid sequence of SEQ ID NO: 13. 12. The CSR according to any one of items 1 to 10, wherein the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 15. 13. The CSR according to any one of items 1 to 10, wherein the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. 14. The CSR according to any one of items 1 to 10, wherein the IL17RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. 15. The CSR according to any one of items 1 to 10, wherein the IL15RA intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. 16. The CSR according to any one of items 1 to 10, wherein the IL21R intracellular domain comprises the amino acid sequence of SEQ ID NO: 23. 17. The CSR according to any one of items 1 to 10, wherein the ICOS intracellular domain comprises the amino acid sequence of SEQ ID NO: 25. 18. The CSR according to any of items 1 to 10, wherein the CD27 intracellular domain comprises the amino acid sequence of SEQ ID NO: 27. 19. The CSR according to any of items 1 to 10, wherein the OX40 intracellular domain comprises the amino acid sequence of SEQ ID NO: 29. 20. The CSR according to any one of items 1 to 10, wherein the GITR intracellular domain comprises the amino acid sequence of SEQ ID NO: 31. 21. The CSR according to any one of items 1 to 20, wherein the signaling domain comprising the CD3ζ protein or a part thereof comprises the amino acid sequence of SEQ ID NO: 11. 22. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 39. 23. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 43. 24. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 47. 25. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 51. 26. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 55. 27. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 59. 28. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 63. 29. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 67. 30. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 71. 31. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 37. 32. The CSR according to any one of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 41. 33. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 45. 34. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 49. 35. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 53. 36. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 57. 37. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 61. 38. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 65. 39. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 69. 40. The CSR according to any of items 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO: 73. 41. A nucleic acid sequence encoding the CSR according to any one of items 1 to 40.

Claims

1. A non-natural chimeric stimulating receptor (CSR) comprising a CD2 extracellular domain having a D111H mutation, wherein the amino acid sequence of the CSR consists of any one of the amino acid sequences of SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69 or SEQ ID NO:

73.

2. A nucleic acid encoding the CSR of claim 1.

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