Allogeneic cell composition and method of use

Abstract

Chimeric stimulating receptors (CSRs), cell compositions containing CSRs, methods for preparing them, and methods for using them for the treatment of a disease or disorder in a subject are disclosed. [Solution] (a) An external domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD2 signal peptide or a CD8α signal peptide, and the activating component comprises the CD2 extracellular domain or a part thereof to which the agonist binds; (b) A transmembrane domain including the CD2 transmembrane domain or a portion thereof; and (c) An internal domain comprising a cytoplasmic domain and a signal transduction domain, wherein the cytoplasmic domain is the CD2 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, IL17RA intracellular domain, IL15RA intracellular domain, IL21R intracellular domain, ICOS intracellular domain, CD27 intracellular domain, OX40 intracellular domain, or GITR intracellular domain, or any combination thereof, and the signal transduction domain comprises the CD3ζ protein or a portion thereof, the internal domain and A non-natural chimeric stimulating receptor (CSR) that includes, The receptor wherein the signal peptide and the cytoplasmic domain do not originate from the same protein.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority and benefit of U.S. Provisional Patent Application No. 62 / 896,495, filed on September 5, 2019, and U.S. Provisional Patent Application No. 62 / 976,536, filed on February 14, 2020. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] Areas of disclosure This disclosure relates to molecular biology, more specifically to chimeric receptors, allogeneic cell compositions, methods for producing the same, and methods for using them.

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

[0004] Background of the Invention There has long been an unmet need in the art for allogeneic cell compositions that overcome the challenges presented by eliminating genes involved in graft-versus-host and host-versus-graft responses. This disclosure provides allogeneic cell compositions, methods for producing these compositions, and methods for using them, including structural modifications not found in nature, in order to restore the responsiveness of allogeneic cells to environmental stimuli and to reduce or prevent rejection due to natural killer cell-mediated cytotoxicity. [Overview of the Initiative]

[0005] This disclosure provides a non-native chimeric-stimulated receptor (CSR) comprising: (a) an external domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD2 signal peptide and the activating component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; and (c) an internal domain comprising a cytoplasmic domain and a signal transduction domain, wherein the cytoplasmic domain is the CD28 intracellular domain, the 4-1BB intracellular domain, the IL17RA intracellular domain, the IL15RA intracellular domain, the IL21R intracellular domain, the ICOS intracellular domain, the CD27 intracellular domain, the OX40 intracellular domain, or the GITR intracellular domain, or any combination thereof, and the signal transduction domain comprises the CD3ζ protein or a portion thereof, wherein the signal peptide and the cytoplasmic domain do not originate from the same protein.

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

[0007] The Disclosure also provides a non-native chimeric-stimulated receptor (CSR) comprising: (a) an external domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD8α signal peptide and the activating component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; and (c) an internal domain comprising a cytoplasmic domain and a signal transduction domain, wherein the cytoplasmic domain is the CD2 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, IL17RA intracellular domain, IL15RA intracellular domain, IL21R intracellular domain, ICOS intracellular domain, CD27 intracellular domain, OX40 intracellular domain, or GITR intracellular domain, or any combination thereof, and the signal transduction domain comprises the CD3ζ protein or a portion thereof, wherein the signal peptide and the cytoplasmic domain do not originate from the same protein.

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

[0009] This disclosure also provides non-natural chimeric-stimulated receptors (CSRs) whose activating components include modifications. In some embodiments, the modifications include mutations or truncations 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. In some embodiments, non-natural CSRs including mutations or truncations of the CD2 extracellular domain or part thereof to which the agonist binds do not bind to CD58. In some embodiments, the CD2 extracellular domain or part thereof including the mutation or truncation contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 3. In preferred embodiments, the CD2 extracellular domain or part thereof including the mutation or truncation contains the amino acid sequence of SEQ ID NO: 3.

[0010] In some embodiments, the CD2 transmembrane domain contains an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to that of SEQ ID NO: 9. In preferred embodiments, the CD2 transmembrane domain or a portion thereof contains the amino acid sequence of SEQ ID NO: 9.

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

[0012] In some embodiments, the signaling domain comprising the CD3ζ protein or a portion thereof contains an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 11. In a preferred embodiment, the signaling domain comprising the CD3ζ protein or a portion thereof contains the amino acid sequence of SEQ ID NO: 11.

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

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

[0015] This disclosure provides nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides vectors comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides transposons comprising nucleic acid sequences encoding any CSR disclosed herein.

[0016] This disclosure provides cells containing any CSR disclosed herein. This disclosure provides cells containing nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides cells containing vectors containing nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides cells containing transposons containing nucleic acid sequences encoding any CSR disclosed herein.

[0017] This disclosure also provides modified T lymphocytes (T cells) comprising: (a) modifications of an endogenous sequence encoding a T cell receptor (TCR) that reduce or eliminate the expression or activity level of the TCR; and (b) any chimeric stimulating receptor (CSR) as disclosed herein. The modified T cells disclosed herein may be allogeneic or autologous. In some preferred embodiments, the modified cells are allogeneic. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0018] This disclosure provides compositions comprising any CSR disclosed herein. This disclosure provides compositions comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising vectors comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising transposons comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising modified cells disclosed herein, or compositions comprising a plurality of modified cells disclosed herein.

[0019] This disclosure provides a modified T lymphocyte (T cell) comprising a chimeric stimulating receptor (CSR) including (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) which reduces or eliminates the expression or activity level of the TCR; and (b) an external domain comprising (i) an activating component, the external domain being isolated or induced from a first protein; (ii) a transmembrane domain; and (iii) an internal domain comprising at least one signaling domain, the internal domain being isolated or induced from a second protein, wherein the first protein and the second protein are not identical.

[0020] Modified T cells may further contain inducible pro-apoptotic polypeptides. Modified T cells may further contain modifications to the endogenous sequence encoding beta-2-microglobulin (B2M), where the modification reduces or eliminates the level of expression or activity of major histocompatibility complex (MHC) class I (MHC-I).

[0021] Modified T cells may further comprise a non-natural polypeptide containing an HLA class I histocompatibility antigen, an alpha-chain E (HLA-E) polypeptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise a B2M signal peptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise a linker, where the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise the peptide and the B2M polypeptide. The non-natural polypeptide containing HLA-E may further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding HLA-E.

[0022] Modified T cells may further include non-native antigen receptors, sequences encoding therapeutic polypeptides, or combinations thereof. Non-native antigen receptors may include chimeric antigen receptors (CARs).

[0023] CSR can be transiently expressed in modified T cells. CSR can be stably expressed in modified T cells. Polypeptides containing HLA-E polypeptides can be transiently expressed in modified T cells. Polypeptides containing HLA-E polypeptides can be stably expressed in modified T cells. Inducible pro-apoptotic polypeptides can be transiently expressed in modified T cells. Inducible pro-apoptotic polypeptides can be stably expressed in modified T cells. Sequences encoding non-natural antigen receptors or therapeutic proteins can be transiently expressed in modified T cells. Sequences encoding non-natural antigen receptors or therapeutic proteins can be stably expressed in modified T cells.

[0024] Modified T cells can be autologous cells. Modified T cells can be allogeneic cells. Modified T cells include early memory T cells, stem cell-like T cells, and stem memory T cells (T SCM ), central memory T cells (TCM ), or stem cell-like T cells.

[0025] This disclosure provides compositions comprising any modified T cells as disclosed herein. This disclosure also provides compositions comprising a population of modified T lymphocytes (T cells), wherein the multiple modified T cells of the population comprise the CSRs disclosed herein. This disclosure also provides compositions comprising a population of T lymphocytes (T cells), wherein the multiple T cells of the population comprise the modified T cells disclosed herein.

[0026] This disclosure provides a method for treating a disease or disorder, comprising administering to a subject in need any of the compositions disclosed herein, or a therapeutically effective amount of a composition for use in treating a disease or disorder. In one embodiment, the composition is a modified T cell or population of modified T cells as disclosed herein. This disclosure also includes a method for treating a disease or disorder, comprising administering to a subject in need any of the compositions disclosed herein, and a therapeutically effective amount of the composition of at least one non-natural molecule bound to CSRs.

[0027] This disclosure provides a method for producing a population of modified T cells, comprising, essentially comprising, introducing a composition comprising the CSR of this disclosure or a sequence encoding it into a plurality of primary human T cells under conditions that stably express CSR in a plurality of modified T cells and maintain the desired stem-like characteristics of the plurality of modified T cells. This disclosure provides a composition comprising the population of modified T cells produced by this method. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population comprising the CSR are stem memory T cells (T SCM ) or T SCMThe cells express one or more cell surface markers, where one or more cell surface markers include CD45RA and CD62L. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CM The composition expresses one or more cell surface markers of similar cells, wherein the one or more cell surface markers include CD45RO and CD62L. The composition can be used to treat a disease or disorder. The disclosure also provides the use of a composition produced by a method for treating a disease or disorder. The disclosure further provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of the composition produced by the method to a subject in need thereof. The therapeutic method may further include administering the activator composition to a subject to activate a population of modified T cells in vivo, to induce cell division of a population of modified T cells in vivo, or a combination thereof.

[0028] The present disclosure provides a method for producing a population of modified T cells, comprising introducing into a plurality of primary human T cells, a composition comprising the CSR of the present disclosure or a sequence encoding the same, under conditions that transiently express CSR in the plurality of modified T cells and maintain desirable stem-like properties of the plurality of modified T cells, or consisting essentially of or consisting of. The present disclosure provides a composition comprising a 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 CSR expresses one or more cell surface markers of stem memory T cells (T SCM ) or T SCM -like cells, wherein the one or more cell surface markers include CD45RA and CD62L. 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 is central memory T cells (T CM ) or T CMThe composition expresses one or more cell surface markers of similar cells, wherein the one or more cell surface markers include CD45RO and CD62L. The composition can be used to treat a disease or disorder. The disclosure also provides the use of a composition produced by a method for treating a disease or disorder. The disclosure further provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of the composition produced by the method to a subject in need thereof. In some embodiments, the modified T cells in a population of modified T cells administered to the subject no longer express CSR.

[0029] This disclosure provides a method for expanding a population of modified T cells, comprising introducing a composition comprising the CSR of this disclosure or a sequence encoding it into a plurality of primary human T cells under conditions in which the CSR is stably expressed in the plurality of modified T cells and the desired stem-like characteristics of the plurality of modified T cells, and then 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 twice as high as the expansion of a plurality of wild-type T cells that do not stably express CSR under the same conditions. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population containing CSR are stem memory T cells (T SCM ) or T SCMThe cells express one or more cell surface markers, where one or more cell surface markers include CD45RA and CD62L. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population are central memory T cells (T CM ) or T CM The cells express one or more cell surface markers, wherein the one or more cell surface markers include CD45RO and CD62L. This disclosure provides a composition comprising a population of modified T cells expanded by this method. This composition can be used to treat a disease or disorder. This disclosure also provides the use of the composition expanded by the method for the treatment of a disease or disorder. This disclosure further provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of the composition expanded by the method to a subject in need thereof. This therapeutic method may further include administering an activator composition to a subject to activate the population of modified T cells in vivo, to induce cell division of the population of modified T cells in vivo, or a combination thereof.

[0030] This disclosure provides a method for expanding a population of modified T cells, comprising introducing a composition containing the CSR of this disclosure or a sequence encoding it into a plurality of primary human T cells under conditions in which CSR is transiently expressed in a plurality of modified T cells and the desired stem-like characteristics of the plurality of modified T cells are maintained, and then 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 twice as high as the expansion of a plurality of wild-type T cells that do not transiently express CSR under the same conditions. This disclosure provides a composition containing the population of modified T cells expanded by the method described above. 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 containing CSRs are stem memory T cells (T SCM ) or T SCM The cells express one or more cell surface markers, where one or more cell surface markers include CD45RA and CD62L. 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 a type of cell, where the one or more cell surface markers include CD45RO and CD62L. This composition can be used to treat a disease or disorder. The disclosure also provides the use of the composition extended by a method for the treatment of a disease or disorder. The disclosure further provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of the composition extended by the method to a subject in need thereof. In some embodiments, the modified T cells in a population of modified T cells administered to the subject no longer express CSR.

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

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure belongs. In this specification, unless otherwise specified in the context, singular terms also include plural terms. For example, the terms “a,” “an,” and “the” are understood to be singular or plural, and the term “or” is understood to be inclusive. For example, “element” means one or more elements. Throughout this specification, the word “comprising,” or variations such as “comprises” or “comprising,” is understood to mean including the elements, integers, or processes, or groups of elements, integers, or processes described herein, but not to exclude other elements, integers, or processes, or groups of elements, integers, or processes. "Approximately" can be understood to mean 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 specified in the context, all figures provided herein are modified by the term "approximately."

[0033] Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein in their entirety by reference. References cited herein are not considered to be prior art of the claimed invention. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope. Other features and advantages of this disclosure will become apparent from the detailed description and claims below.

[0034] This patent or application file includes at least one color drawing. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 is a schematic diagram showing an example of a CSR CD2z-D111H mutant for enhancing the production of allogeneic or autologous CAR-T cells. The CSR CD2z-D111H mutant can be delivered to allogeneic or autologous CAR-T cells during production to enhance cell proliferation and expansion, quality, viability, phenotype, function, subset composition, and gene editing efficiency. These mutant CSRs can be delivered transiently by encoding them in mRNA, or stably by encoding them in transposons.

[0036] [Figure 2]Figure 2 is a schematic diagram showing an example of a CSR CD2z-D111H mutant possessing the CD8a signal peptide for enhancing the production of allogeneic or autologous CAR-T cells. The CSR CD2z-D111H mutant can be delivered to allogeneic or autologous CAR-T cells for production to enhance cell proliferation and expansion, quality, viability, phenotype, function, subset composition, and gene editing efficiency. These mutant CSRs can be delivered transiently by encoding them in mRNA, or stably by encoding them in transposons.

[0037] [Figure 3] Figure 3 is a graph showing that CSR delivery enhances the expansion of CAR T cells in 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 CAR and the selected gene, mRNA encoding CSR, mRNA encoding the super piggyBac® transposase enzyme, mRNA encoding Cas-CLOVER®, and multiple guide RNAs (gRNAs) targeting TCRb and b2M to knock out TCR and MHCI (double knockout; DKO). Cells were then stimulated with monoclonal antibodies anti-CD2, anti-CD3, and anti-CD28, which are agonists, and selected for genetic modification over a 14-day culture period. At the end of the first culture period, all T cells expressed CAR, indicating successful selection of genetically modified cells. A more pronounced expansion of DKO cells was observed in samples expressing CSR.

[0038] [Figure 4]Figure 4 is a schematic diagram showing the experimental protocol for evaluating in vivo tumor control by CAR T cells produced using different CSRs. Allogeneic CAR-T cells were produced using different additional immunoassays, as described in Figures 1 and 3 and Tables 1 and 2. Using a mouse xenograft model of multiple myeloma, the in vivo antitumor effects of allo-CAR T cells produced with 10 different additional immunoassays were evaluated. Specifically, the RPMI-8226 cell line was subcutaneously injected (SC) into female NSG mice at a dose of 1 × 10⁷ cells (-7 days), and then, on day 0 when the tumor was established (75–125 mm³ by caliper measurement [target mean, approximately 100 mm³]), allo-CAR T cells were treated with intravenous (IV) injection 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 produced with different additional immunoassays.

[0039] [Figure 5] Figure 5 is a graph showing tumor volume over time after alloCAR T cell treatment. In vivo tumor control was evaluated using “stress” doses of CAR T cells produced using different CSRs, following the protocol shown in Figure 4. Tumor volume assessments by caliper measurements for all animals are presented as SEM (standard error of mean) as group mean with error bars.

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

[0041] [Figure 7]Figure 7 is a graph showing peak T cell levels (T cell Cmax) in the blood. In vivo tumor control was evaluated using “stress” doses of CAR T cells produced using different CSRs, following the protocol shown in Figure 4. For all animals, peak T cell levels in the blood, measured by TruCount staining of human CD45+ (hCD45+) cells, are shown as SEM with group mean values ​​and error bars.

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

[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 evaluated using “stress” doses of CAR T cells produced using different CSRs, according to the protocol shown in Figure 4. For all animals, the phenotype of CD8+ T cells in the blood, measured by FACS staining, is shown as SEM as group mean with error bars at 14 and 35 days post-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), TEFF (CD45RA+CD45RO-CD62L-; purple).

[0044] All documents referenced herein, including patents or patent applications that are cross-referenced or related, are incorporated herein by reference in their entirety for all purposes unless expressly excluded or otherwise limited. Reference of a document does not constitute prior art relating to the disclosed or claimed invention, or teaching, suggesting, or disclosing the invention of this Specified, either alone or in combination with other references. Furthermore, in the event of any conflict between the meaning or definition of a term in that document and the meaning or definition of the same term in this Specified as incorporated by reference, the meaning or definition assigned to that term in this Specified shall prevail. [Modes for carrying out the invention]

[0045] Detailed description of the invention This disclosure provides allogeneic cell compositions, methods for preparing these compositions, and methods for using them, which include structural modifications not found in nature, for restoring the responsiveness of allogeneic cells to environmental stimuli and reducing or preventing rejection due to cytotoxicity mediated by natural killer cells.

[0046] Chimeric stimulating receptors (CSRs) and recombinant HLA-E polypeptides

[0047] Adoptive cell compositions that are "universally" safe for administration to any patient require a significant reduction or elimination of alloreactivity. For this purpose, the cells of the Disclosure (e.g., allogeneic cells) may be modified to interfere with the expression or function of classes of T cell receptors (TCRs) and / or major histocompatibility complexes (MHCs). TCRs mediate graft-versus-host (GvH) responses, while MHCs mediate host-versus-graft (HvG) responses. In a preferred embodiment, the expression and / or function of TCRs are eliminated to prevent T cell-mediated GvH that could cause death in the subject. Thus, in a preferred embodiment, the Disclosure provides a pure TCR-negative allogeneic T cell composition (e.g., each cell in the composition is expressed at a level so low that it is undetectable or absent).

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

[0049] The above strategy presents further challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), a component of the TCR complex. The loss of CD3ζ in TCR-KO T cells dramatically reduces their ability to optimally activate and expand using standard stimuli / activators, including, but not limited to, the agonist anti-CD3mAb. When the expression or function of any component of the TCR complex is disrupted, all components of the complex, including TCR-alpha (TCRα), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ), are lost. Both CD3ε and CD3ζ are required for T cell activation and expansion. The agonist anti-CD3mAb typically recognizes CD3ε and possibly another protein within the complex, which then signals CD3ζ. CD3ζ provides the primary stimulus (along with secondary costimulatory signals) for T cell activation for optimal activation and expansion. Under normal conditions, complete T cell activation depends on the involvement of the TCR, combined with a secondary signal mediated by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL) that enhance the immune response. However, in the absence of a TCR, stimulation with standard activation / stimulation reagents containing the agonist anti-CD3 mAb results in significantly reduced T cell expansion. In fact, stimulation with standard activation / stimulation reagents containing the agonist anti-CD3 mAb reduces T cell expansion to only 20-40% of normal levels.

[0050] Accordingly, this disclosure provides a non-native chimeric-stimulated receptor (CSR) comprising (a) an external domain containing a signal peptide and an activating component; (b) a transmembrane domain; and (c) an internal domain containing a cytoplasmic domain and a signal transduction domain, wherein the signal peptide and the cytoplasmic domain do not originate from the same protein.

[0051] The activating component may include one or more parts of components of T cell receptors (TCRs), TCR complexes, TCR coreceptors, TCR costimulatory proteins, TCR inhibitory proteins, cytokine receptors, and chemokine receptors to which the agonist of the activating component binds. The activating component may also include the extracellular domain of CD2 or a portion thereof to which the agonist binds.

[0052] The signaling domain may include one or more components of human signaling domains, T cell receptors (TCRs), TCR complexes, TCR coreceptors, TCR costimulatory proteins, TCR inhibitory proteins, cytokine receptors, and chemokine receptors. The signaling domain may include the CD3 protein or a portion thereof. The CD3 protein may include the CD3ζ protein or a portion thereof.

[0053] The activation domain can be isolated or derived from the first protein. The signal peptide can be isolated or derived from the second protein. The transmembrane domain can be isolated or derived from the third protein. The cytoplasmic domain can be isolated or derived from the fourth protein. The signal transduction domain can be isolated or derived from the fifth protein. The first and second proteins may be identical. The first and third proteins may be identical. The first and fourth proteins cannot be identical. The first and fifth proteins cannot be identical. The second and third proteins may be identical. The second and fourth proteins cannot be identical. The second and fifth proteins cannot be identical. The third and fourth proteins cannot be identical. The third and fifth proteins cannot be identical. The fourth and fifth proteins cannot be identical.

[0054] In some embodiments, the activating component does not bind to the native molecule. In some embodiments, the activating component binds to the native molecule, but the CSR does not transmit a signal when the activating component binds to the native molecule. In some embodiments, the activating component binds to a non-natural molecule. In some embodiments, the activating component does not bind to the native molecule, but it does bind to a non-natural molecule. The CSR can selectively transmit a signal when the activating component binds to a non-natural molecule.

[0055] This disclosure provides nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides transposons or vectors comprising nucleic acid sequences encoding any CSR disclosed herein.

[0056] This disclosure provides cells containing any CSR disclosed herein. This disclosure provides cells containing nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides cells containing vectors containing nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides cells containing transposons containing nucleic acid sequences encoding any CSR disclosed herein.

[0057] The modified cells disclosed herein may be allogeneic cells 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] This disclosure provides compositions comprising any CSR disclosed herein. This disclosure provides compositions comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising vectors comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising transposons comprising nucleic acid sequences encoding any CSR disclosed herein. This disclosure provides compositions comprising modified cells disclosed herein, or compositions comprising a plurality of modified cells disclosed herein.

[0059] This disclosure provides a modified T lymphocyte (T cell) comprising (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the expression or activity level of the TCR; and (b) a chimeric stimulating receptor (CSR) comprising (i) an external domain containing an activating component isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an internal domain containing at least one signaling domain isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

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

[0061] Modified T cells may further comprise a non-natural polypeptide containing an HLA class I histocompatibility antigen, an alpha-chain E (HLA-E) polypeptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise a B2M signal peptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise a linker, where the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-natural polypeptide containing the HLA-E polypeptide may further comprise the peptide and the B2M polypeptide. The non-natural polypeptide containing HLA-E may further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding HLA-E.

[0062] Modified T cells may further include non-native antigen receptors, sequences encoding therapeutic polypeptides, or combinations thereof. Non-native antigen receptors may include chimeric antigen receptors (CARs).

[0063] CSR can be transiently expressed in modified T cells. CSR can be stably expressed in modified T cells. Polypeptides containing HLA-E polypeptides can be transiently expressed in modified T cells. Polypeptides containing HLA-E polypeptides can be stably expressed in modified T cells. Inducible pro-apoptotic polypeptides can be transiently expressed in modified T cells. Inducible pro-apoptotic polypeptides can be stably expressed in modified T cells. Sequences encoding non-natural antigen receptors or therapeutic proteins can be transiently expressed in modified T cells. Sequences encoding non-natural antigen receptors or therapeutic proteins can be stably expressed in modified T cells.

[0064] As described in detail herein, but not limited to, gene editing compositions comprising an RNA-induced fusion protein containing dCas9-Clo051 can be used to target, reduce, or eliminate the expression of endogenous T cell receptors. In preferred embodiments, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) that encodes an endogenous T cell receptor. Non-limiting examples of primers (including a T7 promoter, a genomic target sequence, and a gRNA scaffold) for generating guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α), TCR-beta (TCR-β), and beta-2-microglobulin (β2M) are disclosed in PCT application number PCT / US2019 / 049816.

[0065] Gene editing compositions comprising an RNA-induced fusion protein containing dCas9-Clo051 can be used to target, reduce, or eliminate the expression of endogenous MHCI, 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 of a gene (such as a promoter) that encodes one or more components of an endogenous MHCI, MHCII, or MHC activator. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT application number PCT / US2019 / 049816 (which is incorporated herein by reference in its entirety).

[0066] A detailed description of non-natural polypeptides, including non-natural chimeric stimulatory receptors, TCR-alpha (TCR-α), TCR-beta (TCR-β), and / or beta-2-microglobulin (β2M), and genetic modifications of endogenous sequences encoding HLA class I histocompatibility antigens, alpha-E (HLA-E) polypeptides, is disclosed in PCT application number PCT / US2019 / 049816 (which is incorporated herein by reference in its entirety).

[0067] Chimeric stimulating receptors of the present disclosure

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

[0069] In some embodiments, the extracellular domain of CD2 to which the agonist binds, or a portion thereof, [ka] The sequence is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the extracellular domain of CD2 to which the agonist binds, or a portion thereof, is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 2.

[0070] This disclosure provides a chimeric stimulating receptor (CSR) comprising an external domain containing an activating component which includes, is essentially derived from, or comprises a non-natural CD2 extracellular domain. In some embodiments, the external domain of the CSR of this disclosure may include modifications. The modifications may include mutations or terminal breaks in the amino acid sequence of the activating component compared to the wild-type amino acid sequence of the activating component. Mutations or terminal breaks in the amino acid sequence of the activating component may include mutations or terminal breaks in the CD2 extracellular domain to which the agonist binds or in part thereof. Mutated or cleaved CD2 extracellular domains bind to anti-CD2 activating agonists and anti-CD2 activating molecules but do not bind to natural CD58. In some embodiments, a mutation present in a CD2 extracellular domain that binds to an anti-CD2 activating agonist but does not bind to CD58 is a D111H mutation. A CD2 extracellular domain having a D111H mutation is [ka] The extracellular domain of CD2 having the D111H mutation comprises, essentially comprises, or consists of the same amino acid sequence by at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between).

[0071] In some embodiments, the extracellular domain of CD2 having the D111H mutation is [ka] The extracellular domain of CD2 having the D111H mutation is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence.

[0072] The internal domains of the CSR of this disclosure may further include, be essentially derived from, or consist of a signal peptide. In some embodiments, the signal peptide may include, be essentially derived from, or consist of a CD2 signal peptide or a portion thereof. In some embodiments, the signal peptide may include, be essentially derived from, or consist of a CD8a signal peptide or a portion thereof.

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

[0074] In some embodiments, the CD2 signal peptide is [ka] The CD2 signal peptide is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD2 signal peptide is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 6.

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

[0076] In some embodiments, the CD8a signal peptide is [ka] The signal peptide is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD8a signal peptide is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 8.

[0077] This disclosure provides CSRs including a transmembrane domain. In some embodiments, the transmembrane domain may include, be essentially derived from, or consist of a CD2 transmembrane domain or a portion thereof. In some embodiments, the CD2 transmembrane domain or a portion thereof may include, be essentially derived from, or consist of an amino acid sequence identical to IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 9) by at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between). In a preferred embodiment, the CD2 transmembrane domain or a portion thereof may include, be essentially derived from, or consist of the amino acid sequence of SEQ ID NO: 9.

[0078] In some embodiments, the CD2 transmembrane domain or a portion thereof is [ka] The CD2 transmembrane domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD2 transmembrane domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 10.

[0079] This disclosure provides a CSR comprising an internal domain containing at least one signaling domain. In some embodiments, the signaling domain may include, essentially consist of, or comprise a CD3ζ intracellular domain or a portion thereof. In some embodiments, the CD3ζ intracellular domain or a portion thereof [ka] The CD3ζ intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the CD3ζ intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 11.

[0080] In some embodiments, the CD3ζ intracellular domain or a part thereof [ka] The CD3ζ intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD3ζ intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 12.

[0081] The internal domains of the CSRs of this disclosure may further include, be essentially derived from, or consist of a cytoplasmic domain. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of a CD2 intracellular domain (ICD) or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of a CD28 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of a 4-1BB intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of an IL17RA intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of an IL15RA intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of an IL21R intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, be essentially derived from, or consist of an ICOS intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain includes, essentially comprises, or consists of the CD27 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, essentially comprises, or consists of the OX40 intracellular domain or a portion thereof. In some embodiments, the cytoplasmic domain may include, essentially comprises, or consists of the GITR intracellular domain or a portion thereof.

[0082] In some embodiments, the intracellular domain of CD2 or a part thereof is [ka] The CD2 intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the CD2 intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 13.

[0083] In some embodiments, the intracellular domain of CD2 or a part thereof is [ka] The CD2 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD2 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 14.

[0084] In some embodiments, the CD28 intracellular domain or a portion thereof is [ka] The CD28 intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the CD28 intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 15.

[0085] In some embodiments, the CD28 intracellular domain or a portion thereof is [ka] The CD28 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD28 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 16.

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

[0087] In some embodiments, the 4-1BB intracellular domain or a portion thereof is [ka] The 4-1BB intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the 4-1BB intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 18.

[0088] In some embodiments, the intracellular domain of IL17RA or a portion thereof is [ka] The IL17RA intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the IL17RA intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 19.

[0089] In some embodiments, the intracellular domain of IL17RA or a portion thereof is [ka] The IL17RA intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the IL17RA intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 20.

[0090] In some embodiments, the intracellular domain of IL15RA or a portion thereof is [ka] The IL15RA intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the IL15RA intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 21.

[0091] In some embodiments, the intracellular domain of IL15RA or a portion thereof is [ka] The IL15RA intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the IL15RA intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 22.

[0092] In some embodiments, the intracellular domain of IL21R or a portion thereof is [ka] The IL21R intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the IL21R intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 23.

[0093] In some embodiments, the intracellular domain of IL21R or a portion thereof is [ka] The IL21R intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the IL21R intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 24.

[0094] In some embodiments, the intracellular domain of ICOS or a part thereof is [ka] The ICOS intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the ICOS intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 25.

[0095] In some embodiments, the intracellular domain of ICOS or a part thereof is [ka] The ICOS intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the ICOS intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 26.

[0096] In some embodiments, the intracellular domain of CD27 or a portion thereof is [ka] The CD27 intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the CD27 intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 27.

[0097] In some embodiments, the intracellular domain of CD27 or a portion thereof is [ka] The CD27 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the CD27 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 28.

[0098] In some embodiments, the OX40 intracellular domain or a portion thereof is [ka] The OX40 intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the OX40 intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 29.

[0099] In some embodiments, the OX40 intracellular domain or a portion thereof is [ka] The OX40 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the OX40 intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of SEQ ID NO: 30.

[0100] In some embodiments, the GITR intracellular domain or a portion thereof is [ka] The GITR intracellular domain or a portion thereof contains, essentially comprises, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the same amino acid sequence. In a preferred embodiment, the GITR intracellular domain or a portion thereof contains, essentially comprises, or comprises the amino acid sequence of SEQ ID NO: 31.

[0101] In some embodiments, the GITR intracellular domain or a portion thereof is [ka] The GITR intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same nucleic acid sequence. In a preferred embodiment, the GITR intracellular domain or a portion thereof is encoded by a polynucleotide that contains, essentially consists of, or comprises the nucleic acid sequence of Sequence ID No. 32.

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[0144] The compositions of this disclosure (e.g., CSR) bind to anti-CD2 activating agonists and anti-CD2 activating molecules, but do not bind to natural CD58.

[0145] The compositions comprising CSRs of this disclosure can be incorporated into cell delivery compositions (e.g., transposons or vectors) as described in detail herein, and can be optionally incorporated into cells.

[0146] Cells and modified cells of this disclosure

[0147] The cells and modified cells of this disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of this disclosure may be immune cells. The immune cells of this disclosure include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), and stem memory T cells (T SCM cells), central memory T cells (T CM ), may include 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 cell capable of differentiating into one or more types of immune cells. Immune progenitor cells can include pluripotent stem cells capable of self-replicating and developing into immune cells. Immune progenitor cells can include hematopoietic stem cells (HSCs) or their offspring. Immune progenitor cells can include progenitor cells capable of developing into immune cells. Immune progenitor cells can include hematopoietic progenitor cells (HPCs).

[0149] Hematopoietic stem cells (HSCs) are pluripotent self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages originate 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 cells. Non-limiting examples of HSC progeny cells include multipotent stem cells, lymphocyte progenitor cells, natural killer (NK) cells, T lymphocyte cells (T cells), B lymphocyte cells (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[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 being involved in a single lineage. For example, the "primitive" HSCs generated by the disclosed methods retain their "stemness" and do not differentiate even after division. As a result, as adoptive cell therapy, the "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. The "primitive" HSCs generated by the disclosed methods can be therapeutically effective when administered as a single dose.

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

[0154] Primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the disclosed methods to express an exogenous sequence (such as a chimeric antigen receptor or a therapeutic protein). Without being particularly limited, the modified primitive HSCs, modified HSCs, and / or modified HSC progeny cells can be differentiated forward to produce modified immune cells including, without being particularly limited, 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 that differentiate from lymphocyte progenitor cells. Modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some embodiments, the inactivated NK cells are derived from CD3 depleted leukapheresis (including CD14 / CD19 / CD56+ cells).

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

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

[0158] Development of antigen receptor molecules that do not cause T cell depletion via 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, including stem cell-like T cells. The stem cell-like modified T cells of this disclosure exhibit maximum capabilities in self-renewal and pluripotency, and central memory (T CM ) T cells or T CM Cellular, effector memory (T EM ) and effector T cells (T E This induces ) and thereby achieves better tumor eradication and long-term modified T cell engraftment. The linear pathway of differentiation involves these cells (naive T cells (T N )>T SCM >>T EM >T E >T TE ) may be involved in the generation of, and here, T N is, T SCM These are parental progenitor cells that directly produce T CM Directly generates T cells. The T cell composition of this disclosure may contain one or more of each parental T cell subset. SCM Cells are 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 naive T cells (T N), T SCM , TCM, T EM , T E , or T TE They may or may differentiate into these. Immune cell precursors may be primitive HSCs, HSCs, or HSC progeny cells of this disclosure. Immune cells include early memory T cells, stem cell-like T cells, and naive T cells (T). N ), T SCM , T CM , T EM , T E , or T TE It is possible.

[0160] The method of the present disclosure can modify and / or produce a population of modified T cells, where at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, of the 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 multiple modified stem cell-like T cells. SCM Includes cells. A population of modified early memory T cells consists of multiple modified T cells. CM Contains cells.

[0161] The method of the present disclosure can modify and / or produce a population of modified T cells, where at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage in between, of the multiple modified T cells in the population express one or more cell surface markers of stem cell-like T cells. The population of modified stem cell-like T cells is composed of multiple modified T SCM Includes cells. A population of modified stem cell-like T cells consists of multiple modified T cells. 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 in between, are stem memory T cells (T SCM ) or T SCM The cells express one or more cell surface markers; and here, the one or more cell surface markers include CD45RA and CD62L. The cell surface markers may include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. The cell surface markers may include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[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 in between, are central memory T cells (T CM ) or T CM The cells express one or more cell surface markers; and the one or more cell surface markers include CD45RO and CD62L. The cell surface markers may include one or more of CD45RO, IL-2Rβ, CCR7, and CD62L.

[0164] The method of the present 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 between these, of the multiple modified T cells in the population are naive T cells (T N The cells express one or more of the following cell surface markers. The cell surface markers may include one or more of CD45RA, CCR7, and CD62L.

[0165] The method disclosed herein 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 between these, of the multiple modified T cells in the population are effector T cells (T EFF The cells express one or more of the following cell surface markers. The cell surface markers may include one or more of CD45RA, CD95, and IL-2Rβ.

[0166] The method disclosed herein 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 between these, of the multiple 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 It expresses one or more cell surface markers.

[0167] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where at least 70%, at least 75%, at least More than 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 the population of modified cells, at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% express one or more cell surface markers including CD34 (e.g., including cell surface marker phenotype CD34+).

[0168] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where 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% 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% express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38, or where 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] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where 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 Cells express one or more cell surface markers including CD4 and CD90, and do not express one or more cell surface markers including CD38, or, where, at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers 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] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where 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 one or more types of cells containing CD34 and CD90. Cells express a surface marker and do not express one or more cell surface markers including CD38 and CD45RA, or, where, at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including CD38 and CD45RA (e.g., cell surface marker phenotype CD34+, CD38-, CD90+, CD45RA-).

[0171] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where 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, where, at least about 0.02% to about 30%, 0.02% to about 2%, about 0.04% to about 2%, or about 0% of the population of modified cells.Approximately 0.4% to 1% of cells 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] Multiple modified cells in a population contain a trans gene or a sequence encoding a trans gene (e.g., CAR), where 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 multiple cells in the population contain a trans gene or a sequence encoding a trans gene, where at least one of the populations of modified cells contains a trans gene or a sequence encoding a trans gene. 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%, and at least 99.9% or 100% express one or more cell surface markers including CD34 and CD90, and do not express one or more cell surface markers including CD45RA, or, where, at least about 0.2% to about 5%, about 0.2% to about 3%, or about 0.4% to about 3% of the modified cell population express one or more cell surface markers including CD34 and CD90, and do not express one or more cell surface markers including one or more cell surface markers including CD45RA (e.g., cell surface marker phenotype CD34+, CD90+, and CD45RA-).

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

[0174] The cells and engineered cells of the disclosure can be somatic cells. The cells and engineered cells of the disclosure can be differentiated cells. The cells and engineered cells of the 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 can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0175] Method for expressing chimeric antigen receptors

[0176] The disclosure provides a method of expressing a CAR on the surface of a cell. The method includes: (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 transfer the CAR across the cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration 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 the 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+ leukocytes to CD8+ leukocytes does not occur naturally in vivo. The cell population can comprise tumor cells.

[0178] In some embodiments, conditions sufficient for transporting a CAR, or a sequence encoding a CAR, transposon, or vector, across the cell membrane of at least one cell in a cell population include the application of one or more electrical pulses of a specific voltage, a buffer, and at least one of one or more supplemental factors. In some embodiments, conditions suitable for incorporating a sequence encoding a CAR include at least one buffer and one or more supplemental factors.

[0179] The buffer may include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. One or more supplementary factors may include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, inorganic substances, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cellular DNA sensing, metabolism, differentiation, signaling, one or more apoptotic pathways, or combinations thereof; and (e) reagents for modifying or stabilizing one or more nucleic acids. 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 This may include 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-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANKL, or any combination thereof.Salts, inorganic substances, metabolites, or any combination thereof may include hepes, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earl's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCl, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. Cell culture media may include PBS, HBSS, OptiMEM, DMEM, RPMI1640, AIM-V, X-VIVO15, CellGro DC medium, CTS OpTimizer T cell-strengthened SFM, TexMACS medium, PRIME-XV T cell-strengthened medium, ImmunoCult-XFT cell-strengthened medium, or any combination thereof. Inhibitors of cellular DNA sensing, metabolism, differentiation, signaling, one or more apoptotic 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, glycogen synthase kinase-3β (GSK-3β) inhibitors (e.g., TWS119), or any combination thereof. Examples of such inhibitors may include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.Reagents for modifying or stabilizing one or more nucleic acids may include pH modifiers, DNA-binding proteins, lipids, phospholipids, CaPO4, net-neutral charged DNA-binding peptides containing or not containing NLS sequences, TREX1 enzymes, or combinations thereof.

[0180] The expansion and selection processes can be performed simultaneously or sequentially. Expansion may occur before selection. Expansion may occur after selection, and optionally, further (i.e., a second) selection may occur after expansion. Collaborative expansion and selection can be performed simultaneously. The expansion and / or selection process can continue for 10 to 14 days (including both end values).

[0181] Expansion may include contacting at least one cell of a modified cell population with an antigen to stimulate that at least one cell via CAR, thus generating an expanded cell population. The antigen may be presented on the surface of a substrate. The substrate may be in any form, but is not limited, and may include a surface, wells, beads, or a combination thereof, and a matrix. The substrate may further include paramagnetic or magnetic components. The antigen may be presented on the surface of a substrate, where the substrate is magnetic beads, and the magnetic beads can be removed or separated from the modified and expanded cell population using a magnet. The antigen may be presented on the surface of cells or on artificial antigen-presenting cells. Artificial antigen-presenting cells may include, but are not limited, tumor cells and stem cells.

[0182] In some embodiments in which the transposon or vector comprises a selection gene, the selection step involves contacting at least one cell of a modified cell population with a compound in which the selection gene confers resistance, thereby identifying cells that express the selection gene as surviving the selection and cells that cannot express the selection gene as not surviving the selection step.

[0183] This disclosure provides compositions comprising modified, extended, and selected cell populations of the methods described herein.

[0184] A more detailed description of the method for expressing CAR on the surface of cells is disclosed in PCT publication numbers WO2019 / 049816 and PCT / US2019 / 049816.

[0185] This disclosure provides cells or a 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 when constructs (a) and (b) are incorporated into the genomic sequence of the cells, the exogenous receptor is expressed, wherein, upon binding to a ligand or antigen, the exogenous receptor transmits an intracellular signal that directly or indirectly targets the inducible promoter, thereby modifying gene expression by regulating the expression of the inducible transgene (a).

[0186] A composition can modify gene expression by reducing it. A composition can modify gene expression by temporarily modifying it (e.g., while the ligand is bound to the exogenous receptor). A composition can rapidly modify gene expression (e.g., the ligand reversibly binds to the exogenous receptor). A composition can chronically modify gene expression (e.g., the ligand irreversibly binds to the exogenous receptor).

[0187] Exogenous receptors may include endogenous receptors in relation to the cell's genomic sequence. 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] Exogenous receptors may include non-native receptors. Non-native receptors may be synthetic, modified, recombinant, mutant, or chimeric receptors. Non-native receptors may include one or more sequences isolated or derived from T cell receptors (TCRs). Non-native receptors may include one or more sequences isolated or derived from scaffold proteins. In some embodiments, including those in which the non-native receptor does not contain a transmembrane domain, the non-native receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor, which transmits intracellular signals after contact with the non-native receptor. Non-native receptors may include a transmembrane domain. Non-native receptors may interact with intracellular receptors that transmit intracellular signals. Non-native receptors may include an intracellular signaling domain. Non-native receptors may be chimeric ligand receptors (CLRs). CLRs may be chimeric antigen receptors (CARs).

[0189] Sequences encoding inducible promoters include sequences encoding NFκB promoters, interferon (IFN) promoters, or interleukin-2 promoters. In some embodiments, the IFN promoter is an IFNγ promoter. Inducible promoters can be isolated or derived from cytokine or chemokine promoters. Cytokines or chemokines may 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-motif chemokine ligand 5 (CCL5), CC-motif chemokine ligand 4 (Ccl4), CC-motif chemokine ligand 3 (Ccl3), XC-motif chemokine ligand 1 (Xcl1), or LIF interleukin-6 family cytokines (Lif).

[0190] Inducible promoters can be isolated or induced from promoters of genes containing 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] Inducible promoters can be isolated or induced from promoters of genes involved in the metabolism and differentiation of C. spp. (CD). Inducible promoters can be isolated or induced from the promoters of Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0192] In some embodiments, inducible transgene constructs include or drive the expression of signaling components downstream of repressive 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 treatment, and oncogenes or tumor suppressor genes. Non-limiting examples are disclosed in PCT publication number WO2019 / 173636 and PCT application number PCT / US2019 / 049816.

[0193] armored cells

[0194] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to enhance their therapeutic potential. Alternatively, modified cells can be further modified to reduce their sensitivity to immunological and / or metabolic checkpoints. This type of modification “armors” the cells, and the modified cells are referred to herein as “armored” cells (e.g., armored T cells). Armored cells can be generated, for example, by blocking and / or diluting (e.g., checkpoint inhibition) specific checkpoint signals that are naturally delivered to the cells, within the tumor immunosuppressive microenvironment.

[0195] The armored cells of this disclosure may 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 induced from G-CSF-mobilized peripheral blood), or umbilical cord blood (UCB)-derived T cells. Armored cells (e.g., armored T cells) may contain one or more of the following: chimeric ligand receptors (CLRs including protein scaffolds, antibodies, ScFv, or antibody mimes), chimeric antigen receptors (CARs including protein scaffolds, antibodies, ScFv, or antibody mimetics), CARTyrin (CARs including centinrin), and / or VCARs (CARs including camel VHH or single-domain VH). Armored cells (e.g., armored T cells) may contain inducible pro-apoptotic polypeptides as disclosed herein. Armored cells (e.g., armored T cells) may contain exogenous sequences. The exogenous sequences may include sequences encoding therapeutic proteins. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell surface-bound, or secreted proteins. Exemplary therapeutic proteins expressed by armored cells (e.g., armored T cells) may modify the activity of armored cells or the activity of a second cell. Armored cells (e.g., armored T cells) may contain select genes or select markers. Armored cells (e.g., armored T cells) may contain synthetic gene expression cassettes (also called inducible trans gene constructs).

[0196] The modified cells described herein (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding inhibitory checkpoint signal receptors to generate armored cells (e.g., armored CAR T cells). Inhibitory checkpoint signal receptors are expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing the expression of genes encoding inhibitory checkpoint signal receptors results in loss of inhibitory checkpoint receptor protein expression on the surface or in the cytoplasm of armored cells. Thus, armored cells in which the expression of one or more genes encoding inhibitory checkpoint receptors is silenced or reduced are resistant, nonreceptive, or insensitive to checkpoint signals. The resistance or reduced sensitivity of armored cells to inhibitory checkpoint signals enhances the therapeutic potential of armored cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and immunosuppressive-inducing proteins) are disclosed in PCT publication number 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 of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding intracellular proteins involved in checkpoint signaling, thereby producing 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 the checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference to one or more checkpoint pathways. A non-limiting example of intracellular signaling proteins involved in checkpoint signaling is disclosed in PCT publication number WO2019 / 173636.

[0198] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding transcription factors that interfere with therapeutic efficacy, thereby producing armored cells (e.g., armored CAR T cells). The activity of the modified cells can be enhanced or regulated by silencing or reducing (or suppressing the function of) the transcription factors that interfere with therapeutic efficacy. Non-limiting examples of transcription factors that can be modified to silence or reduce their expression or suppress their function include, but are not limited to, exemplary transcription factors disclosed in PCT Publication No. WO2019 / 173636.

[0199] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding cell death or apoptosis receptors to produce armored cells (e.g., armored CAR T cells). The interaction between the cell death receptor and its endogenous ligand leads to the initiation of apoptosis. Disruption of the expression, activity, or interaction of the cell death and / or apoptosis receptor and / or ligand reduces the sensitivity of the modified cells to the cell death signal, thereby making the armored cells more effective in the tumor environment. Non-limiting examples of cell death and / or apoptosis receptors and ligands are disclosed in PCT publication number WO2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas(CD95).

[0200] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding metabolism-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 leads to an extended retention of T cell function, resulting in the killing of more tumor cells per cell. Non-limiting examples of metabolism-sensing genes and proteins are disclosed in PCT publication number WO2019 / 173636. Preferred 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 of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding proteins that confer sensitivity to cancer therapies, including monoclonal antibodies, in order to produce armored cells (e.g., armored CAR T cells). Thus, armored cells can function and may exhibit superior function or efficacy in the presence of cancer therapies (e.g., chemotherapy, monoclonal antibody therapy, or other antitumor therapies). A non-limiting example of proteins involved in conferring sensitivity to cancer therapies is disclosed in PCT publication number WO2019 / 173636.

[0202] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding growth advantage factors to produce armored cells (e.g., armored CAR T cells). Silencing or reducing the expression of oncogenes can confer growth advantage to cells. For example, silencing or reducing (e.g., disrupting) the expression of the TET2 gene during the CAR T cell production process results in the production of armored CAR T cells with a significantly greater ability to expand and subsequently eradicate tumors compared to unarmored CAR T cells lacking expansion capacity. This strategy can be combined with a safety switch (e.g., the iC9 safety switch described herein) to enable targeted destruction of armored CAR T cells in the event of adverse reactions from the subject or uncontrolled proliferation of armored CAR T cells. Non-limiting examples of growth advantage factors are disclosed in PCT publication number WO2019 / 173636.

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

[0204] Modified / chimeric checkpoint receptors may include null receptors, decoy receptors, or dominant-negative receptors. Null receptors, decoy receptors, or dominant-negative receptors may be modified / chimeric receptors / proteins. Null receptors, decoy receptors, or dominant-negative receptors may be cleaved at their terminal ends for the expression of an intracellular signaling domain. Alternatively, null receptors, decoy receptors, or dominant-negative receptors may be mutated within their intracellular signaling domain at one or more amino acid positions that are deterministic or necessary for effective signaling. Terminal cleavage or mutation of a null receptor, decoy receptor, or dominant-negative receptor may result in a loss of the receptor's ability to send or transmit checkpoint signals to or within a cell.

[0205] For example, dilution or blockade of immunosuppressive checkpoint signaling from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing modified / chimeric PD-1 null receptors on the surface of armored cells (e.g., armored CAR T cells), which effectively compete with endogenous (unmodified) PD-1 receptors also expressed on the surface of armored cells, thereby reducing or inhibiting the transmission of immunosuppressive checkpoint signaling via the endogenous PD-1 receptors of armored cells. In this non-limiting example, competition between 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 may include transmembrane receptors, membrane-bound or membrane-linked receptors / proteins, or null receptors, decoy receptors, or dominant-negative receptors that are 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 treatment, and oncogenes or tumor suppressor genes. Non-exclusive examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT publication number WO2019 / 173636.

[0207] Modified / chimeric checkpoint receptors can include switch receptors. An exemplary switch receptor includes a modified / chimeric receptor / protein, where the native or wild-type intracellular signaling domain is switched or substituted with a different intracellular signaling domain that is not specific to the protein and / or is not the wild-type domain. For example, substituting an inhibitory signaling domain with a stimulating signaling domain switches an immunosuppressive signal to an immunostimulatory signal. Alternatively, substituting an inhibitory signaling domain with another inhibitory domain can reduce or enhance the level of inhibitory signaling. Expression or overexpression of a switch receptor may result in dilution and / or blockade of the congenital checkpoint signal through competition with endogenous wild-type checkpoint receptors (not switch receptors) for binding to congenital checkpoint receptors expressed within the immunosuppressive tumor microenvironment. Armored cells (e.g., armored CAR T cells) may contain sequences encoding switch receptors, resulting in the expression of one or more switch receptors and consequently altering the activity of armored cells. Armored cells (e.g., armored CAR T cells) can express checkpoint receptors, downstream intracellular expression proteins, transcription factors, cytokine receptors, death receptors, metabolic sensing molecules, cancer therapies, oncogenes, and / or tumor suppressor proteins or gene-targeting switch receptors.

[0208] Exemplary switch receptors may include, or be derived from, proteins comprising, 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 treatment, and oncogenes or tumor suppressor genes.

[0209] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to express CLR / CAR, which mediates conditional gene expression, thereby generating armored T cells. The combination of CLR / CAR and the conditional gene expression system in the nucleus of armored T cells constitutes a synthetic gene expression system that is conditionally activated by the binding of a congeneral ligand to CLR or a congeneral antigen to CAR. This system may help "armor" or enhance the therapeutic potential of modified T cells by reducing or restricting synthetic gene expression, for example, at ligand or antigen binding sites or within the tumor environment.

[0210] Gene editing compositions and methods

[0211] Modified cells are generated by introducing transgenes into cells. The introduction process may include the delivery of nucleic acid sequences, transgenes, and / or genome editing constructs via a non-translocation delivery system.

[0212] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ may include one or more of the following methods: local delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, ultrasonic delivery, vibrational delivery, magnetofection, or nanoparticle-mediated delivery. The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ may include liposome transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells by mechanical transfection ex vivo, in vivo, in vitro, or in situ may include cell compression, cell shock, or gene gun technology. The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells by nanoparticle-mediated transfection ex vivo, in vivo, in vitro, or in situ may 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 may include nonviral vectors. Nonviral vectors may include nucleic acids. Nonviral vectors may include plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone® DNA, nanoplasmids, mini-circular DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). Nonviral vectors may 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 may involve viral vectors. Viral vectors may be non-integrated, non-chromosomal vectors. Non-limiting examples of non-integrated, non-chromosomal vectors may include adeno-associated viruses (AAVs), adenoviruses, and herpesviruses. Viral vectors may also be integrated, chromosomal vectors. Non-limiting examples of integrated, chromosomal vectors may include adeno-associated vectors (AAVs), lentiviruses, and gamma-retroviruses.

[0215] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ may include vector combinations. Non-limiting examples of vector combinations include viral vectors and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Non-limiting examples of vector combinations include DNA-derived vectors and RNA-derived vectors, RNA and reverse transcriptase combinations, transposons and transposase combinations, non-viral vectors and endonuclease combinations, and viral vectors and endonuclease combinations.

[0216] Genome modification involves introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably incorporate nucleic acid sequences, transiently incorporate nucleic acid sequences, generate site-specific integration of nucleic acid sequences, or generate biased integration of nucleic acid sequences. Nucleic acid sequences can be transgenes.

[0217] Genome modification involves introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably incorporate nucleic acid sequences. Stable chromosomal integration can be random, site-specific, or biased. Site-specific integration may be unsupported or supported. Supported site-specific integration is co-delivered with site-specific nucleases. Site-specific nucleases include transgenes with 5' and 3' nucleotide sequence elongations containing homologous percentages to the upstream and downstream regions of the genomic integration site. Transgenes with homologous nucleotide elongations enable genomic integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration may occur at safe harbor sites. Safe harbor sites in the genome can accommodate the integration of new genetic material in a way that ensures the newly inserted gene element functions (e.g., is expressed at therapeutically effective levels) and does not cause harmful changes to the host genome that pose a risk to the host organism. Non-limiting examples of potential genome-safe harbors include intron sequences in the human albumin gene, adeno-associated virus site 1 (AAVS1), native integration sites of the AAV virus on chromosome 19, sites in the chemokine (CC motif) receptor 5 (CCR5) gene, and sites of human orthologs of the mouse Rosa26 locus.

[0218] Site-specific transgene integration can occur at sites that interfere with the expression of a target gene. Disruption of target gene expression can occur through site-specific integration at introns, exons, promoters, gene elements, enhancers, suppressors, start codons, stop codons, and response elements. Non-exclusive examples of target genes that can be targeted by site-specific integration include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allorejection.

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

[0220] Enzymes can be used to create strand breaks in the host genome, facilitating the delivery or integration of transgenes. Enzymes can create single-strand or double-strand breaks. Non-exclusive 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 as DNA-encoded, mRNA-encoded, proteins, or as nuclear protein complexes with guide RNA (gRNA).

[0221] Site-specific transgene integration can be controlled by biasing the integration site via the vector. This biasing can be controlled by a selected lentiviral vector or a selected gamma-retroviral vector.

[0222] Site-specific transgene integration sites can be unstable chromosomal insertions. The integrated transgene may be silenced, removed, excised, or further modified. Genomic modifications can result in unstable integration of the transgene. Unstable integration can be transient non-chromosomal integration, semi-stable non-chromosomal integration, semi-persistent non-chromosomal insertion, or unstable chromosomal insertion. Transient non-chromosomal insertions can be episomal or cytoplasmic. In one embodiment, a transient non-chromosomal insertion of a transgene is not integrated into a chromosome, and the modified genetic material is not replicated during cell division.

[0223] Genome modifications can result in semi-stable or persistent non-chromosomal integration of transgenes. DNA vectors encode scaffold / matrix-binding region (S-MAR) modules that bind to nuclear matrix proteins for episome retention of nonviral vectors, enabling autonomous replication in the nucleus of dividing cells.

[0224] Genome modification can result in unstable chromosomal integration of transgenes. Integrated transgenes may be silenced, removed, excised, or further modified.

[0225] Genomic modifications by transgene insertions can occur via host cell-directed double-strand break repair (homologous-directed repair) through homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or recombinant-mediated modification. Genomic modifications by transgene insertions can also occur via CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER®, and cpf1.

[0226] In gene editing systems involving the insertion of novel or existing nucleotides / nucleic acids, in addition to the cutting enzyme (e.g., nuclease, recombinase, integrase, or transposase), it is necessary to deliver the insertion tool (e.g., DNA template vector, transposer (transposon or retrotransposon)) to the cell. An example of such an insertion tool for recombinase is a DNA vector. Other gene editing systems require the delivery of integrase along with the insertion vector, or transposons along with transposons / retrotransposons. 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 as insertion tools include virus-based enzymes derived from one of many viruses, such as AAV, gamma retrovirus, and lentivirus. Examples of transposons / retrotransposons that can be used as insertion tools are described in more detail herein.

[0227] Cells with ex vivo, in vivo, in vitro, or in situ genome modifications may be germline cells or somatic cells. Modified cells may be human, non-human, mammalian, rat, mouse, or canine cells. Modified cells may be differentiated, undifferentiated, or immortalized. Modified undifferentiated cells may be stem cells. Modified undifferentiated cells may be induced pluripotent stem cells. Modified cells may be immune cells. Modified cells may be T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts. Modified cells may be modified while the cell is in a quiescent, activated, resting, interphase, prophase, metaphase, anaphase, or telophase state. Modified cells may be fresh, cryopreserved, bulk, from whole blood, from leukocyte apheresis, or from immortalized cell lines, and may be classified into subpopulations. Detailed instructions for isolating cells from leukocyte apheresis products or blood are disclosed in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816.

[0228] This disclosure provides a gene editing composition and / or cells containing 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 its 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 CRISPR / Cas proteins, activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases.

[0229] A nuclease or its nuclease domain may include a nuclease-inactivating Cas(dCas) protein and an endonuclease. The endonuclease may include a Clo051 nuclease or its nuclease domain. The gene editing composition may include a fusion protein. The fusion protein may include a nuclease-inactivating Cas9(dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition may further include a guide sequence. The guide sequence includes an RNA sequence.

[0230] This disclosure provides compositions comprising a small Cas9 (Cas9) conjugated in a functional manner to an effector. This disclosure provides fusion proteins comprising, essentially comprising, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 constructs of this disclosure may include an effector comprising an IIS-type endonuclease. Staphylococcus aureus Cas9 having an active catalytic site comprises the amino acid sequence of SEQ ID NO: 79.

[0231] This disclosure provides compositions comprising an inactivated small Cas9 (dSaCas9) bound in a functional manner to an effector. This disclosure provides fusion proteins comprising, essentially comprising, 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) constructs of this disclosure may include an effector comprising an IIS-type endonuclease. dSaCas9 comprises the amino acid sequence of SEQ ID NO: 80, which includes D10A and N580A mutations for inactivating the catalytic site.

[0232] This disclosure provides compositions comprising inactivated Cas9 (dCas9) conjugated in a functional manner to an effector. This disclosure provides fusion proteins comprising, essentially comprising, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of this disclosure may include an effector comprising an IIS-type endonuclease.

[0233] dCas9 can be isolated or derived from Streptococcus pyogenes. dCas9 can include dCas9 having 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 contains, essentially consists of, or comprises the amino acid sequence of Sequence ID No. 83.

[0235] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may contain, essentially consist of, or comprise the amino acid sequence of SEQ ID NO: 84. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide containing, essentially consisting of, or comprising the nucleic acid sequence of SEQ ID NO: 85. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0236] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may contain, essentially consist of, or comprise the amino acid sequence of SEQ ID NO: 86. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide containing, essentially consisting of, or comprising the nucleic acid sequence of SEQ ID NO: 87. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0237] Cells containing a gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is expressed transiently. The guide RNA may include a sequence complementary to the target sequence in the genomic DNA sequence. The target sequence in the genomic DNA sequence may be a target sequence within a safe harbor region of the genomic DNA sequence.

[0238] Gene editing compositions containing Cas-CLOVER, and methods for 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 an imidazole ring that provides a lone pair of electrons on an unsaturated nitrogen. That is, poly(histidine) has amphoteric properties due to protonation-deprotonation. In particular, at certain pH levels, poly(histidine)-containing triblock copolymers assemble to form micelles with positively charged poly(histidine) units on their surface, thus enabling complex formation with negatively charged gene editing molecules. Using these nanoparticles, an efficient and selective mechanism for performing desired gene modifications can be provided by binding and releasing proteins and / or nucleic acids in a pH-dependent manner. In particular, this micelle-based delivery system offers substantial flexibility with respect to 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 a nuclease using poly(histidine)-based micelles. While we do not wish to be bound by any particular theory, it is thought that in micelles formed by various triblock copolymers, hydrophobic blocks aggregate to form a core, leaving hydrophilic blocks and poly(histidine) blocks at the ends, and forming one or more surrounding layers.

[0240] In one embodiment, the disclosure provides a triblock copolymer comprising a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be poly(ethylene oxide) (PEO), and the charged block may 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 of 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) (which is chemically synonymous with PEG) bound to a variety of hydrophobic aliphatic poly(anhydride), poly(nucleic acid), poly(ester), poly(orthoester), poly(peptide), poly(phosphazene), and poly(saccharides) [but not limited to poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic acid-coglycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC)]. Polymeric micelles composed of 100% PEGylated surfaces exhibit improved in vitro chemical stability, improved in vivo bioavailability, and extended blood circulation half-life.

[0242] Polymer vesicles, polymerosomes, and poly(histidine)-based micelles, including those containing triblock copolymers, and methods for producing them are described in detail in U.S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Patent Application Publications 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication WO2019 / 126589.

[0243] Transposons and vector compositions

[0244] This disclosure provides compositions and methods for delivering antibodies (e.g., scFv) or CARs (e.g., including scFv) to cells or populations of cells. Non-limiting examples of compositions for delivering the compositions of this disclosure to cells or populations of cells include transposons or vectors. Accordingly, this disclosure provides transposons containing antibodies (e.g., scFv) or CARs (e.g., including scFv), or vectors containing antibodies (e.g., scFv) or CARs (e.g., including scFv).

[0245] A transposon or vector containing the CAR of the Disclosure may further include a sequence encoding an inducible pro-apoptotic polypeptide. Alternatively, one transposon or vector may further include the CAR of the Disclosure, and a second transposon or vector may include a sequence encoding the inducible pro-apoptotic polypeptide of the Disclosure. The inducible pro-apoptotic polypeptide is described in more detail herein.

[0246] A transposon or vector containing a CAR of the Disclosure may further include a sequence encoding a chimeric stimulating receptor (CSR). Alternatively, one transposon or vector may further include a CAR of the Disclosure, and a second transposon or vector may include a sequence encoding a CSR of the Disclosure. Chimeric stimulating receptors are described in more detail herein.

[0247] A transposon or vector containing the CAR of the Disclosure may further include a sequence encoding a recombinant HLA-E polypeptide. Alternatively, one transposon or vector may further include the CAR of the Disclosure, and a second transposon or vector may include a sequence encoding a recombinant HLA-E polypeptide. Recombinant HLA-E polypeptides are described in more detail herein.

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

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

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

[0251] A transposon containing a CAR of the present disclosure or a vector containing a CAR of the present disclosure may further comprise at least two self-cleaving peptides. For example, a first self-cleaving peptide is located upstream or immediately upstream of the CAR, and a second self-cleaving peptide is located downstream or immediately downstream of the CAR. Alternatively, the first and second self-cleaving peptides are adjacent to the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of an 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 and second self-cleaving peptides are adjacent to an inducible apoptosis-promoting polypeptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of a protein encoded by a selection gene, and the second self-cleaving peptide is located downstream or immediately downstream of a protein encoded by a selection gene. Alternatively, the first and second self-cleaving peptides are adjacent to a protein encoded by a selection gene.

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

[0253] Dislocation system

[0254] This disclosure provides transposons comprising a protein scaffold as disclosed herein, or provides transposons comprising an antibody (e.g., scFv) or a CAR (e.g., including scFv) as disclosed herein. In a preferred embodiment, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding scFv or a CAR (e.g., including scFv) as disclosed herein, with two cis-regulatory insulator elements adjacent to each other. This disclosure also provides compositions comprising transposons. In a preferred embodiment, the composition comprising a transposon further comprises a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0255] The transposons of this disclosure may be PiggyBac®(PB) transposons. 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. Patents 6,218,182; 6,962,810; 8,399,643 and PCT Publication No. WO2010 / 099296.

[0257] PB, PBL, and SPB transposases recognize transposon-specific reverse terminal repeat sequences (ITRs) at the ends of transposons and insert their contents between ITRs of the chromosomal sequence 5'-TTAT-3' (TTAT target sequence) or the chromosomal sequence 5'-TTAA-3' (TTAA target sequence). 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' -Includes or consists of 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'. There are no payload limitations on the target gene that can be included between ITRs in PB or PBL transposon systems.

[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 a preferred embodiment, a PB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 99.

[0259] PB or PBL transposases may contain or consist of an amino acid sequence having two or more, three or more, or each of, amino acid substitutions at positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 99. The transposase may be an SPB transposase containing or consisting of the amino acid sequence of the sequence of SEQ ID NO: 99, where the amino acid substitution at position 30 may be a substitution of isoleucine (I) by valine (V), the amino acid substitution at position 165 may be a substitution of glycine (G) by serine (S), the amino acid substitution at position 282 may be a substitution of methionine (M) by valine (V), and the amino acid substitution at position 538 may be a substitution of asparagine (N) by lysine (K). In a preferred embodiment, the SPB transposase contains or comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 100.

[0260] In certain embodiments in which the transposase contains the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may further contain one or more amino acid substitutions at 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, which are described in more detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816.

[0261] PB, PBL, or SPB transposases can be isolated or derived from insects, vertebrates, crustaceans, or urochordates, as described in detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, PB, PBL, or SPB transposases are isolated or derived from the insect Trichoplusia ni (GenBank accession number AAA87375) or the silkworm Bombyx mori (GenBank accession number BAD11135).

[0262] Hyperactive PB or PBL transposases are transposases that are more active than the naturally occurring mutants from which they are derived. In preferred embodiments, hyperactive PB or PBL transposases are isolated or derived from silkworms or the tropical frog (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, PB or PBL transposases are integration-deficient. Integration-deficient PB or PBL transposases are transposases that can cleave the corresponding transposon but incorporate the cleaved 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, PB or PBL transposases are fused to nuclear localization signals. 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 transposons of this disclosure may be the “Sleeping Beauty” transposon. In some embodiments, when the transposon is the “Sleeping Beauty” transposon, the transposase is the “Sleeping Beauty” transposase (for example, disclosed in U.S. Patent No. 9,228,180) or the hyperactive “Sleeping Beauty” (SB100X) transposase. In a preferred embodiment, the “Sleeping Beauty” transposase contains or comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same amino acid sequence as SEQ ID NO: 101. In a preferred embodiment, the hyperactive "Sleeping Beauty" (SB100X) transposase contains or comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same amino acid sequence as SEQ ID NO: 102.

[0266] The transposons of this disclosure may be Helliser transposons. An exemplary Helliser transposon includes Helibat 1, which contains or comprises Nagareyama Central Park, which is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same amino acid sequence as SEQ ID NO: 103. In some embodiments, when the transposon is a Helliser transposon, the transposase is a Helitron transposase (disclosed, e.g., in WO2020 / 173636). In a preferred embodiment, the Helitron transposase contains or comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same amino acid sequence as SEQ ID NO: 104.

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

[0268] The transposons of this disclosure may be TcBuster transposons. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactivated TcBuster transposase (as disclosed, e.g., in WO2019 / 173636). The TcBuster transposase may contain or consist of a native amino acid sequence or an amino acid sequence that does not exist in nature. In a preferred embodiment, the TcBuster transposase may contain or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) the same as SEQ ID NO: 107. The polynucleotide encoding the TcBuster transposase may contain or consist of a native nucleic acid sequence or an amino acid sequence that does not exist in nature. In a preferred embodiment, the TcBuster transposase is encoded by a polynucleotide containing or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 108.

[0269] In some embodiments, mutant TcBuster transposases contain one or more sequence changes compared to wild-type TcBuster transposases, as described in detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816.

[0270] A transposon may be a nanotransposon. A nanotransposon comprises, or essentially consists of, (a) a sequence encoding a transposon insert, including a sequence encoding a first reverse-end repeat (ITR), a sequence encoding a second reverse-end repeat (ITR), and an intra-ITR sequence; (b) a sequence encoding a skeleton, the sequence encoding the skeleton including a sequence encoding a replication origin having 1 to 450 nucleotides (including an endpoint) and a sequence encoding a selection marker having 1 to 200 nucleotides (including an endpoint); and (c) an inter-ITR sequence. In some embodiments, the inter-ITR sequence of (c) includes the sequence of (b). In some embodiments, the inter-ITR sequence of (a) includes the sequence of (b).

[0271] The sequence encoding the skeleton may contain 1 to 600 nucleotides (including the endpoint). In some embodiments, the sequence encoding the skeleton 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, and 550 to 600 nucleotides (each including the endpoint).

[0272] The inter-ITR sequence may include 1 to 1000 nucleotides (including the endpoint). In some embodiments, the inter-ITR sequence may consist of 1 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, 600 to 650 nucleotides, 650 to 700 nucleotides, 700 to 750 nucleotides, 750 to 800 nucleotides, 800 to 850 nucleotides, 850 to 900 nucleotides, 900 to 950 nucleotides, or 950 to 1000 nucleotides (each range including the endpoint).

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

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

[0275] A sequence encoding a replication origin having 1 to 450 nucleotides (including the endpoint) may include a sequence encoding a mini replication origin. In some embodiments, a sequence encoding a replication origin having 1 to 450 nucleotides (including the endpoint) may include a sequence encoding an R6K replication origin. An R6K replication origin may include an R6K gamma replication origin. An R6K replication origin may include an R6K mini replication origin. An R6K replication origin may include an R6K gamma mini replication origin. An R6K gamma mini replication origin may contain or consist of 281 base pairs (bp).

[0276] In some embodiments of the nanotransposon, the sequence encoding the skeleton does not contain recombinant sites, excised sites, linking sites, or combinations thereof. In some embodiments, neither the nanotransposon nor the sequence encoding the skeleton contains products of recombinant sites, excised sites, linking sites, or combinations thereof. In some embodiments, neither the nanotransposon nor the sequence encoding the skeleton originates from recombinant sites, excised sites, linking sites, or combinations thereof.

[0277] In some embodiments of the nanotransposon, the recombination site includes a sequence resulting from a recombination event. In some embodiments, the recombination site includes a sequence that is a product of the recombination event. In some embodiments, the recombination event includes the activity of a recombinase (e.g., a recombinase site).

[0278] In some embodiments of nanotransposons, the sequence encoding the backbone further does not contain a sequence encoding foreign DNA.

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

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

[0281] Vector System

[0282] The vectors of this 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 (AAVs). Viral vectors may include recombinant AAVs (rAAVs). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include two or more reverse-terminal repeat (ITR) sequences located cis-adjacent to the sequence encoding the scFv or CAR of this 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 AAVs (scAAVs) and AAV hybrids containing the genome of one serotype and the 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 vectors of this disclosure may be nanoparticles. 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., polymerosomes), 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 transported passively or actively across the cell membrane.

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

[0285] Inducible apoptosis-promoting polypeptides

[0286] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing pro-apoptotic polypeptides because they have far lower immunogenicity. The pro-apoptotic polypeptides are recombinant polypeptides and therefore do not exist in nature. Furthermore, they can be recombined to produce pro-apoptotic polypeptides that do not contain non-human sequences that the host's human immune system can recognize as "non-self," thereby inducing an immune response in subjects administered with the pro-apoptotic polypeptide, cells containing the pro-apoptotic polypeptide, or a composition containing the pro-apoptotic polypeptide, or cells containing the pro-apoptotic polypeptide.

[0287] This disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and an apoptosis-promoting peptide, wherein the pro-apoptotic polypeptide does not contain a non-human sequence. In certain embodiments, the non-human sequence includes a restriction site. In certain embodiments, the ligand-binding region may 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 may be a terminally cleaved caspase 9 polypeptide. Inducible pro-apoptotic polypeptides may not exist naturally. When the caspase is caspase 9 or terminally cleaved caspase 9, the pro-apoptotic polypeptide may be called an "iC9 safety switch".

[0288] The inducible caspase polypeptide may comprise (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible apoptosis-promoting polypeptide does not contain a non-human sequence. In a particular embodiment, the inducible caspase polypeptide comprises (a) a ligand-binding region, (b) a linker, and (c) a terminally cleaved caspase 9 polypeptide, wherein the inducible apoptosis-promoting polypeptide does not contain a non-human sequence.

[0289] The ligand-binding region may contain the FK506-binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand-binding region containing the FK506-binding protein 12 (FKBP12) polypeptide may contain a modification at position 36 of the sequence. The modification may be a substitution of phenylalanine (F) with valine (V) at position 36 (F36V). The FKBP12 polypeptide contains, essentially consists of, or comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 109. The FKBP12 polypeptide contains, essentially consists of, or comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 110. It may be encoded by polynucleotides.

[0290] The linker region may consist of, essentially consist of, or be composed 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 may be encoded by a polynucleotide consisting of, or essentially consisting of, a nucleic acid sequence 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 a restriction site.

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

[0292] In a particular embodiment, the polypeptide comprises a terminally cleaved caspase-9 polypeptide, the inducible apoptosis-promoting polypeptide contains, essentially consists of, or comprises an amino acid sequence identical to, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of SEQ ID NO: 115, or the inducible apoptosis-promoting polypeptide is encoded by a polynucleotide containing, or comprising a nucleic acid sequence identical to, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of SEQ ID NO: 116.

[0293] Inducible pro-apoptotic polypeptides can be expressed intracellularly under transcriptional regulation of any promoter known in the art that can initiate and / or regulate the expression of the inducible pro-apoptotic polypeptide in the cell.

[0294] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, by chemically induced dimerization (CID) mediated by an inducer for producing a conditionally controlled protein or polypeptide. Pro-apoptotic polypeptides are not only inducible, but their induction is also reversible, by degradation of unstable dimerizing agents or administration of monomeric competitive inhibitors.

[0295] In a particular embodiment, if the ligand-binding domain comprises an FKBP12 polypeptide having a substitution of phenylalanine (F) with valine (V) at position 36 (F36V), the inducer is a 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)propyridene]] ester, [2S-[1(R * ), 2R * [S * [S * [1(R * ), 2R * ]]]]]-(9Cl)CAS Registry No.: 195514-63-7; Molecular formula: C78H98N4O20; Molecular weight: 1411.65); AP20187 (CAS Registry No.: 195514-80-8, and Molecular formula: C82H107N5O20), or an analog of AP20187, such as AP1510. The inducers AP20187, AP1903, and AP1510 used herein are interchangeable.

[0296] Inducible apoptosis-promoting 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] Formulation, dosage, and method of administration

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

[0299] The disclosed compositions and pharmaceutical compositions may further include, but are not limited to, at least one of any suitable auxiliary substances, such as diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, and other auxiliary substances. pharmaceutically acceptable auxiliary substances are preferred. Non-limited examples of such sterile solutions and methods for preparing them are well known in the art, for example, but are 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 that are well known in the art or suitable for the solubility and / or stability of the administration methods, protein scaffolds, fragments, or variant compositions 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., alditol, aldonic acid, esterified sugars, etc.; and polysaccharides or sugar polymers), which may exist alone or in combination, and may be present alone or in combination in amounts of 1 to 99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. 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, etc. One preferred amino acid is glycine.

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

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

[0303] Furthermore, the disclosed compositions may include polymeric excipients / additives, such as polyvinylpyrrolidone, Ficol (a high-molecular-weight sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "Tween 20" and "Tween 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0304] Many known and developed modes of administration can be used to administer therapeutically effective doses of the compositions disclosed herein or any disclosed pharmaceutical compositions. Non-limiting examples of modes of administration include bolus, intrabuccal, injection, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intraventricular, intracolon, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal cord, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intrabladder, oral, parenteral, intrarectal, sublingual, subcutaneous, percutaneous, or vaginal means.

[0305] The compositions of this disclosure may be used for parenteral (subcutaneous, intramuscular, or intravenous) administration or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly, but not limited to, a semi-solid form such as a cream or suppository; for oral or sublingual administration, but not limited to, a tablet or capsule; or nasally, for example, but not limited to, a powder, nasal spray, or aerosol, or in the form of a specific drug; or transdermally, for example, but not limited to, a gel, lotion, or suspension, or with a chemical enhancer such as dimethyl sulfoxide to modify the skin structure or increase the drug concentration in a transdermal patch (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, DS, Eds., pp. 59-90, (Marcel Dekker, Inc. New York) It can be prepared for use in patch delivery systems, together with (1994)) or with an oxidizing agent (WO98 / 53847) that enables the application of formulations containing proteins or peptides to the skin, or with the application of an electric field to create a temporary transport pathway, such as electroporation, or with iontophoresis to enhance the mobility of charged drugs through the skin, or with the application of ultrasound such as sonophoresis (U.S. Patents 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0306] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, particles, powder, or lyophilized powder in combination with a pharmaceutically acceptable parenteral vehicle, or provided separately. Formulations for parenteral administration may include, as common excipients, sterile water or saline solution, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogenated naphthalene, etc. Aqueous or oily suspensions for injection may be prepared by using appropriate emulsifiers or humectants and suspension agents according to known methods. Injectable agents may be non-toxic, non-oral diluents, such as aqueous solutions, sterile injection solutions, or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline solution, etc. Sterile non-volatile oils may be used as ordinary solvents or suspension agents. For these purposes, all kinds of non-volatile oils and fatty acids, such as natural, synthetic, or semi-synthetic fatty oils or fatty acids, natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides may be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, gas-operated needleless injectors such as those described in U.S. Patent No. 5,851,198, and laser puncture devices as described in U.S. Patent No. 5,839,446.

[0307] Formulations for oral administration rely on the co-administration of adjuvants to artificially increase intestinal permeability (e.g., resorcinol and nonionic surfactants, e.g., polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether), and the co-administration of enzymatic inhibitors to inhibit enzymatic degradation (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF) and transilol). Formulations for delivering hydrophilic agents containing proteins and protein scaffolds, as well as combinations of at least two surfactants for oral, buccal, mucosal, nasal, pulmonary, transvaginal, or rectal administration, are described in U.S. Patent No. 6,309,663. The active ingredient compounds in solid dosage forms for oral administration may be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants, such as magnesium stearate, parabens, preservatives, such as sorbic acid, ascorbic acid, α-tocopherol, antioxidants, such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and fragrances.

[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 may 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. Patent No. 4,239,754). More recently, microspheres (proteinoids), artificial polymers of mixed amino acids, have been used to deliver pharmaceuticals (U.S. Patent No. 4,925,673). Furthermore, carrier compounds used for orally delivering bioactive drugs are known in the art, as described in U.S. Patents No. 5,879,681 and 5,871,753.

[0309] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size effective for reaching the lungs or the lower airways of the sinuses. The compositions or pharmaceutical compositions can be delivered by any of the various inhalation or nasal devices known in the art for the administration of therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into the sinuses or alveoli of a patient, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices can use formulations suitable for administration for the distribution of the compositions or pharmaceutical compositions described herein in an aerosol. Such aerosols can consist of either a solution (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered-dose inhaler (MDI), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture containing liquefied compressed gas. The operation of the metering valve releases the mixture as an aerosol containing particles in a size range of preferably 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, formulation, and associated apparatus is disclosed in PCT publication number WO2019 / 049816.

[0310] For absorption via the mucosal surface, the composition comprises an emulsion containing a plurality of submicron particles, a mucosal-adhering polymer, a bioactive peptide, and an aqueous continuous phase that promotes absorption via the mucosal surface by achieving mucosal adhesion of the emulsion particles (U.S. Patent No. 5,514,670). Suitable mucosal surfaces for application of the emulsion formulations of this disclosure may include the cornea, conjunctiva, oral cavity, sublingual, nasal, vaginal, lung, stomach, intestine, and rectal administration routes. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycol, petrolatum, and cocoa butter. Formulations for intranasal administration are solid and may contain excipients such as lactose, or may be an aqueous or oily solution of the nasal spray. For oral administration, excipients may include sugars, calcium stearate, magnesium stearate, and pregelatinized starch (U.S. Patent No. 5,849,695). More detailed information regarding mucosal administration and formulations is disclosed in PCT publication number WO2019 / 049816.

[0311] For transdermal administration, the compositions disclosed herein or pharmaceutical compositions are encapsulated in delivery devices, such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as 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 the subject over a long period, for example, from a single dose to a period of one week to one year. Various sustained-release, depot, or implantable dosage forms can be used. For example, dosage forms may include pharmaceutically acceptable nontoxic salts of compounds with low solubility in body fluids, such as (a) acid addition salts with polybasic acids, such as phosphoric acid, sulfuric acid, citrate, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono or disulfonic acid, polygalacturonic acid, etc.; (b) salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or with organic cations formed from, for example, N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), such as zinc tannate salt. Furthermore, the disclosed compounds, or preferably relatively insoluble salts such as those described above, can be incorporated into a gel, for example, an aluminum monostearate gel 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 may contain a compound or salt dispersed in a slowly degrading, non-toxic, non-antigenic polymer, such as 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 incorporated into 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”, JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0313] Appropriate dosages are well known in the art. For example, see 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. Preferred doses may optionally include about 0.1 to 99 and / or 100 to 500 mg / kg / administered, or any range, value, or fraction thereof, or include serum concentrations of about 0.1 to 5000 μg / ml per single or multiple doses, or any range, value, or percentage thereof. Preferred dose ranges for the compositions disclosed herein or pharmaceutical compositions are about 1 mg / kg to a maximum of about 3, about 6, or about 12 mg / kg per kg of body weight of the subject.

[0314] Alternatively, the dose administered may vary depending on known factors, such as the pharmacodynamic properties of a particular drug and its mode and route of administration; the recipient's age, health condition, and weight; the nature and severity of 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. Usually, 0.1 to 50, preferably 0.1 to 10 mg per kilogram of body weight per single dose or in sustained-release form is effective in obtaining the desired result.

[0315] As a non-limiting example, treatment for humans or animals may be provided in a single or periodic dose of the compositions or pharmaceutical compositions disclosed herein in an amount of approximately 0.1 to 100 mg / kg per day or any range, value, or fraction thereof, for at least one day in 1 to 40 days, or alternatively or additionally for at least one week in 1 to 52 weeks, or alternatively or additionally for at least one year in 1 to 20 years, or any combination thereof, in a single injection or repeated doses.

[0316] Dosage forms suitable for internal administration generally contain approximately 0.001 mg to 500 mg of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is typically present in an amount of approximately 0.5 to 99.999% by weight based on the total weight of the composition.

[0317] When measured using known methods, such as those described herein or known in related technologies, an effective dose to achieve a serum concentration of 0.01 to 5000 μg / ml in a single, multiple, or sequential dose may include an amount of approximately 0.001 to approximately 500 mg / kg per single (e.g., bolus), multiple, or sequential dose.

[0318] In an embodiment in which the composition administered to a subject requiring it is modified cells as disclosed herein, about 1 × 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 × 10⁻⁶ 6 ~25×10 6 In one embodiment, cells can be administered in a quantity of approximately 5 × 10⁻⁶. 6 ~25×10 6 The cells are administered.

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

[0320] Method of using the composition disclosed herein

[0321] This disclosure provides the use of the disclosed compositions and pharmaceutical compositions for the treatment of diseases or disorders in cells, tissues, organs, animals, or subjects, for example, by administering or contacting a therapeutically effective amount of the compositions or pharmaceutical compositions to cells, tissues, organs, animals, or subjects, as known in the art or as described herein. In one embodiment, the subject is a mammal. Preferably, the subject is a human. The terms “subject” and “patient” are used synonymously.

[0322] This disclosure provides methods for modulating or treating at least one malignant disease or disorder in cells, tissues, organs, animals, or subjects. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, B cell, T cell, or FAB. This includes ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), pilosemic leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, cervical cancer, hereditary nonpolyposis cancer, Hodgkin lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, etc.

[0323] In a preferred embodiment, the treatment of a malignant disease or disorder includes adoptive cell therapy. For example, in one embodiment, the disclosure provides modified cells expressing a CAR comprising at least one disclosed antibody (e.g., scFv) and / or antibody (e.g., scFv), selected and / or expanded for administration to subjects in need. 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 direct administration to subjects from sterile packaging. The modified cells can be prepared in a pharmaceutically acceptable carrier having an indicator of cell viability and / or CAR expression level to ensure minimum levels of cell function and CAR expression. The modified cells can be prepared in a pharmaceutically acceptable carrier at a predetermined density using one or more reagents to inhibit further expansion and / or prevent cell death.

[0324] Any method may include administering an effective amount of any composition or pharmaceutical composition disclosed herein to cells, tissues, organs, animals, or subjects requiring such modification, treatment, or therapy. Such methods may optionally further include concurrent or combination therapy for the treatment of such disease or disorder, wherein the administration of any composition or pharmaceutical composition disclosed herein is performed before, concurrently with, and / or after at least one chemotherapeutic agent (e.g., alkylating agents, mitotic inhibitors, radiopharmaceuticals).

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

[0326] In some embodiments, the 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, which are manufactured simultaneously. In some embodiments where the composition is autologous cells or allogeneic cells, the dose is sufficient for the cells to engraft and / or lasts long enough to treat the disease or disorder.

[0327] In one example, the present disclosure provides a method for treating cancer in a subject requiring treatment, comprising administering to the subject a composition comprising an antibody (e.g., scFv) or a CAR comprising an antibody (e.g., scFv), wherein the antibody or CAR specifically binds to an antigen on tumor cells. In embodiments in which the composition comprises modified cells or a population of cells, the cells or population of cells may be autologous or allogeneic.

[0328] In some embodiments of the therapeutic methods described herein, the 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 within cells by contacting the cells with the inducer. The treatment can be modified or terminated in response to, for example, signs of recovery or a decrease in the severity / progression of the disease, signs of remission / cessation of the disease, and / or the occurrence of adverse events. In some embodiments, the method includes a step of administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (for example, if signs or symptoms of the disease recur, or if the severity increases and / or adverse events are resolved).

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

[0330] At least one antibody of this disclosure (e.g., a monoclonal antibody, a chimeric antibody, a single-domain antibody, VHH, VH, a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), or a Fab fragment) may be produced selectively by cell lines, mixed cell lines, immortalized cells, or clonal populations 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 scFv can be modified, added, and / or deleted, as is known in the art, to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, onate, offate, binding strength, specificity, half-life, stability, solubility, or other desirable characteristics.

[0332] Optionally, scFvs can be engineered to retain high affinity for antigens and other desirable biological properties. To achieve this goal, scaffold proteins can be optionally prepared by analytical processes of the parent sequence and various conceptual engineered products using three-dimensional models of the parent sequence and the engineered sequence. Three-dimensional models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display possible three-dimensional conformational structures of selected candidate sequences and measure possible immunogenicity (e.g., the Immunofilter program from Xencor, Inc. of Monrovia). Examination of these displays allows for analysis of the possible roles 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 can be selected and combined from the parent sequence and reference sequence, resulting in the achievement of desired properties such as affinity for the target antigen. Alternatively, in addition to the above procedure, other suitable engineering methods can be used.

[0333] Screening of scFv for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA representation) or peptide representation libraries, e.g., in vitro representation. This method involves screening a large collection of peptides for individual members having a desired function or structure. The represented nucleotide or peptide sequence may be nucleotides or amino acids of length 3 to 5000 or more, 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 representing peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a specific represented peptide sequence. Such methods are described in PCT patent publication numbers WO91 / 17271, WO91 / 18980, WO91 / 19818, and WO93 / 08278.

[0334] Other systems for creating peptide libraries include both in vitro chemical synthesis and recombinant methods. See PCT patent publications WO92 / 05258, WO92 / 14843, and WO96 / 19256. Also see U.S. patents 5,658,754 and 5,643,768. Peptide-represented libraries, vectors, and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, Calif.) and Cambridge Antibody Technologies (Cambridgeshire, UK). For example, 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, all transferred to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500, all transferred to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717, all transferred to Affymax; Cambridge Antibody See U.S. Patent No. 5,885,793, assigned to Technologies; U.S. Patent No. 5,750,373, assigned to Genentech; U.S. Patents No. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417, Colligan, cited above; Ausubel, cited above; Sambrook, cited above.

[0335] The scFvs of this disclosure can bind to human or other mammalian proteins with a wide range of affinity (KD). In a preferred embodiment, at least one scFv of this disclosure can be optionally bound to a target protein with high affinity, as measured by surface plasmon resonance or Kinexa, for example, as practiced by those skilled in the art, at about 10 -7 KD of M or less, for example, not limited to, but 0.1 to 9.9 (or any range or value within that range) × 10 -8 , 10-9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , or can be combined in any suitable range or value therein.

[0336] The affinity or binding force of the scFv to the antigen can be determined experimentally using any suitable method (e.g., Berzofsky, et al., “Antibody - Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and the methods described herein). The measured affinity of a particular scFv - antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Thus, the measurement of affinity and other antigen - binding parameters (e.g., KD, Kon, Kof) is preferably carried out using a standardized solution of the protein scaffold and antigen, and a standardized buffer such as the buffer described herein.

[0337] Competitive assays can be performed using the scFv of this disclosure to determine which proteins, antibodies, and other antagonists compete with the scFv of this disclosure for binding to target proteins 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. Proteins and / or antibodies are immobilized or insolubilized before and after competition, and samples bound to the target protein are isolated from unbound samples, for example, by decantation (where the protein / antibody is insolubilized beforehand) or centrifugation (where the protein / antibody is precipitated after the competitive reaction). Competitive binding may also be determined by whether the function of the scFv changes due to its binding to or lack thereof to the target protein, for example, whether the scFv molecule inhibits or enhances the enzymatic activity of a labeled substance, for example. ELISA and other functional assays can be used as are well known in the art.

[0338] nucleic acid molecule

[0339] The nucleic acid molecules of this disclosure encoding scFv may be in the form of RNA, e.g., mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by synthetic methods through cloning, or any combination thereof. The DNA may be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand, also known as a sense strand, or a non-coding strand, also known as an antisense strand.

[0340] The isolated nucleic acid molecules of this disclosure may optionally include nucleic acid molecules comprising an open reading frame (ORF) having one or more introns, e.g., at least one specific portion of at least one scFv; nucleic acid molecules comprising a coding sequence of a protein scaffold or loop region that binds to a target protein; and nucleic acid molecules comprising a substantially different nucleotide sequence from those described above, but which, due to the degeneracy of the genetic code, still encode a protein scaffold described herein and / or known in the art. Of course, the genetic code is well known in the art. Therefore, it would be commonplace for those skilled in the art to generate such degenerate nucleic acid variants encoding a particular scFv of this disclosure. See, for example, Ausubel, et al. above, and such nucleic acid variants are included in this disclosure.

[0341] As described herein, nucleic acid molecules of this disclosure, including nucleic acids encoding scFv, include, but are not limited to, sequences that themselves encode the amino acid sequence of an scFv fragment; a coding sequence for the entire protein scaffold or a portion thereof; a coding sequence for scFv, a fragment or a portion thereof; and additional sequences, for example, a coding sequence for at least one signal reader or fusion peptide, which may or may not include the aforementioned additional coding sequence, for example, at least one intron together with an additional non-coding sequence, including non-coding 5' and 3' sequences, for example, a transcribed untranslated sequence that plays 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 providing additional functional groups. Thus, a sequence encoding a protein scaffold can be fused to a marker sequence, for example, a sequence encoding a peptide that facilitates the purification of a fusion protein scaffold including a protein scaffold fragment or a portion thereof.

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

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

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

[0345] Optionally, the polynucleotide encodes at least a portion of the protein scaffold encoded by the polynucleotide described herein. The polynucleotide comprises nucleic acid sequences that can be used for selective hybridization to the polynucleotide encoding the protein scaffold of this disclosure. See, for example, Ausubel, above; Colligan, above. These are each incorporated herein in their entirety by reference.

[0346] Nucleic acid construction

[0347] The isolated nucleic acids of this disclosure can be prepared using (a) recombinant methods, (b) synthesis techniques, (c) purification techniques, and / or (d) a combination thereof, as is well known in the art.

[0348] Nucleic acids may conveniently contain nucleotide sequences in addition to the polynucleotides of the Disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of polynucleotides. Alternatively, a translatable sequence can be inserted to aid in the isolation of translated polynucleotides of the Disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the Disclosure. The nucleic acids of the Disclosure, excluding the coding sequence, are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides of the Disclosure.

[0349] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in the isolation of polynucleotides, or improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, for example, Ausubel, mentioned above; or Sambrook, mentioned above).

[0350] Recombination methods for constructing nucleic acids

[0351] The isolated nucleic acid compositions of this disclosure, e.g., RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some embodiments, desired sequences in cDNA or genomic DNA libraries are identified using oligonucleotide probes that selectively hybridize to the polynucleotides of this disclosure under strict conditions. The isolation of RNA, as well as the construction of cDNA and genomic libraries, are well known to those skilled in the art (see, for example, Ausubel, above; or Sambrook, above).

[0352] Nucleic acid screening and isolation methods

[0353] The polynucleotide sequence-based probes of this disclosure can be used to screen cDNA or genomic libraries. The 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 varying degrees of hybridization rigor can be used in assays, and that either the hybridization medium or the washing medium can be the rigor. As the hybridization conditions become more rigorous, a higher degree of complementarity is required between the probe and the target for double-strand formation to occur. The degree of rigor can be controlled by temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the rigor of hybridization can be conveniently altered by manipulating the concentration of formamide, for example, within the range of 0% to 50%, to change the polarity of the reactant solution. The degree of complementarity (sequence identity) required for detectable binding varies depending on the rigor of the hybridization medium and / or washing medium. The degree of complementarity will ideally be 100%, or 70-100%, or any range or value in between. However, it should be understood that slight changes in the sequence of the probe and primer can compensate for this by reducing the strictness of hybridization and / or washing media.

[0354] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure without excessive experimentation, based on the teachings and guidelines presented herein.

[0355] Known methods for DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (e.g., Mullis et al. U.S. Patents 4,683,195, 4,683,202, 4,800,159, and 4,965,188; Tabor et al. 4,795,699, and 4,921,794; Innis 5,142,033; Wilson et al. 5,122,464; Innis 5,091,310; Gyllensten et al. 5,066 This includes U.S. Patent No. 584; Gelfand et al. No. 4,889,818; Silver et al. No. 4,994,370; Biswas No. 4,766,067; Ringold No. 4,656,134), and RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (Maled et al., U.S. Patent No. 5,130,238, trade name NASBA), the entire contents of these references are incorporated herein by reference (see, for example, Ausubel, above; or Sambrook, above).

[0356] For example, polymerase chain reaction (PCR) technology can be used to amplify the sequences of the polynucleotides and related genes of this disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful for purposes such as cloning nucleic acid sequences encoding proteins to be expressed, preparing 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 technicians in in vitro amplification methods can be found in Berger, (previously), Sambrook, (previously), and Ausubel, (previously), as well as Mullis, et al., U.S. Patent 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). Furthermore, the yield of long PCR products can be improved by using, for example, the T4 gene 32 protein (Boehringer Mannheim).

[0357] Synthesis methods for constructing nucleic acids

[0358] The isolated nucleic acids of this disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., above). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with complementary sequences or by polymerization using DNA polymerase with a single strand as a template. Those skilled in the art will recognize that while the chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences can be more easily obtained by concatenating shorter sequences.

[0359] Recombinant expression cassette

[0360] The Disclosure further provides recombinant expression cassettes comprising the nucleic acids of the Disclosure. Recombinant expression cassettes can be constructed using nucleic acid sequences of the Disclosure, for example, cDNA or genomic sequences encoding a protein scaffold of the Disclosure, and introduced into at least one desired host cell. The recombinant expression cassette would typically contain polynucleotides of the Disclosure bound in a manner that can function with a transcription initiation regulatory sequence that directs the transcription of the polynucleotides in the host cell of interest. Expression of the nucleic acids of the Disclosure can be directed using both heterogeneous and non-heterogeneous (i.e., endogenous) promoters.

[0361] In some embodiments, isolated nucleic acids functioning as promoters, enhancers, or other elements can be introduced at appropriate locations (upstream, downstream, or within introns) of the non-heterogeneous forms of the polynucleotides of the Disclosure to upregulate or downregulate the expression of the polynucleotides of the Disclosure. For example, an endogenous promoter can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0362] Expression vectors and host cells

[0363] This disclosure also relates, as is well known in the art, to vectors containing isolated nucleic acid molecules of this disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one protein scaffold by recombinant technology. See, for example, Sambrook, et al., hereafter; and Ausubel, et al., hereafter (each of which is incorporated herein by reference in its entirety).

[0364] Polynucleotides can be selectively bound to vectors containing a selection marker for replication in a host. Plasmid vectors are generally introduced into precipitates, such as calcium phosphate precipitates, or in complex with charged lipids. 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 bound to an appropriate promoter in a functional manner. The expression construct will further include sites for transcription initiation and termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct preferably includes a leading translation initiation codon and a stop codon (e.g., UAA, UGA, or UAG) appropriately placed at the end of the mRNA being translated, with UAA and UAG preferred for expression in mammalian or eukaryotic cells.

[0366] The expression vector preferably, but optionally, includes at least one select marker. Such markers include, for example, but are not limited to, ampicillin, zeosin (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. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blasticidine (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, zeosin (Sh The vectors include the bla gene, puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culture in Escherichia coli and other bacteria or prokaryotic cells (the above patents are incorporated herein by reference in their entirety). Suitable media and conditions for the above host cells are known in the art. Suitable vectors will be immediately apparent to those skilled in the art. Introduction of the vector construct into host cells can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described, for example, in Sambrook, hereafter, Chapters 1-4 and 16-18; and Ausubel, hereafter, Chapters 1, 9, 13, 15, 16.

[0367] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for the isolation of cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure include a group of surface proteins, glycoproteins, or proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the compositions or methods of the present disclosure from cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, “designated cluster” or “classification determinant” proteins (often abbreviated as “CD”), e.g., CD19, CD271, CD34, CD22, CD20, CD33, CD52, truncated or full-length versions, 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. The selectable drug resistance markers of the present disclosure may 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 this disclosure can be expressed in a modified form, such as a fusion protein, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification, subsequent handling, and storage. Alternatively, peptide portions can be added to the protein scaffold of this disclosure to facilitate purification. Such regions can be removed before the final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in standard laboratory manuals, e.g., Sambrook, cited above, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, cited above, Chapters 16, 17, and 18.

[0370] Those skilled in the art are familiar with numerous expression systems available for expressing the nucleic acids encoding the proteins of the Disclosure. Alternatively, the nucleic acids of the Disclosure may be expressed in a host cell by being (operationally) turned on in a host cell containing endogenous DNA encoding the protein scaffold of the Disclosure. 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 entirety.

[0371] Examples of cell cultures useful for producing protein scaffolds, or specific parts or variants thereof, are bacterial, yeast, and mammalian cells known in the art. Mammalian cell lines are often in the form of a single layer of cells, but mammalian cell suspensions and bioreactors can also be used. Many suitable host cell lines capable of expressing undenatured 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, etc., which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include lymphoid cells such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a preferred embodiment, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0372] These cell expression vectors may include one or more of the following expression regulatory sequences, for example, replication origins; promoters (e.g., late or early SV40 promoter, CMV promoter (US Patent No. 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerin kinase) promoter, EF-1 alpha promoter (US Patent No. 5,266,491), or at least one human promoter); enhancer and / or processing information sites, for example, ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag poly-A addition site); and transcription terminator sequences. See, for example, Ausubel, et al., and Sambrook, et al., above. Other cells useful for producing the nucleic acids or proteins of this 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 commercially available sources.

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

[0374] scFv purification

[0375] scFv 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, for example, Colligan, *Current Protocols in Immunology*, or *Current Protocols in Protein Science*, John Wiley & Sons, NY, NY, (1997–2001), e.g., Chapters 1, 4, 6, 8, 9, 10 (each of which is incorporated herein in its entirety by reference).

[0376] The scFv of this disclosure includes purified products, products of chemosynthetic procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, Escherichia coli, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production method, the protein scaffolds of this disclosure may be glycosylated or deglycosylated. Such methods are described in many standard experimental manuals, e.g., Sambrook, hereafter, Sections 17.37–17.42; Ausubel, hereafter, Chapters 10, 12, 13, 16, 18 and 20; Colligan, Protein Science, hereafter, Chapters 12–14, all of which are incorporated herein by reference in their entirety.

[0377] Amino acid code

[0378] The amino acids constituting the protein scaffolds of this disclosure are often abbreviated. Amino acid designation is performed by specifying the amino acid by its single-letter code, its three-letter code, its name, or its 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). The protein scaffolds of this disclosure may include one or more amino acid substitutions, deletions, or additions, either naturally occurring or induced by mutation and / or human manipulation, as specified herein. Amino acids in the protein scaffolds of this 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, ibid., Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). In the latter procedure, a single alanine mutation is introduced into all residues in the molecule. Next, the resulting mutant molecules are tested for biological activity, including, but not limited to, at least one neutralizing activity. Sites crucial for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0379] As those skilled in the art will understand, this disclosure comprises at least one biologically active protein scaffold. The 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 an undenatured (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying enzyme activity and substrate specificity are well known to those skilled in the art.

[0380] In another embodiment, the disclosure relates to protein scaffolds and fragments described herein that are modified by covalent bonding of organic moieties. Such modifications can produce protein scaffold fragments having improved pharmacokinetic properties (e.g., extension of in vivo serum half-life). The organic moieties may be linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. In certain embodiments, the hydrophilic polymer groups may have a molecular weight of about 800 to about 120,000 daltons and may be polyalkane glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester groups may contain about 8 to about 40 carbon atoms.

[0381] The modified protein scaffolds and fragments of this disclosure may include one or more organic moieties directly or indirectly covalently bound to an antibody. Each organic moiety bound to a protein scaffold or fragment of this disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term “fatty acid” includes monocarboxylic acids 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. Therefore, a protein scaffold modified by covalent bonding of polylysine is included in this disclosure. Suitable hydrophilic polymers for modifying the protein scaffolds of this disclosure may be linear or branched and may 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 this 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 dalton units. The hydrophilic polymer groups can be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by using appropriate methods. For example, polymers containing amine groups can be bonded to carboxylates of fatty acids or fatty acid esters, and activated carboxylates on fatty acids or fatty acid esters (e.g., activated with N,N-carbonyldiimidazole) can be bonded to hydroxyl groups on the polymer.

[0382] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of this disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds of this 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, tetradecanediic acid, octadecanediic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

[0383] Modified protein scaffolds and fragments can be prepared by appropriate methods, such as reactions with one or more modifiers. As used herein, the term “modifier” refers to appropriate organic groups containing activating groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters). An “activating group” is a chemical moiety or functional group that, under appropriate conditions, reacts with a second chemical group to form a covalent bond between the modifier and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylates, mesylates, halo(chloro, bromo, fluoro, iodine), and N-hydroxysuccinimidyl esters (NHS). Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloryl, pyridyl disulfide, and 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol). Aldehyde functional groups can bond to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Appropriate 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 directly bonded to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or bonded via a linker moiety, such as a divalent C1-C12 group (where one or more carbon atoms can be substituted with 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-. Modifiers containing a linker moiety can be produced, 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 bonded to another carboxylate as described, or the product obtained by reaction with maleic anhydride can be cyclized to produce activated maleimide derivatives of fatty acids (see, for example, Thompson, et al., WO92 / 16221, all of which are incorporated herein by reference).

[0384] The modified protein scaffolds of this disclosure can be generated by reacting a protein scaffold or fragment with a modifier. For example, an organic moiety can be attached to the protein scaffold in a non-site-specific manner using an amine-reactive modifier, such as an NHS ester of PEG. Modified protein scaffolds and fragments containing organic moieties bound to specific sites of the protein scaffolds of this disclosure can be prepared using appropriate methods such as reverse proteolysis (as described in 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 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” refer to multiple subjects unless the context clearly indicates otherwise. Thus, for example, a reference to “method” includes multiple such methods, and a reference to “dosage” includes one or more doses and their equivalents known to those skilled in the art, and so on.

[0387] The terms “about” or “approximately” mean an acceptable range of error for a particular value, as determined by those skilled in the art, which in part depends on how the value is measured or determined, for example, the limits of the measuring system. For example, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably up to five times, and more preferably up to two times. When a particular value is stated in a patent application and claims, unless otherwise specified, the term “about” should be inferred to mean within an acceptable range of error for that particular value.

[0388] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. “Isolated” or “purified” polynucleotides or proteins, or their biologically active portions, substantially or essentially contain components that would normally accompany or interact with polynucleotides or proteins as they would in their natural environment. Therefore, isolated or purified polynucleotides or proteins, if produced by recombinant technology, substantially contain no other cell material or culture medium, or, if chemically synthesized, substantially contain no chemical precursors or other chemicals. Optimally, “isolated” polynucleotides do not contain sequences (optimally protein-coding sequences) that naturally adjacency to 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 originates. For example, in various embodiments, isolated polynucleotides may contain nucleotide sequences of approximately 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or less than 0.1kb that naturally adjacency to the polynucleotide in the genomic DNA of the cell from which the polynucleotide originates. Substantially cellular protein-free proteins include protein preparations containing approximately 30%, 20%, 10%, 5%, or less than 1% (by dry weight) of contaminating protein. When the proteins of this disclosure or their biologically active portions are produced by recombinant methods, the optimal culture medium contains approximately 30%, 20%, 10%, 5%, or less than 1% (by dry weight) of chemical precursors or unintended proteins.

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

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

[0391] The term “antibody” is used in its broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions having polyepitope specificity. The use of natural or synthetic analogs, variants, cultivars, alleles, homologs, and orthologues (collectively referred to as “analogs”) of the antibodies defined herein is also within the scope of this specification. Therefore, in one aspect of this specification, the term “antibody as defined herein” also encompasses such analogs in its broadest sense. 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, are defined as part of an undenatured antibody comprising an antigen-binding site or variable region of the undenatured antibody, wherein some parts do not include the constant heavy chain domains of the Fc region of the undenatured antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, (1) single-chain Fv(scFv) molecules, (2) single-chain polypeptides comprising only one light chain variable domain, or fragments thereof comprising three CDRs of a light chain variable domain that do not include the relevant heavy chain portion, and (3) single-chain polypeptides comprising only one heavy chain variable domain, or fragments thereof comprising three CDRs of a heavy chain variable domain that do not include the relevant light chain portion; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chains may include any constant domain sequence found in the non-Fc region of an undenatured antibody (e.g., CHI of an IgG isotype), and / or a hinge region sequence found in an undenatured antibody, and / or a leucine zipper sequence fused to or located within the hinge region sequence or the constant domain sequence of the heavy chain. The term further includes single-domain antibodies ("sdAB"), which generally refer to antibody fragments having a single monomeric variable antibody domain (e.g., camel-derived). Such fragment antibody types will be readily understood by those skilled in the art.

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

[0394] The term “contains” is intended to mean that compositions and methods include the listed elements but do not exclude others. “Essentially consisting of” as used to define compositions and methods means excluding other elements that, when used for their intended purpose, have any essential importance to the combination. Thus, compositions essentially consisting of the elements defined herein do not exclude trace amounts of contaminants or inert carriers. “Consists of” means excluding trace elements of other components or anything other than the substantial steps of the method. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0395] The term "epitope" refers to the antigenic determinant of a polypeptide. An epitope may contain three amino acids in a spatial conformation specific to the epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more generally, 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 polynucleotides are transcribed into mRNA, and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells.

[0397] "Gene expression" refers to the conversion of information contained in a gene into a gene product. A gene product can be a direct transcript 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 the translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0398] The "modulation" or "regulation" of gene expression refers to a change in gene activity. While not strictly limited, gene regulation may include gene activation and gene repression.

[0399] The terms "operatively linked" or their equivalent (e.g., "linked operatively") mean that two or more molecules are positioned relative to each other in such a way that they can interact and influence the function of one or both molecules or a combination thereof.

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

[0401] A method for directing a protein to a specific gene locus in the genome of an organism is disclosed. This method may include the steps of providing a DNA localization component and an effector molecule, where the DNA localization component and the effector molecule can be bound together in a manner that allows them to function via non-covalent ligation.

[0402] The term "scFv" refers to a single-chain variable fragment. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, linked by a linker peptide. The linker peptide can be approximately 5–40 amino acids long, or approximately 10–30 amino acids long, or approximately 5, 10, 15, 20, 25, 30, 35, or 40 amino acids long. Because single-chain variable fragments lack the constant Fc region found in complete antibody molecules, they lack the common binding site (e.g., protein G) used for antibody purification. The term further includes scFv intrabodies, which are antibodies that are stable within the cytoplasm of a cell and capable of binding to intracellular proteins.

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

[0404] As used herein, the terms “specifically binding” 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, the specific binding interaction distinguishes between desirable and undesirable antigens in a sample. In some embodiments, this can be approximately 10 to 100 times or more (e.g., approximately 1,000 times or 10,000 times or more). “Specificity” refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as an immunoglobulin or nanobody, to preferentially bind to a certain 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 the portion of the nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding are present.

[0406] The terms “nucleic acid,” “oligonucleotide,” or “polynucleotide” refer to at least two nucleotides that are covalently linked to each other. A single-stranded description also defines the sequence of a complementary strand. Thus, a nucleic acid may also encompass the complementary strand of the single-stranded sequence described. The nucleic acids of this disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0407] The probes of this disclosure may include single-stranded nucleic acids that can hybridize to a target sequence under strict hybridization conditions. Accordingly, the nucleic acids of this disclosure may refer to probes that hybridize under strict hybridization conditions.

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

[0409] The nucleic acids disclosed may have sequences, either as a whole or in any part thereof, that do not exist in nature. The nucleic acids disclosed may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, making the entire nucleic acid sequence non-natural. The nucleic acids disclosed may contain one or more replicated, inverted, or repeat sequences, the resulting sequences not existing in nature, making the entire nucleic acid sequence non-natural. The nucleic acids disclosed may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that do not exist in nature, making the entire nucleic acid sequence non-natural.

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

[0411] As used throughout this disclosure, the term “functionally bound” refers to the expression of a gene under the control of a promoter to which it is spatially bound. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it originates. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

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

[0413] As used throughout this disclosure, the term “substantially complementary” means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the 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 exact hybridization conditions.

[0414] As used throughout this disclosure, the term “substantially identical” means that the first sequence and the second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical across regions 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 first sequence is substantially complementary to the complement of the second sequence with respect to nucleic acids.

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

[0416] As used throughout this disclosure, the term “vector” refers to a nucleic acid sequence containing a replication origin. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may also be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may contain a combination of amino acids and a DNA sequence, an RNA sequence, or both DNA and RNA sequences.

[0417] As used throughout this disclosure, the term “variant” used to describe a peptide or polypeptide means a peptide or polypeptide having a different amino acid sequence due to an insertion, deletion, or conserved substitution of amino acids, but retaining at least one biological activity. A variant may also mean a protein having a substantially identical amino acid sequence 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 typically recognized in the art as involving small changes. These small changes can, in part, be identified by considering the hydroxyl index of amino acids, as understood in the art. (Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydroxyl index of amino acids is based on consideration of their hydrophobicity and charge. Amino acids with similar hydroxyl indices maintain protein function even when substituted. In one embodiment, amino acids with hydroxyl indices of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in proteins that retain biological function. Considering amino acid hydrophilicity in relation to peptides allows for the calculation of the peptide's maximum local mean hydrophilicity, which is a useful indicator reported to correlate well with antigenicity and immunogenicity. (U.S. Patent No. 4,554,101, incorporated herein by reference in its entirety).

[0419] Substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be carried out with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity of an amino acid are influenced by its specific side chain. Consistent with this observation, functionally compatible amino acid substitutions depend on the relative similarity of the amino acids, particularly their side chains, as evidenced by their 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 include conservative substitutions introduced by modifications of the polynucleotides encoding the polypeptides of this disclosure. Amino acids can be classified according to their physical properties and contributions to secondary and tertiary protein structures. A conservative substitution is the substitution of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are shown in Table A.

[0421] [Table 1]

[0422] Alternatively, conserved amino acids can be grouped as described in Table B, as in 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 this 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 polypeptides having modifications other than insertions, deletions, or substitutions of amino acid residues. The polypeptides or nucleic acids of this disclosure may contain one or more conservative substitutions.

[0427] As used throughout this disclosure, the term “two or more” of the aforementioned amino acid substitutions refers to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, four, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more of the listed amino acid substitutions. The term “two or more” may also refer to two, three, four, or five of the listed amino acid substitutions.

[0428] The polypeptides and proteins of this disclosure may have sequences, either entirely or in any part thereof, that do not exist in nature. The polypeptides and proteins of this disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, which may result in an entire amino acid sequence that does not exist in nature. The polypeptides and proteins of this disclosure may contain one or more replicated, inverted, or repeatable sequences, the resulting sequences that do not exist in nature, which may result in an entire amino acid sequence that does not exist in nature. The polypeptides and proteins of this disclosure may contain modified amino acids, artificial amino acids, or synthetic amino acids that do not exist in nature, which may result in an entire amino acid sequence that does not exist 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) that can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; this is incorporated herein by reference in its entirety). As used in the context of two or more nucleic acid or polypeptide sequences, the terms “identical” or “identical” refer to a specific percentage of residues that are identical across a particular region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences across a particular region, determining the number of positions where identical residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths, or if alignment generates one or more misaligned 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 sequencing algorithms such as BLAST or BLAST 2.0.

[0430] As used throughout this disclosure, the term “endogenous” 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] As used throughout this disclosure, the term “exogenous” means 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 native nucleic acids, such as multiple copies of a DNA sequence that do not exist naturally, or native nucleic acid sequences that exist at genomic locations that do not exist naturally.

[0432] This disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. “Introducing” means presenting the polynucleotide construct to the cell so that the construct can access the interior of the host cell. The method of this disclosure does not depend on any specific method for introducing the polynucleotide construct into a host cell, but only on the polynucleotide construct gaining access to the interior of a single host cell. 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 methods, transient transformation methods, and virus-mediated methods.

[0433] Examples

[0434] Example 1 - Construction of a Chimeric Stimulating Receptor (CSR)

[0435] Stimulation is enhanced by the expression of chimeric stimulatory receptors (CSRs) in the presence or absence of TCRs. When T cells are treated with a reagent displaying an agonist mAb in the presence of transiently or stably expressed surface-expressed CSRs / s, enhanced primary and secondary costimulatory signals are delivered. In one embodiment, this schematic diagram represents allogeneic cells. Since more complete T cell activation is achieved by CSR-mediated stimulatory signals, T cell activation and expansion are promoted.

[0436] Chimeric stimulating receptors (CSRs) were designed to include an antigen-recognition region containing the extracellular domain of CD2. A panel of CSR variants was designed within the extracellular domain of CD2. The aim of this panel was to identify variants 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) CSRs, potentially interfering with the optimal performance of CSRs; and 2) because WT CSRs may function as a natural ligand CAR, T cells expressing CSRs 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 to activated anti-CD2 reagents for optimal cell expansion are desirable.

[0437] The D111H mutation in the extracellular domain of CD2 ("CD2ECD(D111H)") retains the ability to bind to an activated CD2 reagent for optimal cell expansion and does not interact with CD58. Schematic diagrams of the CSRs of this disclosure are shown in Figures 1 and 2. Figure 1 shows a schematic diagram of a CSR with a CD2 signal peptide. Figure 2 shows a schematic diagram of a CSR with a CD8 signal peptide. These CSRs can be used to enhance the production of allogeneic or autologous CAR-T cells. The CSR CD2z-D111H variant can be delivered to allogeneic or autologous CAR-T cells during production to enhance cell proliferation and expansion, quality, viability, phenotype, function, subset composition, gene editing efficiency, etc. These variant CSRs can be delivered transiently encoded in mRNA or stably encoded in transposons.

[0438] Example 2 - Determination of the functional characteristics of chimeric stimulating 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 to knock out TCR and MHCI (double knockout; DKO) using at least the transposon encoding CAR and the selected gene, mRNA encoding CSR, mRNA encoding the super PiggyBac® transposase enzyme, mRNA encoding Cas-CLOVER®, and multi-guide RNA (gRNA) targeting TCRb and b2M. Subsequently, cells were stimulated with the agonist mAbs anti-CD2, anti-CD3, and anti-CD28, and then selected for genetic modification over a 14-day culture period. At the end of the first culture period, all T cells expressed CAR, indicating successful selection of genetically modified cells. Compared to cells that did not express CSR (no booster), cells expressing CSR showed a larger volume of DKO cells (LLC) (Figure 3). Therefore, CSR expression enhances the expansion of CAR-T cells during production.

[0440] The effect of CSR expression on the memory phenotype of DKO CAR-T cells was investigated. 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 cells for DKO CAR-T cells with and without CSR are shown in Tables 1 and 2. Regardless of the presence or absence of CSR, DKO CAR-T cells are mainly composed of very high levels of favorable Tscm and Tcm cells. Therefore, the memory phenotype of DKO CAR-T cells is not significantly affected by co-expression of CSR.

[0441]

Table 4

[0442]

Table 5

[0443] Example 3 - In vivo effect of CAR-T cells expressing chimeric receptor (CSR)

[0444] To test the effect of allogeneic CAR-T cells expressing different CSRs described in Example 2 on the anti-tumor effect, an in vivo experiment was performed using a mouse xenograft model of multiple myeloma. The outline of the experimental procedure is shown in Figure 4, and 10 different allogeneic CAR-T cells were produced using 10 different CSRs. Specifically, the RPMI-8226 cell line was subcutaneously injected into female NSG mice at a dose of 1×10 7 cells (-7 days), and then on day 0 when the tumor was established (75-125 mm by caliper measurement 3 [target average about 100 mm 3 ), the allo CAR-T cells were treated by intravenous (IV) injection at a "stress" dose (5×10 6 ). The "stress" dose was used to obtain a higher resolution when detecting possible functional differences in efficacy between CAR-T cells produced using 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 this disclosure resulted in a reduction in tumor volume compared to PBS controls (Figure 5). Tumor size at 56 days after treatment with allogeneic CAR-T cells expressing CSRs was comparable to that of positive controls. Total T cells in the blood were quantified after treatment with allogeneic CAR-T cells expressing CSRs (Figure 6). Peak levels of T cells in the blood were also quantified after treatment with CAR-T cells expressing CSRs and are shown as the group mean for all animals (Figure 7). All animals showed an increase in peak T cell levels compared to PBS-negative controls. CAR-T cells expressing CD2.DH28z, CD2.DH.BBz, CD2.DH.15z, CD2.DH.Oxz, and CD2.DH.Gz showed an increase in peak T cell levels in the blood compared to CD2.DH.CD2z (also known as CD2.DH.z) controls. 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 an increase in peak levels of serum T cells compared to the CD2.DH.CD2z (also known as CD2.DH.z) control. The effect on the ratios of Teff, Tem, Tcm, and Tscm cells after treatment with each allogeneic CAR-T cell expressing various CSRs was comparable to treatment with the positive control (CAR-T cells expressing CD2.DH.z CSR).

[0446] [Table 6]

Claims

1. (a) an external domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD2 signal peptide or a CD8α signal peptide, and the activating component comprises the CD2 extracellular domain or a portion thereof to which the agonist binds; (b) A transmembrane domain including the CD2 transmembrane domain or a portion thereof; and (c) An internal domain comprising a cytoplasmic domain and a signal transduction 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 signal transduction domain comprises a CD3ζ protein or a part thereof, the internal domain and A non-natural chimeric stimulating receptor (CSR) that includes, The receptor wherein the signal peptide and the cytoplasmic domain do not originate from the same protein.

2. The CSR according to claim 1, wherein the signal peptide comprises a CD2 signal peptide.

3. The CSR according to claim 1, wherein the CD2 signal peptide comprises the amino acid sequence of SEQ ID NO:

5.

4. The CSR according to claim 1, wherein the signal peptide comprises the CD8α signal peptide.

5. The CSR according to claim 1, wherein the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO:

7.

6. The CSR according to any one of claims 1 to 5, wherein the activating component includes modification.

7. The CSR according to any one of claims 1 to 6, wherein the modification includes a mutation or terminal cleavage of the amino acid sequence of the CD2 extracellular domain or a part thereof to which the agonist binds, compared to the wild-type sequence of the CD2 extracellular domain or a part thereof.

8. The CSR according to claim 7, wherein the CSR, which includes a mutation or terminal break in the extracellular domain of CD2 or a part thereof to which the agonist binds, does not bind to CD58.

9. The CSR according to claim 7, wherein the extracellular domain of CD2 including the mutation or terminal cleavage, or a portion thereof, includes the amino acid sequence of SEQ ID NO:

3.

10. The CSR according to any one of claims 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 claims 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 claims 1 to 10, wherein the CD28 intracellular domain comprises the amino acid sequence of Sequence ID No.

15.

13. The CSR according to any one of claims 1 to 10, wherein the 4-1BB intracellular domain comprises the amino acid sequence of Sequence ID No.

17.

14. The CSR according to any one of claims 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 claims 1 to 10, wherein the intracellular domain of IL15RA includes the amino acid sequence of SEQ ID NO:

21.

16. The CSR according to any one of claims 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 claims 1 to 10, wherein the ICOS intracellular domain comprises the amino acid sequence of SEQ ID NO:

25.

18. The CSR according to any one of claims 1 to 10, wherein the intracellular domain of CD27 comprises the amino acid sequence of SEQ ID NO:

27.

19. The CSR according to any one of claims 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 claims 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 claims 1 to 20, wherein the signal transduction domain, which includes the CD3ζ protein or a portion thereof, includes the amino acid sequence of SEQ ID NO:

11.

22. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

39.

23. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

43.

24. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

47.

25. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

51.

26. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

55.

27. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

59.

28. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

63.

29. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

67.

30. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

71.

31. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

37.

32. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

41.

33. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

45.

34. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

49.

35. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

53.

36. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

57.

37. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

61.

38. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

65.

39. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

69.

40. The CSR according to any one of claims 1 to 21, wherein the CSR comprises the amino acid sequence of SEQ ID NO:

73.

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

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