Ror-1 specific chimeric antigen receptors and uses thereof

Abstract

Provided herein are chimeric antigen receptors (CARs) for use in cancer therapy, more specifically, CARs comprising scFv from anti-ROR-1 monoclonal antibodies. Provided are immune effector cells comprising such CARs, and methods of treating proliferative disorders.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 696,075, filed July 10, 2018, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 8, 2019, is named 50471_716_601_SL.txt and is 360,082 bytes in size. Background Technology

[0005] Recombinant peptides, such as chimeric peptides, are valuable for research, diagnostic, manufacturing, and therapeutic applications. Modified effector cells expressing antigen-binding peptides, such as CARs, can be used to treat diseases and conditions, such as infectious diseases, autoimmune diseases, and cancer.

[0006] Incorporation

[0007] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Summary of the Invention

[0008] In some embodiments, this document discloses isolated nucleic acids, vectors, immune effector cells, methods, and systems comprising chimeric antigen receptors (CARs).

[0009] This article provides an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0010] In some embodiments, the CAR comprises at least one of the following: (a) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO: 3-14; or (b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO: 75-82. In some cases, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO: 15-74. In other cases, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73. In some instances, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74. In some embodiments, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 83. In other embodiments, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:84. In some cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:85.In some instances, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:86. In other embodiments, the transmembrane domain is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO:87-88.

[0011] In some cases, the co-stimulatory signaling domain comprises 4-1BB. In other embodiments, the co-stimulatory signaling domain of 4-1BB comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:89. In other cases, the co-stimulatory signaling domain comprises CD28. In some instances, the co-stimulatory signaling domain of CD28 comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:90. In some embodiments, the CD3ζ signaling domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:93. In other embodiments, the isolated nucleic acid further comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of SEQ ID NO:94-108.

[0012] In some instances, the isolated nucleic acid further comprises a cell tag. In some embodiments, the cell tag is a truncated epidermal growth factor receptor. In some cases, the truncated epidermal growth factor receptor is HER1t and comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:109. In other embodiments, the truncated epidermal growth factor receptor is HER1t-1 and comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:110. In some embodiments, the cell tag comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:111. In other cases, the cell tag comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:112. In some instances, the isolated nucleic acid further comprises additional polynucleotides encoding ligand-inducible gene-switching polypeptides for heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker.

[0013] This article provides a vector comprising a backbone and a nucleic acid sequence encoding the following: (1) a cell tag; (2) a cytokine; and (3) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0014] In some embodiments, the vector is a lentiviral vector, a retroviral vector, or a non-viral vector. In some cases, the cell tag is a truncated epidermal growth factor receptor (EGFR). In some embodiments, the truncated EGFR is HER1t, HER1t-1, or a functional variant thereof. In some instances, the truncated EGFR comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:109 or SEQ ID NO:110. In other instances, the cell tag comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:111. In some instances, the cell tag contains a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:112.

[0015] In some embodiments, the cytokine is IL-15. In some embodiments, the IL-15 is membrane-bound IL-15. In some embodiments, the membrane-bound IL-15 comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:113. In other embodiments, the vector further comprises a nucleotide sequence encoding a self-cleaving peptide of the Tsea asigna virus (T2A). In some embodiments, the backbone is a Sleeping Beauty transposon DNA plasmid. In some instances, the vector further comprises a promoter. In some embodiments, the promoter is hEF1a1. In other embodiments, the CAR comprises at least one of the following: (a) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO:3-14; or (b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO:75-82.

[0016] In some embodiments, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO:15-74. In some examples, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO:15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73. In other instances, the ROR-1 antigen-binding domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with at least one of the amino acid sequences shown in SEQ ID NO:16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74. In some cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:83. In other cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:84.

[0017] In some cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:85. In other cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:86. In some embodiments, the transmembrane domain is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO:87-88.

[0018] In some instances, the co-stimulatory signaling domain comprises 4-1BB. In some embodiments, the co-stimulatory signaling domain of 4-1BB comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:89. In other embodiments, the CD3ζ signaling domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:93. In some cases, the vector further comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of SEQ ID NO:94-108.

[0019] In some cases, the vector comprises a plasmid. In some embodiments, each vector comprises an expression plasmid. In some embodiments, the nonviral vector is a Sleeping Beauty transposon. In some instances, the vector further comprises an additional vector containing a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker.

[0020] This article provides an immune effector cell comprising isolated nucleic acids. In some embodiments, the immune effector cell comprises the vector.

[0021] This article further provides an immune effector cell comprising (1) a cell tag; (2) cytokines; and (3) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0022] In some embodiments, the cytokine is IL-15. In some embodiments, the IL-15 is membrane-bound IL-15. In some cases, the membrane-bound IL-15 comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:113. In other cases, the cell tag is a truncated epidermal growth factor receptor. In some embodiments, the truncated epidermal growth factor receptor is HER1t, HER1t-1, or a functional variant thereof. In some cases, the truncated epidermal growth factor receptor comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:109 or SEQ ID NO:110. In some instances, the cell tag comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:111. In other embodiments, the cell tag comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:112.

[0023] In some embodiments, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:83. In other embodiments, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:84. In some cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:85. In other cases, the spacer region is a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:86.

[0024] In some instances, the cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells. In other instances, the CAR comprises at least one polypeptide having the amino acid sequence shown in SEQ ID NO: 3-14 or 75-82. In some cases, the CAR comprises at least one polypeptide having the amino acid sequence shown in SEQ ID NO: 15-74. In other cases, the immune effector cell further comprises a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker.

[0025] This article provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in human subjects in need, comprising administering to the human subject an effective amount of genetically modified cells expressing a CAR, wherein the CAR comprises: (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; (e) a CD3ζ signaling domain; and (f) a truncated epidermal growth factor receptor (HER1t).

[0026] In some implementations, the person has been diagnosed with at least one of lung cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, adrenal cancer, melanoma, uterine cancer, testicular cancer, or bladder cancer. In some cases, the person has been diagnosed with non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0027] This document provides an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a ROR-1 antigen-binding domain having at least one of the amino acid sequences shown in SEQ ID NO: 3-14, 75-82, or 15-74; (b) a spacer region having at least one of the amino acid sequences shown in SEQ ID NO: 83-86; (c) a co-stimulatory signal transduction domain comprising a CD28 having the amino acid sequence shown in SEQ ID NO: 90; (d) a HER1 tag comprising at least one of HER1t having the amino acid sequence shown in SEQ ID NO: 109 and HER1t-1 having the amino acid sequence shown in SEQ ID NO: 110; and (e) a CD3ζ signal transduction domain having the amino acid sequence shown in SEQ ID NO: 93.

[0028] This document provides an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a ROR-1 antigen-binding domain having at least one of the amino acid sequences shown in SEQ ID NO: 3-14, 85-82, or 15-74; (b) a spacer region having at least one of the amino acid sequences shown in SEQ ID NO: 83-86; (c) a co-stimulatory signal transduction domain comprising a 4-1BB having the amino acid sequence shown in SEQ ID NO: 89; (d) a HER1 tag comprising at least one of HER1t having the amino acid sequence shown in SEQ ID NO: 109 and HER1t-1 having the amino acid sequence shown in SEQ ID NO: 110; and (e) a CD3ζ signal transduction domain having the amino acid sequence shown in SEQ ID NO: 93.

[0029] In some instances, the vector comprises any one or more of the isolated polynucleotides. In some embodiments, the vector is a lentiviral vector, a retroviral vector, or a nonviral vector. In some embodiments, the nonviral vector is a Sleeping Beauty transposon. In other instances, the vector is multiple vectors. In some embodiments, the vector further comprises an additional vector containing a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker.

[0030] This document provides a system for expressing a CAR in immune effector cells, the system comprising a vector encoding one or more isolated nucleic acids. In some instances, the immune effector cells are T cells or NK cells. In some embodiments, the system further comprises a nucleic acid encoding at least one additional gene. In some embodiments, the additional gene comprises a cytokine. In some cases, the cytokine comprises at least one of IL-2, IL-15, IL-12, IL-21, and a fusion of IL-15 and IL-15Rα. In other embodiments, the cytokine is in a secreted form. In some embodiments, the cytokine is in a membrane-bound form. In some embodiments, the system comprises at least one vector. In other cases, the at least one vector is a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the non-viral vector is a Sleeping Beauty transposon. In some embodiments, the system further comprises a Sleeping Beauty transposon. In some instances, the Sleeping Beauty transposon is SB11, SB100X, or SB110. In other instances, the system further comprises an additional vector containing a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker. In some embodiments, the immune effector cells are mammalian cells.

[0031] This article provides a system for expressing a CAR in immune effector cells, the system comprising (1) a first vector comprising a backbone and a nucleic acid sequence encoding: (a) a cell tag; (b) a cytokine; and (c) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a ROR-1 antigen-binding domain; (ii) a spacer region; (iii) a transmembrane domain; (iv) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (v) a CD3ζ signaling domain; and (2) a second vector comprising a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain; and (b) a second gene-switching polypeptide comprising a transactivation domain fused to a second nuclear receptor ligand-binding domain; wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker.

[0032] This article provides a method for expressing CAR in immune effector cells, which includes contacting the immune effector cells with a system.

[0033] This article provides a method for stimulating the proliferation and / or survival of engineered T cells, comprising: (a) obtaining a cell sample from a subject, the sample containing T cells or T cell progenitor cells; (b) transfecting the cells with one or more vectors encoding isolated nucleic acids provided in any one of claims 1-22 and 79-80 and vectors encoding transposases to provide an engineered population of T cells expressing ROR-1 CAR; and (c) optionally, culturing the ROR-1 CAR T cell population in vitro for 2 days or less.

[0034] In some embodiments, the vector further encodes cytokines and cell tags. In some cases, the cytokines are fusion proteins comprising IL-15 and IL-15Rα. In other embodiments, the method further includes an additional vector comprising a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker.

[0035] In some embodiments, the DNA-binding domain comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. In other embodiments, the DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO:134. In some embodiments, the transactivation domain comprises at least one of the VP16 transactivation domain and the B42 acid activator transactivation domain. In other cases, the transactivation domain comprises the amino acid sequence shown in SEQ ID NO:131. In some embodiments, at least one of the first nuclear receptor ligand binding domain and the second nuclear receptor ligand binding domain comprises at least one of the following: ecdysone receptor (EcR), ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinol X receptor interactor-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor, liver X receptor α, farnesol X receptor, receptor interactor-14, and famesol receptor.

[0036] In some cases, at least one of the first and second nuclear receptor ligand-binding domains comprises at least one of the amino acid sequences shown in SEQ ID NO:135-136. In some examples, the first gene switch polypeptide comprises a GAL4 DBD fused to the EcR nuclear receptor ligand-binding domain, and the second gene switch polypeptide comprises a VP16 transactivation domain fused to the retinoid receptor X (RXR) nuclear receptor ligand-binding domain. In some embodiments, the Gal4 DBD fused to the EcR nuclear receptor ligand-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:137-138, and the VP16 transactivation domain fused to the retinoid receptor X (RXR) nuclear receptor ligand-binding domain comprises the amino acid sequence shown in SEQ ID NO:133.

[0037] In some embodiments, the linker is a cleavable linker, a ribosomal skipping linker sequence, or an IRES linker. In some instances, the linker is an IRES linker, and the IRES linker has a sequence shown in any of SEQ ID NO: 146-147. In other embodiments, the linker is a cleavable linker or a ribosomal skipping linker sequence. In other cases, the cleavable linker or ribosomal skipping linker sequence comprises one or more of the following: 2A linker, p2A linker, T2A linker, F2A linker, E2A linker, GSG-2A linker, GSG linker (SEQ ID NO: 129), SGSG linker (SEQ ID NO: 130), furinlink linker variants, and derivatives thereof. In some embodiments, the cleavable linker or ribosomal skipping linker sequence has a sequence shown in any of SEQ ID NO: 116-126. In some embodiments, the one or more vectors and at least one of the additional vectors are lentiviral vectors, retroviral vectors, or nonviral vectors. In some cases, the non-viral vector is the Sleeping Beauty transposon. In some embodiments, the method further includes a Sleeping Beauty transpoase. In some embodiments, the Sleeping Beauty transpoase is SB11, SB100X, or SB110.

[0038] This article provides a method for culturing engineered T cells, comprising: (a) obtaining a cell sample from a subject, the sample containing T cells or T cell progenitor cells; (b) transfecting the cells with one or more vectors encoding isolated nucleic acids provided in any one of claims 1-22 and 79-80 and vectors encoding transposases to provide a population of engineered T cells expressing ROR1 CAR; and (c) culturing the population of engineered T cells expressing ROR1 CAR in vitro in a medium that selectively enhances the proliferation of the engineered T cells expressing ROR1 CAR, wherein the engineered T cells expressing ROR1 CAR are cultured for no more than 21 days.

[0039] In some instances, the engineered T cells expressing the ROR1 CAR are cultured for no more than 14 days. In some cases, the one or more vectors further encode cytokines and cell tags. In some embodiments, the method further includes an additional vector comprising a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of heterologous gene expression, wherein the gene-switching polypeptide comprises: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker. In other embodiments, the DNA-binding domain comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. In some embodiments, the DNA-binding domain comprises the amino acid sequence shown in SEQ ID NO:134.

[0040] In other embodiments, the transactivation domain comprises at least one of a VP16 transactivation domain and a B42 acid activator transactivation domain. In some embodiments, the transactivation domain comprises the amino acid sequence shown in SEQ ID NO:131. In some cases, at least one of the first nuclear receptor ligand-binding domain and the second nuclear receptor ligand-binding domain comprises at least one of the following: ecdysone receptor (EcR), ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinoid X receptor-interacting protein-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor, liver X receptor α, farnesol X receptor, receptor-interacting protein 14, and famesol receptor. In other cases, at least one of the first nuclear receptor ligand-binding domain and the second nuclear receptor ligand-binding domain comprises at least one of the amino acid sequences shown in SEQ ID NO:135-136. In some embodiments, the first gene-switching polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand-binding domain, and the second gene-switching polypeptide comprises a VP16 trans-activation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand-binding domain. In some examples, the Gal4 DBD fused to the EcR nuclear receptor ligand-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:137-138, and the VP16 trans-activation domain fused to the retinoid receptor X (RXR) nuclear receptor ligand-binding domain comprises the amino acid sequence shown in SEQ ID NO:133.

[0041] In some embodiments, the linker is a cleavable linker, a ribosomal skipping linker sequence, or an IRES linker. In other embodiments, the linker is an IRES linker, and the IRES linker has a sequence shown in any of SEQ ID NO: 146-147. In some embodiments, the linker is a cleavable linker or a ribosomal skipping linker sequence. In some instances, the cleavable linker or ribosomal skipping linker sequence comprises one or more of the following: 2A linker, p2A linker, T2A linker, F2A linker, E2A linker, GSG-2A linker, GSG linker (SEQ ID NO: 129), SGSG linker (SEQ ID NO: 130), furinlink linker variants, and derivatives thereof. In other instances, the cleavable linker or ribosomal skipping linker sequence has a sequence shown in any of SEQ ID NO: 116-126. In some embodiments, at least one of the one or more vectors, the vector, and the additional vectors is a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the non-viral vector is the Sleeping Beauty transposon. In other embodiments, the method further includes the Sleeping Beauty transposase. In some cases, the Sleeping Beauty transpoase is SB11, SB100X, or SB110. In some embodiments, (c) culturing engineered T cells expressing ROR1 CAR includes culturing engineered T cells expressing ROR1 CAR in the presence of artificial antigen-presenting cells (aAPCs) that stimulate the expansion of engineered T cells expressing ROR1 CAR. In other cases, the aAPC is engineered K562 cells. In some embodiments, the aAPC contains (i) the ROR1 antigen expressed on engineered CAR cells; (ii) CD64; (iii) CD86; (iv) CD137L; and / or (v) membrane-bound IL-15 expressed on the surface of the aAPC.

[0042] This article provides a method for treating cancer in a subject in need, comprising administering one or more doses of an effective amount of engineered T cells to the subject, wherein the engineered T cells comprise ROR-1 CAR and membrane-bound IL-15.

[0043] In some embodiments, the first dose of an effective amount of engineered T cells is administered intraperitoneally. In other embodiments, the second dose of an effective amount of engineered T cells is administered intravenously. In some embodiments, the cancer is non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML). In some cases, the cancer is lung cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, adrenal cancer, melanoma, uterine cancer, testicular cancer, or bladder cancer. In some instances, the ROR-1 CAR is encoded by any of the sequences shown in SEQ ID NO:3-14, 75-82, or 15-74. In some embodiments, the membrane-bound IL-15 is encoded by SEQ ID NO:260. In some embodiments, the effective amount of engineered T cells is at least 10 2 Cells / kg. In other embodiments, the effective quantity of engineered T cells is at least 10. 4 Cells / kg. In some cases, the effective quantity of engineered T cells is at least 10. 5 Cells / kg Attached Figure Description

[0044] The features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be gained by referring to the following detailed description, along with the accompanying drawings, which illustrate embodiments utilizing the principles of this disclosure, in which:

[0045] Figure 1A-1E Exemplary gene expression cassettes depicting different conformations of ROR1 CAR and cytokines are described. In some cases, ROR1 CAR and / or cytokines can be co-expressed with cells or killer tags for conditional in vivo ablation. Exemplary gene expression cassettes or combinations thereof for constitutive expression of ROR1 CAR and mbIL-15 may include c+e or d+e.

[0046] Figures 2A-2E Exemplary gene expression cassettes depicting different configurations of ROR1 CARs and cytokines with gene switch components are described. Exemplary gene expression cassettes or combinations thereof for inducible expression may include a+e, b+e, c+e, or d+e.

[0047] Figure 3 Exemplary ROR1 CAR designs are depicted. ROR1 CARs based on standard CAR designs did not show any expression of the ROR1 CAR after gene transfer. Functional ROR1 CARs with surface expression were developed using various optimization strategies described in Example 1.

[0048] Figure 4 A diagram depicting ROR1 CARs with intervals of different lengths is shown.

[0049] Figure 5 Combinations of signal peptides tested in ROR1 CAR (mouse scFv) constructs utilizing conventional CD8α stems were depicted, and expression of ROR1 CAR (mouse scFv) with different signal peptides was shown. Transgene expression was measured on day 1 post-nuclear transfection. Cells were gated as live CD3+ cells. Short CD8α (conventional) stalks were not permitted to express the signal peptides on the surface regardless of whether they were used (human or mouse).

[0050] Figure 6 Expression of ROR1 CARs (mouse scFv) with different combinations of signal peptides and different spacer regions and spacer lengths was demonstrated. ROR1 CARs with strong surface expression utilizing three different longer spacer regions were identified.

[0051] Figure 7 The expression of ROR1 CARs (mouse scFv) with different signal peptides and CD8α 3X stems was shown. For certain signal peptides (mIgGVH, β2M, and azuril), the expression of ROR1 CARs based on mouse scFv with 3X CD8α stems was improved.

[0052] Figure 8 The study shows the expression of various ROR 1 CARs (mouse scFv) with different combinations of stem, intracellular domain, and VH / VL chain orientations, as well as the expression of ROR 1 CARs (mouse scFv) with different combinations of stem, intracellular domain, and VH / VL chain orientations. As observed, reversing the orientation from VL-VH to VH-VL enabled surface expression of the hGMCSFR and hIGHV3-23 signal peptides.

[0053] Figure 9A and Figure 9B The specificity of ROR1 (mouse scFv)-CD8-3x stem.CD28z CAR-T cells in various tumor cell lines was demonstrated, as measured in the CD107a degranulation assay and IFN-α expression assay.

[0054] Figure 10AThis paper presents a quantitative analysis of JeKo-1 tumor burden as measured by in vivo bioluminescence imaging (IVIS) on day 7 post-CAR-T therapy. On day 0, JeKo tumor cell lines were administered to NSG mice (N = 5–7 mice per group) via intraperitoneal injection. Seven days after tumor cell administration, tumor-bearing mice were treated with different hROR1 CAR-T cells via a single intraperitoneal injection, and tumor burden was quantified by IVIS during treatment. Data shown are mean ± SEM.

[0055] Figure 10B The tumor burden before treatment was displayed.

[0056] Figure 11 The quantitative analysis of JeKo-1 tumor burden as measured by in vivo bioluminescence (IVIS) imaging (as described above) on day 36 is shown.

[0057] Figure 12 The quantification of human CD3+ T cells in the peripheral blood of tumor-bearing NSG mice treated with various hROR1 CAR-T cells is shown. CD3+ T cell proliferation in the blood was measured at days 15, 22, 29, 36, 43, and 50.

[0058] Figure 13A and Figure 13B The results showed that ROR1+ tumor cell lines were killed in vitro by hROR1 CAR T cells co-expressing constitutive or RTS-mbIL-15.

[0059] Figure 14 It was demonstrated that in the JeKo-1 xenograft mouse model, administration of hROR1 CAR RTS-mbIL-15-T cells + Veledimex promoted antitumor activity in a dose-dependent manner. Detailed Implementation

[0060] The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and therefore variations are possible. Those skilled in the art will recognize that many variations and modifications are possible with respect to the present invention, all of which are covered within its scope.

[0061] All terms are intended to be understood as they would be understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0062] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0063] While various features of the invention may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although the invention may be described herein in the context of a single embodiment for clarity, the invention may also be implemented in a single embodiment.

[0064] The following definitions supplement those in the art and are specific to this application, and should not be attributed to any related or unrelated circumstances, such as any jointly owned patents or applications. Preferred materials and methods are described herein; however, any methods and materials similar to or equivalent to those described herein may be used in practice to test the contents of this disclosure. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0065] In this application, unless otherwise specified, the use of the singular form includes the plural form. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein include plural indicators. In this application, unless otherwise specified, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “having” is not restrictive.

[0066] The terms “some embodiments,” “implementation,” “one embodiment,” or “other embodiments” used in this specification mean that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0067] As used in this specification and claims, the terms "comprising" (and any form of inclusion), "having" (and any form of having), "including" (and any form of including), or "containing" (and any form of containing) are inclusive or open-ended and do not exclude other elements or method steps not listed. It is conceivable that any embodiment discussed in this specification can be implemented using any method or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to implement the methods of the invention.

[0068] As used herein, the term “about” and its grammatical equivalents for reference values ​​may include the value itself and a range of values ​​plus or minus 10%. For example, the quantity “about 10” includes 10 as well as any quantity from 9 to 11. For example, the term “about” for reference values ​​may also include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0069] The term "isolated" refers to nucleic acids removed from their natural environment. The term "purified" refers to a given nucleic acid, whether taken from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions, with improved purity. Here, "purity" is a relative term, not "absolute purity." However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants while still remaining isolated for practical purposes. For example, when intended for introduction into cells, nucleic acids are typically mixed with an acceptable carrier or diluent.

[0070] As used herein, “polynucleotide” or “oligonucleotide” refers to a polymeric form of nucleotides—ribonucleotides or deoxyribonucleotides—of any length. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified forms (e.g., through methylation and / or capping) and unmodified forms of polynucleotides. The term is also intended to include molecules containing non-naturally occurring nucleotides or synthetic nucleotides, as well as nucleotide analogs.

[0071] The term "polypeptide" is used interchangeably with the terms "polypeptide" and "protein" and refers to a polymer of amino acid residues. A "mature protein" is a full-length protein that optionally contains glycosylation or other modifications typical of proteins in a given cellular environment.

[0072] Nucleic acids and / or nucleic acid sequences are “homologous” when they are naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences whose encoding DNA is naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence are homologous. Homologous molecules may be called homologs. For example, any naturally occurring protein as described herein can be modified by any available mutagenesis method. When expressed, the polypeptide encoded by the mutagenized nucleic acid is homologous to the protein encoded by the original nucleic acid. Homology is usually inferred from sequence identity between two or more nucleic acids or proteins (or their sequences). The precise percentage of sequence identity that can be used to establish homology varies depending on the nucleic acid and protein in question, but it is routinely used to establish homology as low as 25%. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or higher, can also be used to establish homology.

[0073] In the context of two nucleic acid sequences or amino acid sequences of peptides, the terms "identical" or "sequence identity," as used herein, refer to the identical residues in the two sequences when aligned to obtain maximum correspondence within a specified comparison window. In one embodiment, the peptide described herein is at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference peptide or fragment thereof, for example, as determined by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids may also be described with reference to the starting nucleic acid, for example, they may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference nucleic acid or fragment thereof, for example, as determined by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When describing a molecule as having a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, the percentage of residues in the smaller molecule finds matching residues in the larger molecule according to the order of the optimal alignment.

[0074] Transposons, or transposable elements (TEs), are vector DNA sequences that can change their position within the genome, sometimes generating or reversing mutations and altering the size of a cell's genome. Transposition often leads to the replication of TEs. Class I TEs are copied in two stages: first, they are transcribed from DNA into RNA, and then the resulting RNA is reverse transcribed back into DNA. The copied DNA is then inserted into a new location in the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the TE itself. Retrotransposons are characterized similarly to retroviruses, such as HIV. The cleavage-paste transposition mechanism of class II TEs does not involve RNA intermediates. Transposition is catalyzed by several transposases. Some transposases bind nonspecifically to any target site in DNA, while others bind to specific DNA sequence targets. Transposases perform staggered cleavage at the target site, creating single-stranded 5' or 3' DNA overhangs (sticky ends). This step cleaves the DNA transposon and then ligates it to a new target site; this process involves the activity of DNA polymerases that fill in the gaps and DNA ligases that seal the sugar-phosphate backbone. This leads to the replication of the target site. The insertion site of a DNA transposon can be identified by a short, directional repeat sequence (which can be generated by cross-cutting in the target DNA and then filled in by DNA polymerase) followed by a series of inverted repeat sequences crucial for transposase excision of the TE. If transposition of a cut-and-paste TE occurs when the donor site has been replicated but the target site has not yet been replicated during the S phase of the cell cycle, the cut-and-paste TE can be replicated. In class I and class II TEs, transposition can be classified as “autonomous” or “non-autonomous.” Autonomous TEs can move on their own, while the movement of non-autonomous TEs requires the presence of another TE. This is usually because non-autonomous TEs lack either transposase (for class II) or reverse transcriptase (for class I).

[0075] A transposase is an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome via a cut-and-paste mechanism or a replication-transposition mechanism. In some implementations, the catalytic activity of transposases can be used to move genes from vectors to the genome.

[0076] The nucleic acid sequences and vectors disclosed or considered herein can be introduced into cells via “transfection,” “transformation,” “nuclear transfection,” or “transduction.” As used herein, “transfection,” “transformation,” or “transduction” refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray EJ, Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)); and nuclear transfection (Trompeter et al., J. Immunol. Methods 274:245-256 (2003)). After the infectious particles have grown in suitable packaging cells, phage or viral vectors can be introduced into host cells, many of which are commercially available.

[0077] A promoter is a multinucleotide region that initiates the transcription of a coding sequence. Promoters are located near the transcription start site of a gene, on the same strand of DNA and upstream (towards the 5' region of the sense strand). Some promoters are constitutive because they are active in all conditions within the cell, while others are regulated to become active in response to specific stimuli, such as inducible promoters.

[0078] The term "promoter activity" refers to the degree of expression of a nucleotide sequence operatively linked to a promoter whose activity is being measured. Promoter activity can be determined directly by identifying the amount of RNA transcript produced (e.g., by Northern blotting analysis) or indirectly by identifying the amount of product encoded by the linked nucleic acid sequence (e.g., a reporter nucleic acid sequence linked to the promoter).

[0079] As used herein, an "inducible promoter" is a promoter whose activity is induced by the presence or absence of a transcriptional regulator (e.g., an biotic or abiotic factor). Inducible promoters are useful because the expression of genes operatively linked to them can be turned on or off at certain developmental stages of an organism or in specific tissues. Examples of inducible promoters are alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. In one embodiment, the inducible promoter is part of a genetic switch.

[0080] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription, for example, by an operatively linked nucleic acid sequence. Enhancers can be located thousands of bases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or alterations in DNA structure. A large number of enhancers from a variety of different sources are well known in the art and are available either as cloned polynucleotides (from collections such as ATCC and other commercial or private sources) or within cloned polynucleotides. Many polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, inside, or downstream of the coding sequence. The term “Ig enhancer” refers to an enhancer element derived from an enhancer region located within an immunoglobulin (Ig) gene locus (such enhancers include, for example, heavy chain (μ) 5' enhancers, light chain (κ) 5' enhancers, κ and μ in-cell enhancers, and 3' enhancers) (see generally Paul WE, ed., Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Patent 5,885,827).

[0081] As used in this article, "coding sequence" refers to a multinucleotide region that encodes a polypeptide. This region or sequence is defined by a start codon near the 5' end and a stop codon near the 3' end. A coding sequence can also be referred to as an open reading frame.

[0082] As used herein, "operably linked" refers to the physical and / or functional connection of one DNA segment to another, thereby allowing those segments to function in their intended manner. A DNA sequence encoding a gene product is operably linked to a regulatory sequence, such as a promoter, enhancer, and / or silencer, in a way that allows for direct or indirect regulation of transcription of that DNA sequence. For example, a DNA sequence is operably linked to a promoter when it is linked downstream of the promoter's transcription start site, conforms to the correct reading frame of the transcription start site, and allows transcriptional elongation through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is linked in a way that increases or decreases transcription of that DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of a DNA sequence. If a signal sequence is expressed as a preprotein involved in polypeptide secretion, the DNA of that signal sequence is operably linked to the DNA encoding that polypeptide. The ligation of a DNA sequence to a regulatory sequence is typically accomplished by ligating at a suitable restriction site or by inserting an adaptor or linker into the sequence using a restriction endonuclease known to those skilled in the art.

[0083] The term "transcription regulator" refers to a biochemical element that, under certain environmental conditions, prevents or inhibits the transcription of a promoter-driven DNA sequence (e.g., a repressor or nuclear repressor protein), or under certain environmental conditions, allows or stimulates the transcription of a promoter-driven DNA sequence (e.g., an inducer or enhancer).

[0084] The term "induction" refers to an increase in nucleic acid sequence transcription, promoter activity, and / or expression caused by transcription regulators relative to a given baseline transcription level.

[0085] A "target" gene, "heterologous" gene, or "goal-in-process" gene (GOI) refers to a gene introduced into a host cell through gene transfer.

[0086] As used herein, “recombinase” refers to a group of enzymes that can promote site-specific recombination between defined sites, where these sites are physically separated on a single DNA molecule or located on separate DNA molecules. The DNA sequences of the defined recombination sites are not necessarily identical. Initiation of recombination depends on protein-DNA interactions, and within this group are numerous protein-catalyzed phage integration and excision (e.g., λ integrase, φC31), dissociation of circulating plasmids (e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression of alternative genes (e.g., Hin, Gln, Pin), gene assembly during development (e.g., nitrogen-fixing genes in Anabaena), and transposition (e.g., the IS607 transposon). Based on evolutionary and mechanistic relevance, most site-specific recombinases belong to one of two families. These are the λ integrase family or tyrosine recombinases (e.g., Cre, Flp, Xer D) and the resolvase / integrase family or serine recombinase family (e.g., φC31, TP901-1, Tn3, gamma delta).

[0087] A “recombination attachment site” is a specific polynucleotide sequence recognized by the recombinase described herein. Typically, it involves two distinct sites (called “complementary sites”), one located in the target nucleic acid (e.g., a chromosome or episome in eukaryotes or prokaryotes) and the other on the nucleic acid to be integrated into the target recombination site. The terms “attB” and “attP” are used herein to refer to attachment (or recombination) sites originally derived from the bacterial target and bacteriophage donor, respectively, although recombination sites for specific enzymes may have different names. Recombination sites typically consist of a left arm and a right arm separated by a core or spacer region. Thus, the attB recombination site is composed of BOB’, where B and B’ are the left and right arms, respectively, and O is the core region. Similarly, attP is POP’, where P and P’ are arms, and O is the core region. When recombination occurs between attB and attP sites and the nucleic acid is simultaneously integrated into the target, the recombination sites flanking the integrated DNA are referred to as “attL” and “attR”. Using the above terminology, the attL and attR sites are therefore composed of BOP’ and POB’, respectively. In some representations in this article, “O” is omitted, and attB and attP are represented as BB' and PP', for example.

[0088] An "expression vector" or "vector" is any genetic element, such as a plasmid, chromosome, virus, or transposon, that is an autonomous unit of intracellular polynucleotide replication (i.e., capable of replicating under its own control) or capable of replication by insertion into a host cell chromosome, with an attached polynucleotide segment to enable replication and / or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and granules. Vectors may contain polynucleotide sequences necessary for vector ligation or insertion into the desired host cell and for expression of the attached segment. Such sequences vary depending on the host organism; they include promoter sequences for transcription, enhancer sequences for increased transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, an expression vector may be able to directly express the nucleic acid sequence product encoded therein without vector ligation or integration into the host cell DNA sequence.

[0089] Vectors may also contain “selection marker genes.” As used herein, the term “selection marker gene” refers to a nucleic acid sequence that, in the presence of a suitable selectant, allows cells expressing a nucleic acid sequence to be specifically selected or excluded. Suitable selection marker genes are known in the art and described, for example, in International Patent Application Publications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981). ; Santerre et al., Gene, 30:147 (1984); Kent et al., Science, 237:901-903 (1987); Wigler et al., Cell, 11:223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962); Lowy et al., Cell, 22:817 (1980); and U.S. Patents 5,122,464 and 5,770,359.

[0090] In some embodiments, the vector is an "attachment expression vector" or "attachment" capable of replicating in a host cell and existing within the host cell as an extrachromosomal segment of DNA in the presence of appropriate selective pressure (see, for example, Conese et al., Gene Therapy, 11:1735-1742 (2004)). Representative commercially available attachment expression vectors include, but are not limited to, attachment plasmids utilizing EB nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of attachment vectors using T-antigen and SV40 origin of replication instead of EBNA1 and oriP.

[0091] “Cancer cells” refers to cells that have undergone the early, intermediate, or late stages of the multi-step tumor progression process as previously described (Pitot et al., Fundamentals of Oncology, 15-28 (1978)). This includes cells in the early, intermediate, and late stages of tumor progression, including “pretumor” cells (i.e., “proliferative” cells and dysplastic cells), as well as neoplastic cells in the late stages of tumor progression that are dysplastic cells.

[0092] "Metastatic" cancer cells are cancer cells that have migrated from the primary cancer site (i.e., the location where cancer cells initially formed from normal, proliferating, or dysplastic cells) to a site other than the primary site, where they reside and proliferate.

[0093] "Cancer" refers to multiple cancer cells that may or may not be metastatic, such as ovarian cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, stomach cancer, esophageal cancer, oral cancer, tongue cancer, gingival cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testicular cancer, kidney cancer, endometrial cancer, uterine cancer, bladder cancer, bone marrow cancer, lymphoma, spleen cancer, thymic cancer, and thyroid cancer. Adenocarcinoma, brain cancer, neuronal carcinoma, mesothelioma, gallbladder cancer, eye cancer (e.g., corneal cancer, uveal cancer, choroidal cancer, macular cancer, vitreous humor cancer), joint cancer (e.g., synovial cancer), glioblastoma, lymphoma, leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma (MM), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0094] ROR-1

[0095] ROR1 is a transmembrane protein in the receptor tyrosine kinase (RTK) family. The ROR1 gene encodes two well-defined isoforms: a short 393-amino acid (aa) intracellular protein (isoform 2) and a long 937-aa type 1 transmembrane protein (isoform 1). The long cell surface isoform is expressed in primary human B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, a portion of B-cell acute lymphoblastic leukemia, and many tumors, including those associated with the metastatic phenotype.

[0096] ROR1 is primarily expressed during embryonic development, but its expression decreases during fetal development. However, ROR1 is aberrantly expressed in certain B-cell malignancies (e.g., but not limited to lymphoma, CLL, and B-ALL) and many solid tumor malignancies (e.g., but not limited to adrenal carcinoma, bladder cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, skin cancer, testicular cancer, uterine cancer, and neuroblastoma).

[0097] Chimeric antigen receptor

[0098] In the embodiments described herein, the CAR may include an extracellular antibody-derived single-chain variable domain (scFv) for target identification, wherein the scFv can be linked via flexible linkers to a transmembrane domain and / or an intracellular signaling domain, the latter including, for example, CD3ζ for T cell activation. Under normal circumstances, when T cells are activated in vivo, they receive a primary antigen-induced TCR signal and a secondary co-stimulatory signaling pathway from CD28, which induces the production of cytokines (i.e., IL-2 and IL-21), which are then fed back into the signaling loop in an autocrine / paracrine manner. Thus, the CAR may include a signaling domain, such as a CD28 cytoplasmic signaling domain or other co-stimulatory molecule signaling domains, such as a 4-1BB signaling domain. Chimeric CD28 co-stimulation improves T cell persistence by upregulating anti-apoptotic molecules and producing IL-2, as well as expanding T cells derived from peripheral blood mononuclear cells (PBMCs).

[0099] In one implementation, the CAR is a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for each epitope of ROR-1, for example, a fusion with a transmembrane domain and a CD3-ζ intracellular domain. Such molecules respond to the recognition of their targets by the scFv, resulting in the transmission of ζ signaling.

[0100] In implementations, the CAR may have an extracellular domain, a transmembrane domain, and an intracellular domain. In one implementation of the CAR extracellular domain, the signal peptide guides the nascent protein into the endoplasmic reticulum. This is, for example, when a receptor is glycosylated and anchored to the cell membrane. Any eukaryotic signal peptide sequence is conceivable to be functional. Typically, a signal peptide natively linked to the most N-terminal component is used (e.g., in scFv with a light chain-linker-heavy chain orientation, a native signal peptide using the light chain). In implementations, the signal peptide is GM-CSFRa (SEQ ID NO:94) or IgK (SEQ ID NO:95). Other signal peptides that may be used include those derived from CD8alpha (SEQ ID NO:97) and CD28. In one embodiment, the signal peptide is mouse IgVH region 3 (SEQ ID NO:101), azoxystrobin (SEQ ID NO:103), IGHV3-23 (SEQ ID NO:106), IGKV1-D33 (HuL1) (SEQ ID NO:107), or IGKV1-D33 (L14F) (HuH7) (SEQ ID NO:108).

[0101] The antigen recognition domain can be an scFv. However, alternatives are also possible. Antigen recognition domains derived from the α and β single chains of the natural T cell receptor (TCR) are considered because they possess simple extracellular domains (e.g., the CD4 extracellular domain) as well as other recognition components, such as linked cytokines (leading to recognition of cells carrying cytokine receptors). Almost any substance that binds to a given target with high affinity, such as virus-associated antigens, can be used as an antigen recognition region.

[0102] Typically, CARs exist in a dimerized form and are expressed as fusion proteins connecting the extracellular scFv (VH linked to VL), spacer region, transmembrane domain, and intracellular signaling motifs. First-generation CARs induce T cell activation solely through CD3-ζ signaling in their intracellular domain. Second-generation CARs provide activation signaling via CD3-ζ and CD28 or other intracellular domains such as 4-1BB or OX40. Third-generation CARs activate T cells through a combination of three signaling motifs, including CD28, 4-1BB, or OX40 containing CD3-ζ.

[0103] In embodiments, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In embodiments, the extracellular domain comprises a target-specific binding element, also referred to as an antigen-binding moiety or scFv, and a spacer region. In embodiments, the intracellular signaling domain, or cytoplasmic signaling domain, comprises a co-stimulatory signaling region and a ζ-chain portion.

[0104] The co-stimulatory signal transduction region refers to a portion of the CAR (cellular cellular signaling domain) that contains the intracellular signal transduction domain of co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands, required for an effective lymphocyte response to antigens.

[0105] In the implementation scheme, a stem domain or stem region is incorporated between the extracellular domain and the transmembrane domain of the CAR. As used herein, the term "stem domain" or "stem region" generally refers to any oligonucleotide or polypeptide that serves to link the transmembrane domain to the cytoplasmic domain in the scFv or polypeptide chain. The stem domain may include a flexible hinge such as an Fc hinge and optionally one or both Fc constant domains. In some cases, the stem region contains a hinge region derived from IgG1. Alternatively, the stem region contains the CH2CH3 region of an immunoglobulin and optionally a portion of CD3. In some cases, the stem region contains a CD8α hinge region (SEQ ID NO: 83), an IgG4-Fc 12-amino acid hinge region (ESKYGPPCPPCP (SEQ ID NO: 285)), or an IgG4 hinge region, as described in WO / 2016 / 073755.

[0106] In other embodiments, a spacer region is incorporated between the extracellular domain and the transmembrane domain of the CAR. The spacer region may include a stem region and a stem extension region, such as... Figure 3 and Figure 4 As shown. In some embodiments, compared to peptides that are identical in other respects but lack a spacer region, the spacer region extends the distance between the different domains of the chimeric peptide, thereby leading to improved peptide expression or functional activity. In some cases, the spacer region includes any peptide that functions to connect the transmembrane region to the extracellular region or the cytoplasmic region in the chimeric peptide. In some embodiments, the spacer region is flexible enough to allow the antigen or ligand binding regions to align in different orientations, thereby facilitating antigen or ligand receptor recognition.

[0107] In some embodiments, the length of the stem region may be from about 20 to about 300 amino acids and includes at least one dimerization site, and the stem extension region may have a length of about 1 to about 10 times that of the stem region, as measured by the number of amino acids.

[0108] In some cases, the length of the stem region can be approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids. In other cases, the length of the stem region can be approximately 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids.

[0109] In one embodiment, the spacer region may include a single stem region. In another embodiment, the spacer region may include a stem region (denoted as “S”) and a stem extension region denoted herein as “S’n”. For example, the spacer region may include a stem region (1) and S’n, where n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In a further embodiment, the stem region may be connected to the stem extension region S’n via a connector. In one embodiment, when the CAR is a ROR1 CAR, n is not 0.

[0110] In some cases, as measured by amino acid number, the stem extension region can have a length of about 1 to about 10 times that of the stem region. For example, as measured by amino acid number, the stem extension region can have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times that of the stem region. In some cases, as measured by amino acid number, the stem extension region can have a length of more than 10 times that of the stem region. In some instances, as measured by amino acid number, the stem extension region can have a length of at most 2 times that of the stem region, but contains fewer dimerization sites than the stem region.

[0111] The stem extension region of the antigen-binding polypeptide of the present invention may have at least one less dimerization site compared to the stem region. For example, if the stem region contains two dimerization sites, the stem extension region may contain one or zero dimerization sites. As another example, if the stem region contains one dimerization site, the stem extension region may contain zero dimerization sites. In some instances, the stem extension region lacks dimerization sites. In some instances, as measured by amino acid number, the stem extension region may have at most twice the length of the stem region but does not contain dimerization sites. In some instances, as measured by amino acid number, the stem extension region may have at most three times the length of the stem region but does not contain dimerization sites. In some instances, as measured by amino acid number, the stem extension region may have at most four times the length of the stem region but contains zero dimerization sites. In some cases, one or more dimerization sites may be located proximal to the membrane. In other cases, one or more dimerization sites may be located distal to the membrane.

[0112] In some instances, the length of each stem extension region can be from about 20 to about 60 amino acids. In other instances, the length of the stem extension region can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or more amino acids, or any integer within or outside this range. In some cases, each stem extension region has a sequence that has at least about 60% identity with the stem region. In some instances, each stem extension region has a sequence that has at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity with the stem region.

[0113] The transmembrane domain can be of natural or synthetic origin. In the case of a natural origin, the domain can be derived from any membrane-binding or transmembrane protein. Suitable transmembrane domains may include transmembrane regions of the α, β, or ζ chains of the T cell receptor; or transmembrane regions derived from CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic and may contain hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at one or both ends of the synthetic transmembrane domain. Optionally, in some embodiments, short oligonucleotides or polypeptide linkers of 2 to 10 amino acids in length may form a link between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain or a CD3ζ transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain. In other embodiments, the transmembrane domain comprises a CD3ζ transmembrane domain.

[0114] Intracellular domains may contain one or more co-stimulatory domains. Exemplary co-stimulatory domains include, but are not limited to, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), CD3ζ, or fragments or combinations thereof. In some cases, the CAR described herein contains one or more co-stimulatory domains selected from CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), or fragments or combinations thereof. In some cases, the CAR described herein contains one or more co-stimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS, OX40 (CD134), or fragments or combinations thereof. In some cases, the CAR described herein contains one or more co-stimulatory domains selected from CD8, CD28, 4-1BB (CD137), DAP10, DAP12, or fragments or combinations thereof. In some cases, the CAR described herein contains one or more costimulatory domains selected from CD28, 4-1BB (CD137), or fragments or combinations thereof. In some cases, the CAR described herein contains costimulatory domains CD28 and 4-1BB (CD137), or fragments thereof. In some cases, the CAR described herein contains costimulatory domains CD28 and OX40 (CD134), or fragments thereof. In some cases, the CAR described herein contains costimulatory domains CD8 and CD28, or fragments thereof. In some cases, the CAR described herein contains costimulatory domain CD28, or fragments thereof. In some cases, the CAR described herein contains costimulatory domain 4-1BB (CD137), or fragments thereof. In some cases, the CAR described herein contains costimulatory domain OX40 (CD134), or fragments thereof. In some cases, the CAR described herein contains costimulatory domain CD8, or fragments thereof. In some cases, the CAR described herein contains at least one costimulatory domain DAP10, or a fragment thereof. In some cases, the CAR described herein contains at least one costimulatory domain DAP12, or a fragment thereof.

[0115] The intracellular signaling domain (also called the cytoplasmic domain) of the CAR disclosed herein is responsible for activating at least one normal effector function of the CAR-placed immune cell. The term "effector function" refers to a specialized function of the cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can generally be used, in many cases it is not necessary to use the entire chain. When using a truncated portion of the intracellular signaling domain, such a truncated portion can be used instead of the complete chain, as long as it transduces effector function signals. Therefore, the term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce effector function signals. In some embodiments, the intracellular domain further includes a signaling domain for T cell activation. In some cases, the signal transduction domains for T cell activation include domains derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In other cases, the signal transduction domains for T cell activation include domains derived from CD3ζ.

[0116] In an implementation, this document provides an isolated nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a stem domain; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; (e) a CD3ζ signaling domain; and optionally (f) a truncated epidermal growth factor receptor (HER1t or HER1t1).

[0117] Nucleic acid sequences encoding the functional portions of the CAR described herein are included within the scope of this invention. Functional portions include, for example, those portions of the CAR that retain, to the same degree or greater extent, the ability to recognize target cells or to detect, treat, or prevent disease as the parent CAR. Regarding the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional portion of the CAR may encode, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the protein constituting the parent CAR.

[0118] In an embodiment, the CAR contains additional amino acids at the amino or carboxyl terminus, or at both ends, of the portion, which are not found in the amino acid sequence of the parent CAR. Ideally, these additional amino acids do not interfere with the biological function of the functional portion, such as recognizing target cells, detecting cancer, treating or preventing cancer, etc. More ideally, these additional amino acids enhance the biological activity of the CAR compared to the biological activity of the parent CAR.

[0119] As used herein, the term "functional variant" refers to a polypeptide or protein that has a high or significant sequence identity or similarity to a reference polypeptide and retains the biological activity of the reference polypeptide as a variant. Functional variants include, for example, those variants of the CAR (parental CAR) described herein that retain the same or greater degree of similarity to the parental CAR in recognizing target cells. Regarding the nucleic acid sequence encoding the parental CAR, the nucleic acid sequence of the functional variant encoding the CAR may be approximately 10%, 25%, 30%, 50%, 65%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence encoding the parental CAR.

[0120] The CARs described herein include (including their functional portions and functional variants) glycosylation, amidation, carboxylation, phosphorylation, esterification, N-acylation, for example via disulfide cyclization, or conversion to acid addition salts and / or optionally dimerization, polymerization, or conjugation.

[0121] antigen-binding portion

[0122] In the implementation scheme, the CAR described herein comprises a target-specific binding element, also referred to as an antigen-binding moiety. In the implementation scheme, the CAR described herein is engineered to target a specific antigen by engineering the desired antigen-binding moiety that specifically binds to an antigen on a cell.

[0123] In this embodiment, the antigen-binding portion of the CAR described herein is specific to ROR-1 (ROR-1 CAR). When expressed on a cell surface, the ROR-1-specific CAR redirects T cells specifically to human ROR-1. In this embodiment, the antigen-binding domain comprises a single-chain antibody fragment (scFv) containing a light chain variable domain (VL) and a heavy chain variable domain (VH) of a target antigen-specific monoclonal anti-ROR-1 antibody linked via a flexible linker such as a glycine-serine linker or a Whitlow linker. In this embodiment, the scFv is humanized mROR-1. In some embodiments, the scFv is hROR1(VH-VL)_14, hROR1(VL-VH)_05, hROR1(VH-VL)_14-3, hROR1(VH-VL)_14-4, hROR1(VH_5-VL_14), hROR1(VH_5-VL_16), hROR1(VH_18-VL_04), or hROR1(VH_18-VL_14). In some embodiments, the antigen-binding portion may include directionally linked VH and VL, for example, from the N-terminus to the C-terminus, VH-linker-VL, or VL-linker-VH.

[0124] In the embodiments described herein, the CAR comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74 (hROR1 VL).

[0125] In the embodiments described herein, the CAR comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73 (hROR1 VH).

[0126] In the implementation scheme, the CAR described herein comprises antigen-binding portions VL (SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72 and 74) and VH (SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 73) having a Gly-Ser linker (SEQ ID NO: 127, 129 or 130) or a functional variant of the linker.

[0127] In the embodiments described herein, the CAR comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO:3-14 and 75-82 (VH, VL, and linker).

[0128] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:15 (hROR1 VH_04).

[0129] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:16 (hROR1 VL_04).

[0130] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:17 (hROR1 VH_05).

[0131] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:18 (hROR1 VL_05).

[0132] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:19 (hROR1 VH_06).

[0133] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:20 (hROR1 VL_06).

[0134] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:21 (hROR1 VH_07).

[0135] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:22 (hROR1 VL_07).

[0136] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:23 (hROR1 VH_08).

[0137] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:24 (hROR1 VL_08).

[0138] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:25 (hROR1 VH_09).

[0139] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:26 (hROR1 VL_09).

[0140] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:27 (hROR1 VH_10).

[0141] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:28 (hROR1 VL_10).

[0142] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:29 (hROR1 VH_11).

[0143] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:30 (hROR1 VL_11).

[0144] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:31 (hROR1 VH_12).

[0145] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:32 (hROR1 VL_12).

[0146] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:33 (hROR1 VH_13).

[0147] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:34 (hROR1 VL_13).

[0148] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:35 (hROR1 VH_14).

[0149] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:36 (hROR1 VL_14).

[0150] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:37 (hROR1 VH_14-1).

[0151] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:38 (hROR1 VL_14-1).

[0152] In the embodiments described herein, the CAR comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:39 (hROR1 VH_14-2).

[0153] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:40 (hROR1 VL_14-2).

[0154] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:41 (hROR1 VH_14-3).

[0155] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:42 (hROR1 VL_14-3).

[0156] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43 (hROR1 VH_14-4).

[0157] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44 (hROR1 VL_14-4).

[0158] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45 (hROR1 VH_14-5).

[0159] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46 (hROR1 VL_14-5).

[0160] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:47 (hROR1 VH_15).

[0161] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48 (hROR1 VL_15).

[0162] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:49 (hROR1 VH_16).

[0163] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:50 (hROR1 VL_16).

[0164] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:51 (hROR1 VH_17).

[0165] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:52 (hROR1 VL_17).

[0166] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:53 (hROR1 VH_18).

[0167] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:54 (hROR1 VL_18).

[0168] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:55 (hROR1 VH_19).

[0169] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:56 (hROR1 VL_19).

[0170] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:57 (hROR1 VH_20).

[0171] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:58 (hROR1 VL_20).

[0172] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:59 (hROR1 VH_21).

[0173] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:60 (hROR1 VL_21).

[0174] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:61 (hROR1 VH_22).

[0175] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:62 (hROR1 VL_22).

[0176] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:63 (hROR1 VH_23).

[0177] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:64 (hROR1 VL_23).

[0178] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:65 (hROR1 VH_24).

[0179] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:66 (hROR1 VL_24).

[0180] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:67 (hROR1 VH_25).

[0181] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:68 (hROR1 VL_25).

[0182] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:69 (hROR1 VH_26).

[0183] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:70 (hROR1 VL_26).

[0184] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:71 (hROR1 VH_27).

[0185] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:72 (hROR1 VL_27).

[0186] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:73 (hROR1 VH_28).

[0187] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:74 (hROR1 VL_28).

[0188] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:17 (hROR1 VH_5) and a VL polypeptide having the amino acid sequence SEQ ID NO:18 (hROR1 VL_5).

[0189] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:35 (hROR1 VH_14) and a VL polypeptide having the amino acid sequence SEQ ID NO:36 (hROR1 VL_14).

[0190] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:41 (hROR1 VH_14-3) and a VL polypeptide having the amino acid sequence SEQ ID NO:42 (hROR1 VL_14-3).

[0191] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:43 (hROR1 VH_14-4) and a VL polypeptide having the amino acid sequence SEQ ID NO:44 (hROR1 VL_14-4).

[0192] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:17 (hROR1 VH_5) and a VL polypeptide having the amino acid sequence SEQ ID NO:36 (hROR1 VL_14).

[0193] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:53 (hROR1 VH_18) and a VL polypeptide having the amino acid sequence SEQ ID NO:16 (hROR1 VL_04).

[0194] In the implementation scheme, the CAR described herein comprises an antigen-binding portion comprising a VH polypeptide having the amino acid sequence SEQ ID NO:53 (hROR1 VH_18) and a VL polypeptide having the amino acid sequence SEQ ID NO:36 (hROR1 VL_14).

[0195] In the embodiments, the antigen-binding portion has a GM-CSFRa signal peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:94.

[0196] In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:3. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:4. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:5. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:6. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:7. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:8. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:9. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:10. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:11. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:12. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:13.In the implementation scheme, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:14.

[0197] In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:75. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:76. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:77. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:78. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:79. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:80. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:81. In embodiments, the CAR described herein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:82.

[0198] Interval

[0199] In embodiments, the ROR-1 CAR of this disclosure includes a spacer region comprising one or more stem regions and optionally one or more stem extension regions, which provide a separation between the antigen-binding portion and the T cell membrane. In embodiments, the spacer region establishes an optimal effector-target membrane distance. In embodiments, the stem domain provides flexibility to the antigen-binding domain to reach its target. In one embodiment, the stem domain is a CD8α hinge domain. In some embodiments, the terms "spacer region," "stem region," and "stem domain" are used interchangeably herein.

[0200] In the embodiments, the CD8α hinge domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:88.

[0201] In other embodiments, a spacer region is incorporated between the extracellular domain and transmembrane domain of the CAR. As described herein, the spacer region may include a stem region and a stem extension region, such as... Figure 3 and Figure 4 As shown. In one embodiment, the spacer region may include a single stem region. In another embodiment, the spacer region may include a stem region (denoted as "S") and a stem extension region denoted herein as "S'n". For example, the spacer region may include a (1) stem region and S'n, where n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, when the scFv of the ROR1 CAR is mROR1 (SEQ ID NO: 3-14) or hROR1 (SEQ ID NO: 75-82), where n is not 0.

[0202] In a further embodiment, the stem region may be connected to the stem extension region S'n via a connector. The connectors described herein may include, for example, GSG connectors (SEQ ID NO:129), SGSG connectors (SEQ ID NO:130), (G4S)3 connectors (SEQ ID NO:127), (G4S)4 connectors (SEQ ID NO:294), and / or Whitlow connectors (SEQ ID NO:128).

[0203] In one embodiment, the stem region and stem extension region may be derived from or designed from peptides of natural or synthetic origin. The stem region and / or stem extension region may contain a hinge domain derived from a cell surface protein or a derivative or variant thereof. In some embodiments, the stem region and / or stem extension region may contain a hinge domain derived from CD28 or CD8α (CD8α). In some embodiments, each of the stem region and stem extension region may be derived from at least one of the CD8α hinge domain, CD28 hinge domain, CTLA-4 hinge domain, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge, and CH2-CH3 domain. The stem region and stem extension region may individually be derived from any combination of the CD8α hinge domain, CD28 hinge domain, CTLA-4 hinge domain, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge, or CH2-CH3 domain. For example, the stem region may be derived from the CD8α hinge domain, while at least one stem extension region may be derived from the CD28 hinge domain, thereby creating a heterozygous spacer region. For example, the stem region may originate from the IgG1 hinge or the IgG4 hinge, while at least one stem extension region may originate from the CH2-CH3 domain of IgG.

[0204] In some embodiments, the stem region may contain one or more dimerization sites to form homodimerized or heterodimerized chimeric polypeptides. In other embodiments, the stem region or one or more stem extension regions may contain mutations that completely eliminate dimerization sites. In some embodiments, the stem extension region may have at least one fewer dimerization site than the stem region. For example, if the stem region contains two dimerization sites, the stem extension region may contain one or zero dimerization sites. As another example, if the stem region contains one dimerization site, the stem extension region may contain zero dimerization sites. In some instances, the stem extension region lacks dimerization sites.

[0205] In some aspects of the embodiments disclosed herein, the stem region of the antigen-binding polypeptide of the present invention comprises a sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity with the CD8 hinge domain. The CD8α hinge domain may comprise a polypeptide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity with the sequence shown in SEQ ID NO:83. In some cases, the stem extension region comprises a polypeptide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher identity with the sequence shown in SEQ ID NO:83. In some cases, the stem extension region comprises a nucleotide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher identity with the sequence shown in SEQ ID NO:230. In some instances, the stem region and the stem extension region may together contain a polynucleotide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher identity with any of the sequences shown in SEQ ID NO:230-233.

[0206] Transmembrane domain

[0207] In one embodiment, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR stem domain. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain is a transmembrane hydrophobic α-helix.

[0208] The transmembrane domain can be derived from natural or synthetic sources. In the case of a natural source, the domain can be derived from any membrane-binding or transmembrane protein. The transmembrane region particularly used in this invention can be derived from (i.e., containing at least its transmembrane region) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, in embodiments, short oligonucleotides or polypeptide linkers of 2 to 10 amino acids in length can form a link between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In embodiments, this linker is a glycine-serine linker.

[0209] In the embodiments described herein, the transmembrane domain in the CAR is the CD8α transmembrane domain. In the embodiments, the CD8α transmembrane domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:87.

[0210] In the embodiments described herein, the transmembrane domain in the CAR is the CD28 transmembrane domain. In the embodiments, the CD28 transmembrane domain comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:88.

[0211] Cytoplasmic domains (co-stimulatory domains and signal transduction domains)

[0212] The cytoplasmic domain of the CAR described herein, also known as the intracellular signaling domain, is responsible for activating at least one normal effector function of the CAR-implanted immune cell. The term "effector function" refers to a cell's specialized function. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can often be used, in many cases it is not necessary to use the whole chain. When using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of the complete chain, as long as it transduces effector function signals. Therefore, the term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce effector function signals.

[0213] Examples of intracellular signaling domains used in the CARs described herein may include cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that work together to initiate signal transduction upon antigen-receptor binding, and include any derivatives or variants of these sequences as well as any synthetic sequences having the same functional capabilities.

[0214] Signals generated by the TCR alone are usually insufficient to fully activate T cells; secondary or co-stimulatory signals are also required. Therefore, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0215] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that function in a stimulatory manner may contain signaling motifs, which are referred to as immune receptor tyrosine-based activation motifs or ITAMs.

[0216] Examples of primary cytoplasmic signaling sequences containing ITAM that are particularly used in this invention include those derived from TCRζ, FcRγ, FcR, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In embodiments, the cytoplasmic signaling molecule in the CAR described herein comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0217] In implementations, the cytoplasmic domain of the CAR can be designed to contain a CD3-ζ signaling domain itself, or combined with any other desired cytoplasmic domain useful in the context of the CAR described herein. For example, the cytoplasmic domain of the CAR may contain a portion of the CD3ζ chain and a co-stimulatory signaling region. A co-stimulatory signaling region refers to a portion of the CAR that contains the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, DAP10, DAP12, and ligands that specifically bind to CD83. In implementations, co-stimulatory molecules can be used together, for example, CD28 and 4-1BB or CD28 and OX40. Therefore, although this invention mainly uses 4-1BB and CD28 as co-stimulatory signal transduction elements, other co-stimulatory elements are also within the scope of this invention.

[0218] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR described herein can be linked together in a random or specified order. Optionally, short oligonucleotides or polypeptide linkers of 2 to 10 amino acids in length can form the linkers. Glycine-serine dinucleotides provide particularly suitable linkers.

[0219] In one embodiment, the cytoplasmic domain comprises a CD3-ζ signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain comprises a CD3-ζ signaling domain and a 4-1BB signaling domain. In yet another embodiment, the cytoplasmic domain comprises a CD3-ζ signaling domain as well as signaling domains for CD28 and 4-1BB.

[0220] In one embodiment, the cytoplasmic domain of the CAR described herein comprises a 4-1BB signal transduction domain and a CD3-ζ signal transduction domain, wherein the 4-1BB signal transduction domain comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequence of SEQ ID NO:89, and the CD3-ζ signal transduction domain comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:93.

[0221] In one embodiment, the cytoplasmic domain of the CAR described herein is designed to include a CD28 signaling domain and a CD3-ζ signaling domain, wherein the CD28 signaling domain comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequence of SEQ ID NO:90, and the CD3-ζ signaling domain comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequence of SEQ ID NO:93.

[0222] In one embodiment, the cytoplasmic domain of the CAR described herein is designed to include a signal transduction domain of DNAX activator protein 10 (DAP10), wherein the signal transduction domain of DAP10 includes a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequence of SEQ ID NO:91.

[0223] In one embodiment, the cytoplasmic domain of the CAR described herein is designed to include a signal transduction domain of DNAX activator protein 12 (DAP12), wherein the signal transduction domain of DAP10 includes a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequence of SEQ ID NO:92.

[0224] In one embodiment, the cytoplasmic domain of the CAR described herein comprises a 4-1BB signal transduction domain and a CD3-ζ signal transduction domain, wherein the 4-1BB signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:89, and the CD3-ζ signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:93.

[0225] Other genetic elements

[0226] While cell therapy holds great promise for treating human diseases, the significant toxicity of the cells themselves or their transgenic products hinders clinical research. In the embodiments described herein, immune effector cells containing the CAR described herein, already infused into mammalian subjects such as humans, can be eliminated to modulate the effects of these immune effector cells if their use would be toxic. Therefore, in some embodiments, in addition to the therapeutic ROR-1-specific chimeric antigen receptor described herein, a second gene is introduced into the engineered immune effector cells described herein. In some embodiments, the second gene is a “cell tag.” In some embodiments, the cell tag is a “kill switch.” In some embodiments, the second gene is actually a “kill switch” that allows the consumption of cells containing ROR-1 CAR or ROR-1 CAR / mbIL-15. In some embodiments, the “kill switch” is a HER1 tag or CD20 tag containing a HER1 peptide or a CD20 peptide, said peptide containing at least an antibody-binding epitope of HER1 or CD20 or a functional fragment thereof, and optionally a signaling peptide sequence or a fragment thereof.

[0227] In some embodiments, the second gene is a HER1 tag, which is an epidermal growth factor receptor (HER1) or a fragment or variant thereof. In some embodiments, the second gene is a HER1 tag, which is a truncated human epidermal growth factor receptor 1 (e.g., HER1t or HER1t1). In some cases, the second gene is a variant of a truncated human epidermal growth factor receptor 1. In some embodiments, at least one of HER1, HER1t, and HER1t1 provides a safety mechanism by allowing the consumption of infused CAR-T cells by administration of an FDA-approved cetuximab or any antibody that recognizes HER1, HER1t, and / or HER1t1. In some embodiments, the HER1t gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:256. In the implementation scheme, the HER1t1 gene comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:257. The truncated HER1 sequences, such as HER1t and HER1t1, eliminate the possibility of EGFR ligand binding, EGFR homodimerization and heterodimerization, and EGFR-mediated signal transduction, while maintaining the intact binding of cetuximab to the receptor (Ferguson, K., 2008. A structure-based view of Epidermal Growth Factor Receptor regulation. Annu Rev Biophys, Volume 37, pp. 353-373).

[0228] In some embodiments, the HER1t tag has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:109. In some embodiments, the HER1t1 tag has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:110.

[0229] In a further embodiment, in addition to the therapeutic ROR-1-specific chimeric antigen receptor of the present invention, the introduced second gene also includes a CD20 tag. In some cases, the CD20 tag is a full-length CD20 polypeptide or a truncated CD20 polypeptide (CD20t-1). In some cases, the CD20 tag, such as CD20 or CD20t-1, also provides a safety mechanism by allowing the consumption of infused CAR-T cells by administration of FDA-approved rituximab therapy. In some embodiments, the CD20 tag has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:111. In some embodiments, the CD20 tag is a CD20t-1 tag and has a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of SEQ ID NO:112. In some embodiments, the CD20 tag is encoded by a CD20 gene containing a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:258. In some embodiments, the CD20 tag is encoded by the CD20t-1 gene, which contains a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:259.

[0230] In an implementation scheme, the CAR vector containing the CAR described herein further comprises a full-length CD20 tag, which contains a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:258.

[0231] In one embodiment, the gene encoding a killing tag such as HER1t, HER1t-1, CD20, or CD20t-1 is genetically fused to the ROR-1 CAR at the 3' end of a reading frame using a self-cleaving peptide, such as, but not limited to, the Thoseea asigna virus (T2A) peptide. In another embodiment, the T2A peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:116.

[0232] In one embodiment, the kill tag gene is cloned into a lentiviral plasmid backbone that matches the reading frame of the ROR-1 CAR gene. In other embodiments, the kill tag is cloned into a separate lentiviral vector. In other embodiments, both genes are cloned into a Sleeping Beauty transposon vector. In still other embodiments, the kill tag, such as HER1t, HER1t-1, CD20, or CD20t-1, is cloned into a separate Sleeping Beauty transposon vector. In some embodiments, the kill tag has a signal peptide, such as the GM-CSFRa signal peptide, wherein the GM-CSFRa signal peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:94. In some embodiments, the signal peptide is IgK, which has a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequence of SEQ ID NO:95.

[0233] In one embodiment, the signal peptide is mouse IgVH region 3 (SEQ ID NO:101), azoxystrobin (SEQ ID NO:103), IGHV3-23 (SEQ ID NO:106), IGKV1-D33 (HuL1) (SEQ ID NO:107), or IGKV1-D33 (L14F) (HuH7) (SEQ ID NO:108).

[0234] Figures 1 and 2 show exemplary gene expression cassettes encoding the CAR and kill tags described herein.

[0235] Exemplary CAR Open Reading Frame

[0236] Table 1 shows exemplary CAR and human ROR-1 receptor open reading frames covered by the methods and compositions described herein:

[0237] Table 1.

[0238]

[0239]

[0240] In the embodiments, this document provides an isolated nucleic acid encoding a CAR, wherein the CAR comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acids of SEQ ID NO:3-14 or SEQ ID NO:75-82.

[0241] In the implementation scheme, CAR hROR1(VH-VL)_14.CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:17 (hROR1 VH_5), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:18 (hROR1VL_5).

[0242] In the implementation scheme, CAR hROR1(VL-VH)_05.CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:35 (hROR1 VH_14), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:36 (hROR1VL_14).

[0243] In the implementation scheme, CAR hROR1(VH-VL)_14-3.CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:41(hROR1 VH_14-3), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:42(hROR1 VL_14-3).

[0244] In the implementation scheme, CAR hROR1(VH-VL)_14-4.CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43 (hROR1 VH_14-4), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44 (hROR1 VL_14-4).

[0245] In the implementation scheme, CAR hROR1(VH_5-VL_14).CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:17 (hROR1 VH_5), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:36 (hROR1VL_14).

[0246] In the implementation scheme, CAR hROR1(VH_5-VL_16).CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:17(hROR1 VH_05), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:50(hROR1VL_16).

[0247] In the implementation scheme, CAR hROR1(VH_18-VL_04).CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:53(hROR1 VH_18), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:16(hROR1VL_04).

[0248] In the implementation scheme, CAR hROR1(VH_18-VL_14).CD8a(3x).CD28z comprises an antigen-binding portion comprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:53(hROR1 VH_18), and a VL polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:36(hROR1VL_14).

[0249] In each embodiment in Table 1, the CAR signal peptide may comprise a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of mouse IgVH region 3 (SEQ ID NO:101), azuril (SEQ ID NO:103), IGHV3-23 (SEQ ID NO:106), IGKV1-D33(HuL1) (SEQ ID NO:107), or IGKV1-D33(L14F) (HuH7) (SEQ ID NO:108).

[0250] In each embodiment marked "CD8a" in Table 1, the transmembrane region of the CAR comprises a CD8α transmembrane domain, which contains a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:87, and the spacer region is CD8a, which contains a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the amino acid sequences shown in SEQ ID NO:83-86.

[0251] In each embodiment marked "CD28m" in Table 1, the intracellular domain of the CAR contains CD28, which has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:90.

[0252] In each embodiment marked "T2A" in Table 1, the CAR ORF comprises a self-cleaving Thosea asigna viral (T2A) peptide, which allows for the generation of multiple gene products from a single vector. In each embodiment, the T2A peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:116.

[0253] In the embodiments marked "HER1t" in Table 1, the CAR ORF comprises a truncated human epidermal growth factor receptor 1 (HER1t), which provides a safety mechanism by allowing the consumption of infused CAR-T cells via administration of FDA-approved cetuximab therapy. The HER1t gene, as described herein, may comprise a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:109. Unless otherwise stated in Table 1, the HER1t tag contains the GM-CSFRa signal peptide ("GM-CSFRsp") (SEQ ID NO:94). In one embodiment, the signal peptide is mouse IgVH region 3 (SEQ ID NO:101), azuril (SEQ ID NO:103), IGHV3-23 (SEQ ID NO:106), IGKV1-D33(HuL1) (SEQ ID NO:107), or IGKV1-D33(L14F) (HuH7) (SEQ ID NO:108). In some embodiments, HER1t may be replaced by another tag such as HER1t-1. The HER1t-1 gene as described herein may comprise a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:110. In the embodiments marked “IgKsp” in Table 1, the signal peptide is IgK, which has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:95.

[0254] In the embodiments marked "4-1BB" in Table 1, the CAR ORF comprises a co-stimulatory molecule having a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:89.

[0255] In the embodiments marked "FL CD20" in Table 1, the CAR ORF comprises a full-length CD20 tag having a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:111. CD20 provides a safety mechanism by allowing the consumption of infused CAR-T cells via administration of an FDA-approved rituximab therapy. In other embodiments, FL CD20 may be replaced by CD20t-1, which comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:112.

[0256] In some implementations shown in Table 1, the CAR ORF can be controlled by an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible gene switch based on a dual-peptide ecdysone receptor, such as... Gene switch. In some implementations, it can be as follows: Figure 1A-1E and Figures 2A-2E The configuration shown includes CARORF and gene switch components.

[0257] Cytokines

[0258] In some embodiments, the CAR described herein is administered to a subject together with one or more additional therapeutic agents, including but not limited to cytokines. In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variants or combinations thereof. In some cases, the cytokine is an interleukin. In some cases, the interleukin is at least one of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, and their functional variants and fragments. In some embodiments, the cytokine may be membrane-bound or secreted. In some embodiments, the cytokine is soluble IL-15, a soluble IL-15 / IL-15Rα complex (e.g., ALT-803). In some cases, the interleukin may comprise membrane-bound IL-15 (mbIL-15) or a fusion of IL-15 and IL-15Rα. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15 that may be co-expressed with the modified immune effector cells described herein. In some embodiments, the mbIL-15 comprises a full-length IL-15 (e.g., a native IL-15 polypeptide) or a fragment or variant thereof fused in a frame-bound manner to full-length IL-15Rα, a functional fragment thereof, or a variant thereof. In some cases, the IL-15 is indirectly linked to IL-15Rα via a linker. In some cases, mbIL-15, as described by Hurton et al., "TetheredIL-15 augments antitumor activity and promotes a stem-cell memory subset intumor-specific T cells," PNAS 2016, is expressed in the same immune effector cells as CAR.

[0259] In a further embodiment, immune effector cells expressing the CAR described herein express membrane-bound IL-15 (“mIL-15 or mbIL-15”). In an aspect of the invention, the mbIL-15 comprises a fusion protein between IL-15 and IL-15Rα. In a further embodiment, the mbIL-15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:113. In some cases, the CAR and cytokines are expressed in separate vectors. In specific cases, the vector may be a lentiviral vector, a retroviral vector, or a Sleeping Beauty transposon.

[0260] In some implementations, mbIL-15 is expressed co-with cellular tags such as HER1t, HER-1t-1, CD20t-1, or CD20 as described herein. mbIL-15 can be expressed in a frame-bound manner with HER1t, HER-1t-1, CD20t-1, or CD20.

[0261] In some implementations, mbIL-15 can be controlled by an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible gene switch based on a dual-peptide ecdysone receptor, such as... Gene switch.

[0262] On the other hand, interleukins may include IL-12. In some embodiments, the IL-12 is single-chain IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or intercalated IL-12. In some cases, IL-12 variants are described in WO2015 / 095249, WO2016 / 048903, and WO2017 / 062953, all of which are incorporated herein by reference.

[0263] In some implementations, the cytokines described above can be controlled by an inducible promoter for gene transcription. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible gene switch based on a dual-peptide ecdysone receptor, such as... Gene switch.

[0264] Gene switch

[0265] This article provides gene switch peptides, polynucleotides encoding ligand-inducible gene switch peptides, and methods and systems comprising these peptides and / or polynucleotides. The term "gene switch" refers to a combination of a promoter-associated responsive element, such as with an ecdysone receptor (EcR)-based system, which, in the presence of one or more ligands, regulates the expression of a gene incorporating the responsive element and the promoter. Tightly regulated inducible gene expression systems, or gene switches, can be used in a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and the regulation of traits in transgenic plants and animals. Such inducible gene expression systems may include ligand-inducible heterologous gene expression systems.

[0266] Early forms of EcR-based gene switches used Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) peptides, and these receptors were shown to transactivate reporter genes in mammalian cell lines and transgenic mice in the presence of steroids and ecdysone A (Christopherson et al., 1992; No et al., 1996). Later, Suhr et al., 1998, showed that the nonsteroidal ecdysone agonist tebufenozide induced high levels of transactivation of reporter genes in mammalian cells via Bombyx mori EcR (BmEcR) in the absence of exogenous heterodimer couplers.

[0267] International patent applications PCT / US97 / 05330 (WO 97 / 38117) and PCT / US99 / 08381 (WO99 / 58155) disclose methods for regulating the expression of exogenous genes. A DNA construct containing an exogenous gene and an ecdysone response element is activated by a second DNA construct containing an ecdysone receptor, which, in the presence of its ligand, optionally in the presence of a receptor capable of acting as a silencing partner, binds to the ecdysone response element to induce gene expression. In this example, the ecdysone receptor was isolated from *Drosophila melanogaster*. Typically, such systems require the presence of a silencing partner, preferably a retinoid X receptor (RXR), to provide optimal activation. In mammalian cells, the insect ecdysone receptor (EcR) can heterodimerize with the mammalian retinoid X receptor (RXR) for ligand-dependent regulation of target or exogenous gene expression. International patent application PCT / US98 / 14215 (WO 99 / 02683) discloses that the ecdysone receptor isolated from the silkworm moth is functional in mammalian systems and does not require an exogenous dimer partner.

[0268] U.S. Patent 6,265,173 discloses that members of the steroid / thyroid superfamily receptors can be combined with the Drosophila melanogaster supercoiled receptor (USP) or fragments thereof containing at least the USP dimerized domain for use in gene expression systems. U.S. Patent 5,880,333 discloses a Drosophila melanogaster EcR and supercoiled (USP) heterodimer system for use in plants, wherein the transactivation domain and DNA-binding domain are located on two distinct heterodimeric proteins. In each of these cases, the transactivation domain and DNA-binding domain (such as the native EcR in International Patent Application PCT / US98 / 14215 or the modified EcR in International Patent Application PCT / US97 / 05330) are incorporated into a single molecule, and other heterodimer couples (USP or RXR) are used in their native state.

[0269] International patent application PCT / US01 / 0905 discloses an inducible gene expression system based on the ecdysone receptor, in which the transactivation domain and DNA-binding domain are separated from each other by placing them on two different proteins, resulting in a significant reduction in background activity in the absence of a ligand, and a significant increase in activity compared to the background in the presence of a ligand. This two-hybrid system is a significantly improved inducible gene expression regulation system compared to the two systems disclosed in applications PCT / US97 / 05330 and PCT / US98 / 14215. It is believed that this two-hybrid system utilizes the ability of a pair of interacting proteins to position the transcriptional activation domain in a more advantageous position relative to the DNA-binding domain, such that the transactivation domain more effectively activates the promoter when the DNA-binding domain binds to a DNA-binding site on the gene (see, for example, U.S. Patent 5,283,173). This two-hybrid gene expression system comprises two gene expression cassettes; the first encodes a DNA-binding domain fused to a nuclear receptor polypeptide, and the second encodes a transactivation domain fused to a different nuclear receptor polypeptide. The presence of the ligand is thought to induce a conformational change, which promotes the interaction between the first and second polypeptides, leading to the dimerization of the DNA-binding domain and the trans-activating domain. Since the DNA-binding domain and the trans-activating domain are located on two different molecules, background activity is significantly reduced in the absence of the ligand.

[0270] Certain modifications to the two-hybrid system can also provide improved sensitivity to non-steroidal ligands (e.g., ecdysone A (“PonA”) or muristone A (“MurA”) compared to steroidal ligands. That is, non-steroidal ligands provide higher gene transcriptional activity at lower ligand concentrations compared to steroids. Furthermore, the two-hybrid system avoids some side effects that can occur due to RXR overexpression when using unmodified RXR as the switch partner. In preferred two-hybrid systems, the native DNA-binding domain and transactivation domain of EcR or RXR are eliminated, thus reducing the chances of these hybrid molecules interacting with other steroid hormone receptors present in the cell, resulting in reduced side effects.

[0271] The ecdysone receptor (EcR) is a member of the nuclear receptor superfamily and is classified into subfamily 1, H (referred to as "group H nuclear receptors" in this paper). Members of each group share 40-60% amino acid identity in the E (ligand-binding) domain (Laudet et al., A Unified Nomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163). Other members of this nuclear receptor subfamily 1, H include: ubiquitous receptor (UR), orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXR interactor protein-15 (RIP-15), liver x receptor β (LXRβ), steroid hormone receptor-like protein (RLD-1), liver x receptor (LXR), liver x receptor (LXRα), farnesoid x receptor (FXR), receptor interactor protein 14 (RIP-14), and farnesoid receptor (HRR-1).

[0272] In some cases, inducible promoters (“IPs”) can be small molecule ligand-inducible gene switches based on the dual-peptide ecdysone receptor, such as those from Intrexon Corporation. Gene switches. In some cases, gene switches may be selected from ecdysone-based receptor components, such as those described in, but not limited to, any of the following systems: PCT / US2001 / 009050 (WO 2001 / 070816); US Patents 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT / US2001 / 030608 (WO 2002 / 029075); US Patents 8,105,825; 8,168,426; PCT / 1J52002 / 005235 (WO 2002 / 066613); US Application 10 / 468,200 (US Publication 20120167239); PCT / US2002 / 005706 (WO 2002 / 066614); US Patent 7,531,326; 8,236,556; 8,598,409; PCT / U52002 / 005090 (WO 2002 / 066612); US Patent 8,715,959 (US Publication 20060100416); PCT / US2002 / 005234 (WO 2003 / 027266); US Patent 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT / U52002 / 005708 (WO 2002 / 066615); U.S. Application 10 / 468,192 (U.S. Publication 20110212528); PCT / US2002 / 005026 (WO 2003 / 027289); U.S. Patent 7,563,879; 8,021,878; 8,497,093; PCT / US2005 / 015089 (WO 2005 / 108617); U.S. Patent 7,935,510; 8,076,454; PCT / U52008 / 011270 (WO 2009 / 045370); U.S. Application 12 / 241,018 (U.S. Publication 20090136465); PCT / US2008 / 011563 (WO U.S. Patent Application No. 2009 / 048560; U.S. Application No. 12 / 247,738 (U.S. Publication No. 20090123441); PCT / US2009 / 005510 (WO 2010 / 042189); U.S. Application No. 13 / 123,129 (U.S. Publication No. 20110268766); PCT / US2011 / 029682 (WO 2011 / 119773); U.S. Application No. 13 / 636,473 (U.S. Publication No. 20130195800); PCT / US2012 / 027515 (WO 2012 / 122025); and U.S. Patent No. 9,402,919; each of which is incorporated herein by reference in its entirety.

[0273] A system for regulating CAR and / or cytokine expression in host cells is provided, comprising a polynucleotide expressing a gene-switching polypeptide disclosed herein. Further, a polynucleotide encoding a gene-switching polypeptide for ligand-inducible control of gene expression is provided, wherein the gene-switching polypeptide comprises (a) a first gene-switching polypeptide comprising a DNA-binding domain (DBD) fused to a nuclear receptor ligand-binding domain; and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a nuclear receptor ligand-binding domain; wherein the first and second gene-switching polypeptides are linked by a linker. In some embodiments, the linker is a cleavable or ribosomal skipping linker sequence selected from 2A, GSG-2A, the GSG linker (SEQ ID NO: 129), the SGSG linker (SEQ ID NO: 130), furinlink variants, and derivatives thereof. In some embodiments, the 2A linker is a p2A linker, a T2A linker, an F2A linker, or an E2A linker.

[0274] In some embodiments, the DNA-binding domain (DBD) comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid / thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. In some cases, the transactivation domain comprises at least one of the VP16 transactivation domain and the B42 acid activator transactivation domain. In other cases, the nuclear receptor ligand-binding domain comprises at least one of ecdysone receptor (EcR), ubiquitous receptor, orphan receptor 1, NER-1, steroid hormone nuclear receptor 1, retinol X receptor-interacting protein-15, liver X receptor β, steroid hormone receptor-like protein, liver X receptor, liver X receptor α, farnesol X receptor, receptor-interacting protein 14, and famesol receptor. In some embodiments, the nuclear receptor ligand-binding domain is derived from the ecdysone receptor polypeptide sequences of SEQ ID NO: 135 and 136.

[0275] In yet another embodiment, the first gene-switching polypeptide comprises a GAL4 DBD fused to an EcR nuclear receptor ligand-binding domain, and the second gene-switching polypeptide comprises a VP16 transactivation domain fused to a retinoid receptor X (RXR) nuclear receptor ligand-binding domain. In some cases, the first and second gene-switching polypeptides are linked by a linker sequence selected from 2A, GSG-2A, GSG linker (SEQ ID NO: 129), SGSG linker (SEQ ID NO: 130), furinlink variants, and derivatives thereof.

[0276] In some embodiments, two or more polypeptides encoded by the polynucleotides described herein may be separated by intercalation sequences encoding a linker polypeptide. In some cases, the linker is a readily cleavable linker. In some embodiments, the target polypeptide is expressed as a fusion protein linked by a readily cleavable linker polypeptide. In some embodiments, the readily cleavable linker polypeptide may be any one or more of the following: F / T2A, T2A, p2A, 2A, GSG-p2A, the GSG linker (SEQ ID NO: 129), and furinlink variants. In some embodiments, the linker polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 116-130.

[0277] In some cases, viral 2A sequences can be used. 2A elements can be shorter than IRES, having 5 to 100 base pairs. In some cases, 2A sequences can have lengths of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides. Genes linked by 2A can be expressed in a single open reading frame, and “self-cleavage” can co-translationally occur between the last two amino acids (GP) at the C-terminus of the 2A polypeptide, producing equal amounts of co-expressed protein.

[0278] The viral 2A sequence can be approximately 20 amino acids long. In some cases, the viral 2A sequence may contain the shared motif Asp-Val / Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO:286). The shared motif sequence can function co-translatorily. For example, it can prevent the formation of normal peptide bonds between glycine and proline residues, which can lead to ribosome jumping and cleavage of nascent polypeptides. This effect can produce equimolar levels of multiple genes.

[0279] 2A peptides allow the translation of multiple proteins into a polypeptide within a single open reading frame, which can then be “cleaved” into a single polypeptide via ribosomal jumping mechanisms (Funston, Kallioinen et al. 2008). In some embodiments, the 2A sequence may include: F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, and any combination thereof.

[0280] In some cases, the vector may contain an IRES sequence and a 2A linker sequence. In other cases, the expression of multiple genes linked by a 2A peptide can be promoted by a spacer sequence preceding the 2A peptide (GSG (SEQ ID NO:129)). In some cases, the construct can combine a spacer, linker, adaptor, promoter, or a combination thereof. For example, the construct may have a spacer with different 2A peptides (SGSG (SEQ ID NO:130) or GSG (SEQ ID NO:129)) and a furin protease linker (RAKR (SEQ ID NO:125)) cleavage site. The spacer may be I-Ceui. In some cases, the linker can be engineered. For example, the linker can be designed to include chemical properties such as hydrophobicity. In some cases, at least two linker sequences can produce the same protein. In other cases, multiple linkers can be used in the vector. For example, the target gene can be separated by at least two linkers.

[0281] In some embodiments, two or more polypeptides encoded by the polynucleotides described herein may be separated by intercalation sequences encoding linker polypeptides. In some cases, the linker is a readily cleavable linker. In some embodiments, the target polypeptide is expressed as a fusion protein linked by readily cleavable linker polypeptides. In some embodiments, the readily cleavable linker polypeptide may be any one or both of the following: Furinlink, fmdv, p2a, GSG-p2a, and / or fp2a as described in SEQ ID NO: 119, 120, 121, 125, and 128.

[0282] In some implementations, the linker may be used in the polynucleotide described herein. The linker may be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some cases, the linker may link functional domains together (as in flexible and rigid linkers) or release free functional domains in vivo, as in an in vivo cleavable linker.

[0283] Linkomers can improve biological activity, increase expression yield, and achieve desired pharmacokinetic profiles. Linkomers may also contain hydrazones, peptides, disulfide bonds, or thioesthers.

[0284] In some cases, the linker sequences described herein may include flexible linkers. Flexible linkers can be applied when the linked domains require a certain degree of mobility or interaction. Flexible linkers can consist of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Flexible linkers can have sequences primarily composed of Gly and Ser residues (“GS” linker segments). An example of a flexible linker can have the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO:287). By adjusting the copy number “n”, the length of this exemplary GS linker can be optimized to achieve proper isolation of functional domains or maintain necessary inter-domain interactions. For example, (Gly-Gly-Gly-Gly-Ser)n, where n is 3, is the (G4S)3 linker shown in SEQ ID NO:127. Besides GS linkers, other flexible linkers can also be used for recombinant fusion proteins. In some cases, flexible linkers can be enriched with small amino acids or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[0285] Flexible linkers included in the linker sequences described herein can be enriched with small or polar amino acids, such as Gly and Ser, to provide good flexibility and solubility. Flexible linkers may be a suitable choice when fusion protein domains require specific movement or interactions. Additionally, although flexible linkers lack a rigid structure in some cases, they can serve as inert linkers to maintain distance between functional domains. The length of flexible linkers can be tuned to allow for proper folding or to achieve optimal biological activity of the fusion protein.

[0286] In some cases, the linkers described herein may further include rigid linkers. Rigid linkers can be used to maintain a fixed distance between peptide domains. Examples of rigid linkers include (to name a few): α-helix linkers, Pro-rich sequences, (XP)n, X-Pro backbones, A(EAAAK)nA (n = 2-5) (SEQ ID NO: 288). In some cases, rigid linkers can exhibit a relatively rigid structure by employing an α-helix structure or by containing multiple Pro residues.

[0287] In some cases, the linkers described herein can be cleavable. In others, the linkers are non-cleavable. Non-cleavable linkers can covalently link functional domains together to function as a single molecule throughout in vivo or in vitro processes. Linkers can also be in vivo cleavable. Cleavable linkers can be introduced to release free functional domains in vivo. For example, cleavable linkers can be cleaved by the presence of reducing agents or proteases. For instance, the reduction of disulfide bonds can be used to generate cleavable linkers. In the case of disulfide-bonded linkers, cleavage can occur through a cleavage event involving the exchange of disulfide bonds with thiols such as glutathione. In other cases, in vivo cleavage of linkers in recombinant fusion proteins can also be carried out by proteases that are expressed in vivo in specific cells or tissues or confined to certain cellular compartments under pathological conditions (e.g., cancer or inflammation). In some cases, cleavable linkers can allow targeted cleavage. For example, the specificity of many proteases can provide slower linker cleavage in confined compartments. Cleavable linkers can also contain hydrazones, peptides, disulfide bonds, or thioesthers. For example, hydrazones can confer serum stability. In other cases, hydrazones can allow cleavage in acidic compartments. These acidic compartments can have a pH of up to 7. Linkers can also include thioethers. Thioethers can be non-reducible. Thioethers can be engineered for intracellular proteolytic degradation.

[0288] In some embodiments, the fmdv linker polypeptide comprises a sequence that may be at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:126. In some embodiments, the fmdv linker polypeptide is one or more linkers that link two or more fusion proteins and are encoded in a single vector. In some cases, the fmdv linker polypeptide may be encoded by a multinucleotide open reading frame (ORF) nucleic acid sequence. In some cases, the ORF encoding fmdv comprises or is composed of the sequence of SEQ ID NO:272. In some embodiments, the polynucleotide encoding fmdv is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:272.

[0289] In some cases, the linker polypeptide may be a "p2a" linker. In some embodiments, the p2a polypeptide may contain at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical sequence to SEQ ID NO:119. In some embodiments, the p2a linker polypeptide may be one or more linkers that link two or more fusion proteins and are encoded in a single vector. In some cases, the p2a linker polypeptide may be encoded by a multinucleotide open reading frame (ORF) nucleic acid sequence. In some embodiments, the ORF encoding p2a contains or is composed of the sequence of SEQ ID NO:266. In some cases, the polynucleotide encoding p2a may be or may be approximately at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:266.

[0290] In some embodiments, the linker polypeptide may be a “GSG-p2a” linker. In some embodiments, the GSG-p2a linker polypeptide may contain at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical sequence to SEQ ID NO:120. In some embodiments, the GSG-p2a linker polypeptide may be one or more linkers that link two or more fusion proteins and are encoded in a single vector. In some embodiments, the GSG-p2a linker polypeptide may be encoded by a polynucleotide open reading frame (ORF) nucleic acid sequence. The ORF encoding GSGp2a may contain the sequence of SEQ ID NO:267. In some cases, the polynucleotide encoding GSG-p2a may be or may be approximately at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:267.

[0291] The linker polypeptide can be the “fp2a” linker provided herein. In some embodiments, the fp2a linker polypeptide may contain at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical sequence to SEQ ID NO:121. In some cases, the fp2a linker polypeptide may be one or more linkers that link two or more fusion proteins and encode them in a single vector. In some cases, the fp2a linker polypeptide may be encoded by a polynucleotide open reading frame (ORF) nucleic acid sequence. In some embodiments, the polynucleotide encoding the fp2a linker may be or may be about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:268.

[0292] In some cases, linkers can be engineered. For example, linkers can be designed to include chemical properties such as hydrophobicity. In some cases, at least two linker sequences can produce the same protein. The sequences are identical to or approximately 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences in SEQ ID NO: 116 to SEQ ID NO: 130. In other cases, multiple linkers can be used in the vector. For example, the target gene and one or more gene-switching polypeptide sequences described herein can be separated by at least two linkers. In some cases, the gene can be separated by 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers.

[0293] Linkomers can be engineered. The methods for designing linkers can be computational. In some cases, computational methods may include graphical techniques. Computational methods can be used to search for suitable peptides from three-dimensional peptide structure libraries derived from databases. For example, the Brookhaven Protein Data Bank (PDB) can be used to span the spatial distances between selected amino acids in a linker.

[0294] In some embodiments, the polypeptide construct encodes a polynucleotide comprising a furin protease polypeptide and a 2A polypeptide, wherein the furin protease polypeptide and the 2A polypeptide are linked by a polypeptide linker comprising at least three hydrophobic amino acids. In some cases, the at least three hydrophobic amino acids are selected from a list consisting of glycine (Gly) (G), alanine (Ala) (A), valine (V) (V), leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (Phe) (F), methionine (Met) (M), and tryptophan (Trp) (W). In some cases, the polypeptide linker may also comprise one or more GS linker sequences, such as (GS)n (SEQ ID NO:289), (SG)n (SEQ ID NO:290), (GSG)n (SEQ ID NO:291), and (SGSG)n (SEQ ID NO:292), where n can be any number from zero to fifteen.

[0295] A method for obtaining improved expression of a polypeptide construct is provided, the method comprising: providing a polynucleotide encoding a polypeptide construct comprising a first functional polypeptide and a second functional polypeptide, wherein the first functional polypeptide and the second functional polypeptide are linked by a linker polypeptide comprising a sequence having at least 60% identity with the sequence APVKQ (SEQ ID NO: 293); and expressing the polynucleotide in a host cell, wherein the expression results in improved expression of the polypeptide construct compared to a corresponding polypeptide construct not having a linker polypeptide comprising a sequence having at least 60% identity with the sequence APVKQ (SEQ ID NO: 293).

[0296] In other cases, the linker can be an IRES. The term "internal ribosome entry site (IRES)" as used herein is intended to refer to an internal ribosome entry site. In vectors containing IRES sequences, the first gene can be translated via a cap-dependent ribosome scanning mechanism with its own 5'-UTR, while translation of subsequent genes can be accomplished by directly recruiting ribosomes to the IRES in a cap-independent manner. IRES sequences can allow eukaryotic ribosome binding and initiation of translation without binding to the 5' cap end. IRES sequences can allow the expression of multiple genes from a single transcript (Mountford and Smith 1995).

[0297] Exemplary IRES sequences can be found in SEQ ID NO:146 and 147. In some cases, the polynucleotide encoding 2xRbm3 IRES may be or may be approximately at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:146. In some cases, the polynucleotide encoding EMCV IRES may be or may be approximately at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:147.

[0298] In some embodiments, a system for regulating the expression of CARs and cytokines in host cells comprises a first gene expression cassette containing a first polynucleotide encoding a first polypeptide; a second gene expression cassette containing a second polynucleotide encoding a second polypeptide; and a ligand; wherein the first and second polypeptides comprise one or more of the following: (i) a trans-activation domain; (ii) a DNA-binding domain; and (iii) a ligand-binding domain; (iv) a CAR; (vi) a cytokine; and / or (vii) a cell tag, such that, upon contact of a host cell with the first and second gene expression cassettes in the presence of the ligand, the CAR and cytokine are expressed in the host cell. In some cases, the CAR is a ROR-1 CAR, and the cytokine is mbIL-15. In some cases, the ROR-1 CAR and mbIL-15 are co-expressed with a cell tag. In some cases, the ROR-1 CAR and mbIL-15 are each co-expressed with a cell tag. In other cases, the ROR-1 CAR is expressed with a cell tag, while mbIL-15 is expressed with a second cell tag. Exemplary configurations of the gene expression cassette are depicted in Figures 1 and 2. In other cases, the CAR is the ROR-1 CAR, and the cytokine is IL-12. In some cases, the ROR-1 CAR and IL-12 are co-expressed with one cellular tag. In some cases, the ROR-1 CAR and IL-12 are each co-expressed with a cellular tag. In other cases, the ROR-1 CAR is expressed with the cellular tag, while IL-12 is expressed with a second cellular tag.

[0299] In some embodiments, a system for regulating the expression of CARs and cytokines in host cells comprises a first gene expression cassette containing a first polynucleotide encoding a first polypeptide; a second gene expression cassette containing a second polynucleotide encoding a second polypeptide; and a ligand; wherein the first polypeptide comprises one or more of the following: (i) a trans-activation domain; (ii) a DNA-binding domain; and (iii) a ligand-binding domain, and the second polypeptide comprises one or more of the following: (i) a CAR; (ii) a cytokine; and / or (iii) a cell tag, such that, upon contact between a host cell and the first and second gene expression cassettes in the presence of the ligand, the CAR and / or cytokine are expressed in the host cell. In some cases, the CAR is ROR-1CAR, and the cytokine is mbIL-15. In some cases, ROR-1 CAR and mbIL-15 are each co-expressed with a cell tag. In other cases, ROR-1 CAR is expressed with a first cell tag, while mbIL-15 is expressed with a second cell tag.

[0300] An exemplary configuration of a system for regulating ROR-1 CAR and cytokine expression in host cells is shown in Figure 1A-1E and Figures 2A-2E The description is as follows. In some embodiments, a virus or virus-based system is used to introduce the gene expression cassette into immune effector cells. Examples of non-virus-based delivery systems as described herein include the SB11 transposon system, the SB100X transposon system, the SB110 transposon system, and the piggyBac transposon system. In one embodiment, one or more Sleeping Beauty transposons are used to introduce the gene expression cassette into immune effector cells.

[0301] Figure 1A-1E An exemplary embodiment of a gene expression cassette encoding constitutive expression of ROR-1 CAR, cytokines (such as mbIL-15 or IL-12), and cellular tags is described. Figure 1A-1B Exemplary gene expression cassette designs for ROR-1 CAR, mblL-15, and cell tags with various configurations are depicted. In this embodiment, a Sleeping Beauty transposon is used to introduce the gene expression cassette into immune effector cells. Figure 1C-1D A gene expression cassette configuration was described, in which the ROR-1 CAR can be contained in one gene expression cassette, and mbIL-15 and a cell tag are contained in a second gene expression cassette. In this embodiment, the gene expression cassette is introduced into immune effector cells using one or more Sleeping Beauty transposons.

[0302] Figures 2A-2EExemplary implementations of gene expression cassettes encoding the expression of ROR-1 CAR, inducible cytokines (such as mbIL-15), and / or cell tags are described. Figure 2A-2D Exemplary gene expression cassette designs for ROR-1 CAR, mblL-15, and cell tags with various configurations under the control of an inducible promoter are depicted. Figure 2E This is an exemplary embodiment of a gene expression cassette encoding a gene-switching polypeptide as described herein. In this embodiment, the gene expression cassette is introduced into immune effector cells using one or more SleepingBeauty transposons.

[0303] ligands

[0304] In some embodiments, the ligand used for inducible gene switch regulation may be selected from, but is not limited to, any of the following: N-[(1R)-1-(1,1-dimethylethyl)butyl]-N'-(2-ethyl-3-methoxybenzoyl)-3,5-dimethylbenzohydrazine (also known as veledimex), (2S,3R,5R,9R,10R,13R,14S,17R)-17-[(2S,3R)-3,6-dihydroxy-6-methylheptane-2-yl]-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-6-one; N'-( 3,5-Dimethylbenzoyl)-N'-[(3R)-2,2-dimethyl-3-hexyl]-2-ethyl-3-methoxybenzohydrazine; 5-methyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(3,5-dimethyl-benzoyl)-N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazine; 5-methyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(3,5-dimethoxy-4-methyl-benzoyl)-N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazine; 5-methyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(1-tert-butyl-butyl)-N′ -(3,5-dimethyl-benzoyl)-hydrazide; 5-methyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 5-ethyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(3,5-dimethyl-benzoyl)-N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-ethyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(3,5-dimethoxy-4-methyl-benzoyl)-N′-(1-ethyl-2,2-dimethyl-propyl)-hydrazide; 5-ethyl-2, 3-Dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethyl-benzoyl)-hydrazide; 5-Ethyl-2,3-dihydro-benzo[1,4]dioxin-6-carboxylic acid N′-(1-tert-butyl-butyl)-N′-(3,5-dimethoxy-4-methyl-benzoyl)-hydrazide; 3,5-dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethoxy-4-methyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-butyl)-hydrazide 3,5-Dimethoxy-4-methylbenzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide; 2-methoxy-nicotinic acid N-(1-tert-butyl-pentyl)-N′-(4-ethyl-benzoyl)-hydrazide; 3,5-dimethylbenzoic acid N-(2,2-dimethyl-1-phenyl-propyl)-N′-(4-ethyl-benzoyl)-hydrazide; 3,5-dimethylbenzoic acid N-(1-tert-butyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide; and 3,5-dimethoxy-4-methylbenzoic acid N-(1-tert-butyl-pentyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

[0305] In some cases, the ligand used for dose regulation control of ecdysone receptor-based inducible gene switch regulation can be selected from, but is not limited to, any of the following: ecdysone steroids (such as ecdysone, 20-hydroxyecdysone, ecdysone A, muscosterone A, etc.), 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazine, such as those disclosed in U.S. Patents 6,013,836, 5,117,057, 5,530,028, and 5,378,726 and U.S. Publications 2005 / 0209283 and 2006 / 0020146; oxadiazoline, as described in U.S. Publication 2004 / 0171651; dibenzoylalkylcyanohydrazine, as disclosed in European Application 461,809; N-alkyl-N,N'-diaroylhydrazine, as described in U.S. Patent 5,225,44 Those disclosed in 3; N-acyl-N-alkylcarbonylhydrazine, such as those disclosed in European Application 234,994; N-aramidyl-N-alkyl-N'-aramidylhydrazine, such as those described in U.S. Patent 4,985,461; aminoketones, such as those described in U.S. Publication 2004 / 0049037; each of the above is incorporated herein by reference, as well as other similar materials, including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharbazoside, oxidosterols, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-α-6-α-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-bisphosphonates, juvenile hormone III, etc. Examples of diacylhydrazine ligands that can be used in this disclosure include RG-115819 (N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazine-), RG-115932 ((R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazine), and RG-115830 (N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazine). See, for example, U.S. Patent Application Serial No. 12 / 155,111 and PCT Application PCT / US2008 / 006757, both of which are incorporated herein by reference in their entirety.

[0306] Non-virus-based delivery systems

[0307] Non-virus-based delivery systems such as the "Sleeping Beauty (SB) transposon system" can also be used to introduce nucleic acids encoding the CAR described herein into immune effector cells. The Sleeping Beauty transposon system refers to a synthetic DNA transposon system for introducing DNA sequences into the chromosomes of vertebrates. Exemplary SB transposon systems are described, for example, in U.S. Patents 6,489,458 and 8,227,432. The Sleeping Beauty transposon system comprises a Sleeping Beauty (SB) transposon and an SB transposon. As used herein, the Sleeping Beauty transposon system may comprise a Sleeping Beauty transposon polypeptide and active derivatives, variants, and / or fragments, as well as active Sleeping Beauty transposon polynucleotides, derivatives, variants, and / or fragments. In some embodiments, the Sleeping Beauty transposon is provided as mRNA. In some aspects, the mRNA encodes SB10, SB11, SB100x, or SB110 transposases. In some aspects, the mRNA comprises a cap and a poly-A tail.

[0308] DNA transposons move from one DNA site to another in a simple cut-and-paste manner. Transposition is a precise process in which a defined segment of DNA is cut from one DNA molecule and moved to another site in the same or different DNA molecule or genome. Like other Tc1 / mariner-type transposases, SB transposases insert transposons into TA dinucleotide base pairs in the acceptor DNA sequence. The insertion site can be elsewhere in the same DNA molecule or in another DNA molecule (or chromosome). There are approximately 200 million TA sites in mammalian genomes, including the human genome. TA insertion sites are repeated during transposon integration. This repetition of the TA sequence is a hallmark of transposition and is used to determine mechanisms in some experiments. Transposases can be encoded within the transposon, or they can be provided from another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are most useful as genetic tools because they cannot independently continue to be cut off and re-inserted after insertion. SB transposons are envisioned as non-viral vectors for introducing genes into the genomes of vertebrates and for gene therapy. In short, the Sleeping Beauty (SB) system (Hackett et al., Mol Ther 18:674-83, (2010)) is suitable for the genetic modification of immune effector cells (Cooper et al., Blood 105:1622-31, (2005)). In one implementation, this includes two steps: (i) electrotransfer of a DNA plasmid expressing the SB transposon (i.e., chimeric antigen receptor (CAR) for redirecting T cell-specific T cells (Jin et al., Gene Ther 18:849-56, (2011); Kebriaei et al., Hum Gene Ther 23:444-50, (2012)) and the SB transposase; and (ii) proliferation and expansion of T cells stably expressing the integronosome on designer artificial antigen-presenting cells (AaPCs) derived from the K562 cell line (also known as AaPCs (activated and expanded cells)). In another implementation, step (ii) is omitted, and the genetically modified T cells are cryopreserved or immediately infused into the patient.

[0309] In one implementation, the SB transposon system is described, for example, in Hudecek et al., Critical Reviews in Biochemistry and Molecular Biology, 52:4, 355-380 (2017), Singh et al., Cancer Res (8):68 (2008). April 15, 2008, and Maiti et al., J Immunother. 36(2):112–123 (2013), which are incorporated herein by reference in their entirety.

[0310] In some embodiments, the ROR-1 CAR and mbIL-15 are encoded in a transposon DNA plasmid vector, while the SB transposase is encoded in a separate vector. In some embodiments, the ROR-1 CAR described herein is encoded in a transposon DNA plasmid vector, mbIL-15 is encoded in a second transposon DNA plasmid vector, and the SB transposase is encoded in a third DNA plasmid vector. In some embodiments, the CAR is encoded together with a killing tag, such as HER1t, HER1t1, CD20, or CD20t-1. In some embodiments, mbIL-15 is encoded together with a killing tag, such as HER1t, HER1t1, CD20, or CD20t-1. Exemplary configurations of the ROR-1 CAR, killing tag, and / or mbIL-15 are depicted in Figure 1.

[0311] In one implementation, the ROR-1 CAR can be co-expressed with mbIL-15 and a cell tag from a transposon DNA plasmid vector. In a further implementation, the ROR-1 CAR can be under the control of an inducible promoter. In another implementation, mbIL-15 can be under the control of an inducible promoter. In one aspect, the inducible promoter can be a gene switch ligand-inducible promoter. In some cases, the inducible promoter can be a small molecule ligand-inducible gene switch based on a dual-peptide ecdysone receptor, such as... Gene switch. In some implementations, the ROR-1 CAR, mbIL-15, and kill tag can be configured in one, two, or more transposons. Figure 2 illustrates an exemplary configuration of the ROR-1 CAR or mbIL15 under the control of an inducible promoter.

[0312] In the implementation scheme, the SB11 transposon system, the SB100X transposon system, the SB110 transposon system, the piggyBac transposon system (see, for example, U.S. Patent 9,228,180, Wilson et al., “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecular Therapy 15:139-145 (2007), which is incorporated herein by reference in its entirety) and / or the piggyBat transposon system (see, for example, Mitra et al., “Functional characterization of piggyBat from the bat Myotis lucifugusunveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234-239 (2013)) are used to deliver ROR-1 CAR and other genetic elements into cells. Other transposases and transposon systems are provided in U.S. Patents 7,148,203, 8,227,432, U.S. Patent Publication 2011 / 0117072, Mates et al., Nat Genet, 41(6):753-61 (2009). doi:10.1038 / ng.343. May 3, 2009 (e.g.), Gene Ther., 18(9):849-56 (2011). doi:10.1038 / gt.2011.40. March 31, 2011 (e.g.), and Ivics et al., Cell. 91(4):501-10, (1997), each of which is incorporated herein by reference in its entirety.

[0313] In other embodiments, the ROR-1 CAR and other genetic elements, such as cytokines, mbIL-15, and / or HER1t / HER1t1 / CD20 / CD20t-1 tags, can be integrated into the DNA of immune effector cells via recombinases and integrative expression vectors. These vectors can be randomly integrated into the host cell's DNA or can include recombination sites to achieve specific recombination between the expression vector and the host cell's chromosome. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell's chromosome to achieve the expression of the desired protein. In some embodiments, targeted integration is facilitated by the presence of a sequence on the donor polynucleotide that is homologous to the flanking sequence of the integration site. For example, targeted integration using the donor polynucleotides described herein can be achieved using conventional transfection techniques, such as those used to generate gene knockout or knock-in through homologous recombination. In other embodiments, targeted integration is facilitated by the presence of a sequence on the donor polynucleotide that is homologous to the flanking sequence of the integration site, and by contacting the cell with the donor polynucleotide in the presence of a site-specific recombinase. Site-specific recombinases, or simply recombinases, are polypeptides that catalyze conserved site-specific recombination between compatible recombination sites. As used herein, site-specific recombinases include native polypeptides and their active derivatives, variants, and / or fragments, as well as native polynucleotides, derivatives, variants, and / or fragments encoding active recombinases.

[0314] Recombinases can be introduced into target cells before, simultaneously with, or after the introduction of a targeting vector. Recombinases can be introduced directly into cells as proteins, for example, using liposomes, coated particles, or microinjection. Alternatively, a suitable expression vector can be used to introduce a polynucleotide (DNA or messenger RNA) encoding the recombinase into the cell. The aforementioned targeting vector components can be used to construct expression cassettes containing sequences encoding the recombinase of interest. However, recombinase expression can be regulated in other ways, for example, by placing recombinase expression under the control of an adjustable promoter (i.e., a promoter that can selectively induce or inhibit its expression).

[0315] Recombinases can originate from either fusion or dissociation enzyme families. The integrase recombinase family has over one hundred members and includes, for example, FLP, Cre, and λ integrase. The integrase family, also known as the tyrosine family or λ integrase family, uses the catalytic hydroxyl group of tyrosine residues to perform a nucleophilic attack on the phosphodiester bond of DNA. Typically, members of the tyrosine family initially form a nick in the DNA, which later results in a double-strand break. Examples of tyrosine family integrases include Cre, FLP, SSV1, and lambda(λ) integrase. In the dissociation enzyme family (also known as the serine recombinase family), conserved serine residues form a covalent link with a DNA target site (Grindley et al., (2006) Ann Rev Biochem 16:16).

[0316] In one embodiment, the recombinase is an isolated polynucleotide sequence comprising a nucleic acid sequence encoding the recombinase, selected from SPβc2 recombinase, SF370.1 recombinase, Bxb1 recombinase, A118 recombinase, and φRv1 recombinase. Examples of serine recombinases are described in detail in U.S. Patent 9,034,652, which is incorporated herein by reference in its entirety.

[0317] The recombinant enzyme used to carry out the present invention can be generated or purified as described above. The polypeptide having the desired recombinant enzyme activity can be purified to the desired purity level by methods known in the field of protein ammonium sulfate precipitation and purification, including but not limited to size fractionation, affinity chromatography, HPLC, ion exchange chromatography, and heparin agarose affinity chromatography (e.g., Thorpe & Smith, Proc. Nat. Acad. Sci. 95:5505-5510, 1998).

[0318] In one embodiment, the recombinase can be introduced into a eukaryotic cell containing a recombinant attachment site for which recombination is required by any suitable method. Methods for introducing functional proteins into cells, such as microinjection or other methods, are well known in the art. The introduction of purified recombinase protein ensures the transient existence of the protein and its function, which is generally a preferred embodiment. Alternatively, the gene encoding the recombinase can be included in an expression vector for transforming cells, wherein the polynucleotide encoding the recombinase is operatively linked to a promoter mediating the expression of the polynucleotide in eukaryotic cells. The recombinase polypeptide can also be introduced into eukaryotic cells via messenger RNA encoding the recombinase polypeptide. It is generally preferred that the recombinase exists only for the time necessary to insert the nucleic acid fragment into the modified genome. Therefore, the lack of persistence associated with most expression vectors is not necessarily a disadvantage. The recombinase gene can be introduced into the cell before, after, or simultaneously with the introduction of the exogenous target polynucleotide. In one embodiment, the recombinase gene is contained within a vector carrying the polynucleotide to be inserted; the recombinase gene can even be included within the polynucleotide.

[0319] In one embodiment, the method for site-specific recombination includes providing a first recombination site and a second recombination site; contacting the first and second recombination sites with a prokaryotic recombinase polypeptide to result in recombination between the recombination sites, wherein the recombinase polypeptide mediates recombination between the first and second recombination sites, the first recombination site being attP or attB, the second recombination site being attB or attP, and the recombinase being selected from Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase, and Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attB.

[0320] Further embodiments provide for introducing a site-specific recombinase into a cell whose genome will be modified. One embodiment relates to a method for obtaining site-specific recombination in a eukaryotic cell, comprising providing a eukaryotic cell containing a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide to result in recombination between the recombination attachment sites, wherein the recombinase polypeptide is mediated to recombine between the first and second recombination attachment sites, the first recombination attachment site being a phage genome recombination attachment site (attP) or a bacterial genome recombination attachment site (attB), the second recombination attachment site being attB or attP, and the recombinase being selected from Listeria monocytogenes phage recombinase, Streptococcus pyogenes phage recombinase, Bacillus subtilis phage recombinase, Mycobacterium tuberculosis phage recombinase, and Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP, and when the first recombination attachment site is attP, the second recombination attachment site is attB. In one embodiment, the recombinase is selected from A118 recombinase, SF370.1 recombinase, SPβc2 recombinase, φRv1 recombinase, and Bxb1 recombinase. In one embodiment, the recombination results in integration.

[0321] Regardless of the method used to introduce exogenous nucleic acids into host cells, various tests can be performed to confirm the presence of recombinant DNA sequences in the host cells. These tests include, for example, "molecular biology" tests known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" tests, such as those for detecting the presence of specific peptides, for example by immunological means (ELISA and Western blotting) or by tests described herein for identifying peptides, proteins, or nucleic acids falling within the scope of this invention.

[0322] Virus-based delivery system

[0323] This document also provides a virus-based delivery system in which the nucleic acid of the present invention is inserted. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands),) lentivirus-based vectors (e.g., the lentivirus-based pLPI from Life Technologies (Carlsbad, Calif.),) and retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH), and herpesviruses. In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. Lentiviral vectors also have the additional advantage of low immunogenicity. Typically, and in embodiments, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent 6,326,193).

[0324] In one embodiment, a lentiviral vector is provided comprising a backbone and a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a stem domain; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain. Optionally, the vector further comprises nucleic acid encoding a truncated epidermal growth factor receptor (HER1t or HER1t1), CD20t-1, or full-length CD20.

[0325] In some cases, a vector comprising a backbone and a nucleic acid sequence encoding (1) a truncated epidermal growth factor receptor, such as HER1t or HERt-1 or a functional variant thereof; and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0326] In some cases, a vector comprising a backbone and a nucleic acid sequence is provided, the nucleic acid sequence encoding (1) a full-length CD20, a truncated CD20 or a functional variant thereof, and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0327] In one embodiment, the nucleic acid encoding a ROR-1 specific CAR is cloned into a vector containing a lentiviral backbone component. Exemplary backbone components include, but are not limited to, pFUGW and pSMPUW. The pFUGW lentiviral vector backbone is a self-inactivating (SIN) lentiviral vector backbone and removes unwanted HIV-1 viral sequences, thereby reducing the likelihood of tumorigenesis, harmful mutations, and regeneration of infectious particles. In another embodiment, the vector encoding the ROR-1 CAR also encodes mbIL-15 in a single construct. In another embodiment, the ROR-1 CAR and mbIL-15 are encoded on two separate lentiviral vectors. In some embodiments, mbIL-15 is expressed together with a truncated epidermal growth factor receptor tag. In another embodiment, the ROR-1 CAR may be co-expressed with mbIL-15 and a cellular tag from a single lentiviral vector. In a further embodiment, the ROR-1 CAR may be under the control of an inducible promoter. In yet another embodiment, mbIL-15 may be under the control of an inducible promoter. In one aspect, the inducible promoter may be a gene switch ligand inducible promoter. In some cases, inducible promoters can be small molecule ligand-inducible gene switches based on the dual-peptide ecdysone receptor, such as... Gene switch.

[0328] In one embodiment, the ROR-1 CAR described herein comprises an anti-ROR-1 scFv, a human CD8 hinge and transmembrane domain, and human 4-1BB and CD3ζ signaling domains. In another embodiment, the ROR-1 CAR of the present invention comprises an anti-ROR-1 scFv, a human CD8 hinge and transmembrane domain, human 4-1BB and CD3ζ signaling domains, and optionally, a truncated epidermal growth factor receptor (HER1t or HER1t-1) tag. Other suitable vectors include integrative expression vectors that can be randomly integrated into the DNA of a host cell, or may include recombination sites to achieve specific recombination between the expression vector and the host cell chromosome. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell chromosome to achieve the expression of the desired protein. Examples of vectors integrated in a site-specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNA). TM 5 / FRT), or, for example, the cre-lox system found in the pExchange-6 core vector from Stratagene (La Jolla, Calif.). Examples of vectors that integrate randomly into the host cell chromosome include, for example, pcDNA3.1 from Invitrogen (Carlsbad, Calif.) (when introduced in the absence of T-antigen) and pCI or pFN10A(ACT)FLEXI from Promega (Madison, Wis.). TM Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. Although many promoters have recently been shown to also contain functional elements downstream of the start site, these promoter elements are typically located in a region 30–110 bp upstream of the start site. The spacing between promoter elements is usually flexible, thus preserving promoter function when the elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. It appears that individual elements can act synergistically or independently to activate transcription, depending on the promoter.

[0329] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it.

[0330] Another example of a suitable promoter is human extension growth factor 1α1 (hEF1a1). In implementations, vector constructs comprising the CAR described herein contain a functional variant of hEF1a1.

[0331] However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention described above. The use of inducible promoters provides a molecular switch capable of turning on the expression of an operatively linked polynucleotide sequence (when such expression is desired) or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In one aspect, the inducible promoter may be a gene switch ligand inducible promoter. In some cases, inducible promoters can be small molecule ligand-inducible gene switches based on the dual-peptide ecdysone receptor, such as... Gene switch.

[0332] To assess the expression of the CAR or a portion thereof described herein, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells attempting to be transfected or infected via viral or non-viral vectors. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection procedures. Both the selectable marker and the reporter gene may be side-mounted with appropriate regulatory sequences to achieve expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neomycin resistance genes (neo) and ampicillin resistance genes. In some embodiments, a truncated epidermal growth factor receptor (HER1t or HER1t-1) tag may be used as a selectable marker gene.

[0333] Reporter genes can be used to identify potentially transfected cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, encoding a polypeptide whose expression is indicated by easily detectable properties, such as enzyme activity. Reporter gene expression is determined at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Letters 479:79-82 (2000)). Suitable expression systems are well-known and can be prepared using known techniques or commercially available. Typically, a construct exhibiting the highest expression level of the reporter gene with a minimal 5' flanking region is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription.

[0334] In the implementation scheme, the viral vector described herein may comprise an hEF1a1 promoter to drive transgene expression, a bovine growth hormone polyA sequence to enhance transcription, a marmot hepatitis virus posttranscriptional regulatory element (WPRE), and an LTR sequence derived from the pFUGW plasmid.

[0335] Methods for introducing genes into cells and expressing genes within cells are well known. In the case of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be transferred into host cells by physical, chemical, or biological means.

[0336] Physical methods for introducing polynucleotides into host cells, such as immune effector cells, include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001)). In embodiments, methods for introducing polynucleotides into host cells include calcium phosphate transfection or polyethyleneimine (PEI) transfection. In some embodiments, methods for introducing polynucleotides into host cells include electroporation.

[0337] Biological methods for introducing target polynucleotides into host cells, such as immune effector cells, include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0338] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0339] In the case of viral delivery systems, an exemplary delivery medium is liposomes. Lipid formulations can be used to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within a lipid bilayer of liposomes, attached to liposomes via linkers associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist as bilayers, micelles, or “folded” structures. They can also simply be dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic. For example, lipids include small droplets of fat naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0340] Suitable lipids are available from commercial sources. For example, dimyristicophosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dihexadecyl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY); cholesterol (“Chol”) is available from Calbiochem-Behring; dimyristicophosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a general term encompassing various monolayer and multilayer lipid mediators formed by the formation of closed lipid bilayers or aggregates. Liposomes are characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in excess aqueous solution, multilayered liposomes spontaneously form. The lipid components undergo rearrangement before forming closed structures, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-10 (1991)). However, compositions exhibiting structures in solution that differ from normal vesicle structures are also included. For example, lipids may present as micelle structures or simply as heterogeneous aggregates of lipid molecules. Lipid transfection of amine-nucleic acid complexes has also been considered.

[0341] Cells containing ROR-1 CAR and vector

[0342] This document provides engineered cells expressing the CAR described herein. In some embodiments, the engineered cells described herein are immune effector cells. In one embodiment, this document provides an immune effector cell comprising a vector containing a backbone and a nucleic acid sequence encoding (1) a truncated epidermal growth factor receptor (HER1t or HER1t1), and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain.

[0343] In some embodiments, the immune effector cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; (e) a CD3ζ signaling domain; and (f) a truncated epidermal growth factor receptor (HER1t or HER1t1).

[0344] In an embodiment, this document provides an immune effector cell comprising (1) a cell tag serving as a kill switch, selection marker, biomarker, or a combination thereof, and (2) a chimeric antigen receptor (CAR), wherein the CAR comprises (a) a ROR-1 antigen-binding domain; (b) a spacer region; (c) a transmembrane domain; (d) a co-stimulatory signaling domain comprising 4-1BB or CD28 or both; and (e) a CD3ζ signaling domain. In an embodiment, the cell tag is HER1t, HER1t1, CD20t-1, or CD20.

[0345] In the implementation scheme, the immune effector cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In the implementation scheme, the cell exhibits antitumor activity when the ROR-1 antigen-binding domain binds to ROR-1.

[0346] Modified immune effector cells

[0347] Immune effector cells modified to express one or more of the heterologous genes or peptides described herein are provided. Immune effector cells modified to express at least one of the ROR-1 CAR described herein and the HER1t, HER1t1, CD20, and CD20t-1 tags are provided. In some cases, immune effector cells modified to express at least one of the ROR-1 CAR, mbIL-15, and the HER1t, HER1t1, CD20, and CD20t-1 tags disclosed herein are provided.

[0348] As used in this article, "T cells" or "T lymphocytes" are a type of lymphocyte that plays an important role in cell-mediated immunity. They are distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of T cell receptors (TCRs) on their cell surface.

[0349] In some embodiments, the modified immune effector cells are modified immune cells comprising T cells and / or natural killer cells. T cells or T lymphocytes are leukocyte subtypes involved in cell-mediated immunity. Exemplary T cells include T helper cells, cytotoxic T cells, TH17 cells, memory stem cell-like T cells (TSCM), naive T cells, memory T cells, effector T cells, regulatory T cells, or natural killer T cells.

[0350] Helper T cells (TH cells) assist other white blood cells in the immune process, which includes the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. In some cases, TH cells are referred to as CD4+ T cells due to the expression of the CD4 glycoprotein on their cell surface. Helper T cells are activated by the presentation of peptide antigens via MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells rapidly divide and secrete small proteins called cytokines, which regulate or assist in active immune responses. These cells can differentiate into several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[0351] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated into an unresponsive state through IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases.

[0352] Memory T cells are a subset of antigen-specific T cells that persist long after infection resolution. Upon re-exposure to their homologous antigens, they rapidly proliferate into a large number of effector T cells, thus providing the immune system with a "memory" of past infections. Memory T cells include the following subtypes: stem cell memory T cells (TSCM), central memory T cells (TCM cells), and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells can express the cell surface proteins CD45RO, CD45RA, and / or CCR7.

[0353] Regulatory T cells (Treg cells), formerly known as suppressor T cells, play a role in maintaining immune tolerance. Their main function is to stop T cell-mediated immunity at the end of the immune response and to suppress autoreactive T cells that evade the negative selection process in the thymus.

[0354] Natural killer T cells (NKT cells) link the innate and adaptive immune systems. Unlike regular T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CD1d. Once activated, these cells can perform functions belonging to both Th (helper) cells and Tc (cytotoxic T) cells (i.e., cytokine production and the release of cell-lysing / cytokilling molecules). They are also capable of recognizing and eliminating some tumor cells and cells infected with herpesviruses.

[0355] Natural killer (NK) cells are a type of cytotoxic lymphocyte in the innate immune system. In some cases, NK cells provide first-line defense against viral infections and / or tumor formation. NK cells detect MHC presented on infected or cancerous cells, triggering cytokine release, which subsequently induces lysis and apoptosis. NK cells can further detect stressed cells in the absence of antibodies and / or MHC, allowing for a rapid immune response.

[0356] Modified immune effector cell dose

[0357] In some implementations, a specific amount of modified immune effector cells is administered to a subject in need, and this amount is determined based on efficacy and the potential to induce cytokine-related toxicity. In some cases, the amount of modified immune effector cells comprises approximately 10... 2 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 3 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 4 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 5 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 5 To about 10 8 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 5 To about 10 7 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 6 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 106 To about 10 8 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 7 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 5 To about 10 6 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 6 To about 10 7 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 7 To about 10 8 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 8 To about 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 9 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 8 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 7 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 6 Modified immune effector cells / kg. In some cases, the amount of modified immune effector cells contains approximately 10 5 Modified immune effector cells / kg.

[0358] In some implementations, the CAR-T cells are used as ROR-1-specific CAR-T cells. In some cases, the amount of ROR-1-specific CAR-T cells contains approximately 10 2 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 3 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 4 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 5 To about 10 9CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 5 To about 10 8 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 5 To about 10 7 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 6 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 6 To about 10 8 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 7 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 5 To about 10 6 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 6 To about 10 7 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 7 To about 10 8 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 8 To about 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 9 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 8 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 7 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 6 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 5 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 4CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 3 CAR-T cells / kg. In some cases, the amount of ROR-1 specific CAR-T cells contains approximately 10 2 CAR-T cells / kg.

[0359] Sources of immune effector cells

[0360] In some aspects, the embodiments described herein include methods for preparing and / or expanding antigen-specific redirected immune effector cells (e.g., T cells, Tregs, NK cells, or NK T cells), comprising transfecting cells with an expression vector containing a DNA (or RNA) construct encoding a CAR, and then optionally stimulating the cells with feeder cells, recombinant antigens, or antibodies against the receptor to induce cell proliferation. In some aspects, the cells (or cell populations) engineered to express CAR are stem cells, iPS cells, T cells differentiated from iPS cells, immune effector cells, or precursors of these cells.

[0361] The sources of immune effector cells can include allogeneic and autologous sources. In some cases, immune effector cells can differentiate from stem cells or induced pluripotent stem cells (iPSCs). Therefore, cells for engineering according to the implementation scheme can be isolated from umbilical cord blood, peripheral blood, human embryonic stem cells, or iPSCs. For example, allogeneic T cells can be modified to include chimeric antigen receptors (and optionally lack functional TCRs). In some aspects, the immune effector cells are primary human T cells, such as T cells derived from human peripheral blood mononuclear cells (PBMCs). PBMCs can be collected from peripheral blood, or from bone marrow after stimulation with G-CSF (granulocyte colony-stimulating factor), or from umbilical cord blood. In one aspect, the immune effector cells are pan T cells. After transfection or transduction (e.g., with a CAR expression construct), the cells can be infused immediately or cryopreserved. In some aspects, after transfection or transduction, the cells can be preserved in a cytokine bath that may include IL-2 and / or IL-21 until ready for infusion. In some respects, following transfection, cells can proliferate in vitro as a host population for days, weeks, or months, approximately 1, 2, 3, 4, 5 days, or more after gene transfer. In other respects, after transfection, transfectant clones are cloned, and clones exhibiting expression of a single integrated or episome-retained expression cassette or plasmid, as well as chimeric antigen receptor expression, are expanded in vitro. Clones selected for expansion exhibit the ability to specifically recognize and lyse target cells expressing antigens. Recombinant T cells can be expanded by stimulation with IL-2 or other cytokines binding a common γ chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). Recombinant T cells can also be expanded by stimulation with artificial antigen-presenting cells. Recombinant T cells can be expanded on artificial antigen-presenting cells or with antibodies such as OKT3 cross-linked to CD3 on the surface of T cells. Subgroups of recombinant T cells can be further selected using magnetic bead-based separation methods and / or fluorescence-activated cell sorting techniques, and then further cultured with AaPCs. In another respect, genetically modified cells can be cryopreserved.

[0362] T cells can also be obtained from many sources, including bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art can be used. In some embodiments of the invention, any techniques known to those skilled in the art can be used, such as... T cells are isolated from blood units collected from a subject. In one embodiment, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected via apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment of the invention, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is calcium-deficient and may be magnesium-deficient or may lack many (if not all) divalent cations. The initial activation step in the absence of calcium results in amplified activation. Those skilled in the art will readily understand that the washing step can be performed by methods known to those skilled in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as calcium-free buffers. 2+ Mg-free 2+ Cells can be placed in PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, unwanted components can be removed from single samples, and the cells can be resuspended directly in the culture medium.

[0363] In another embodiment, this is achieved by lysing red blood cells and consuming monocytes (e.g., through...). T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugation and washing. Specific T cell subsets, such as CD3, can be further isolated using positive or negative selection techniques. + CD28 + CD4 + CD8 + CD45RA + and CD45RO + T cells. In another embodiment, CD14+ cells are consumed from the T cell population. For example, in one embodiment, this is achieved by using beads conjugated with anti-CD3 / anti-CD28 (i.e., 3x28) (such as...). Incubation of T cells with M-450 CD3 / CD28 T cells for a sufficient period of time for positive selection of the desired T cells is used to isolate T cells. In one embodiment, this period of time is approximately 30 minutes. In a further embodiment, this period of time ranges from 30 minutes to 36 hours or longer and all integer values ​​in between. In a further embodiment, this period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, this period of time is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Using a longer incubation time (such as 24 hours) can increase cell yield for isolating T cells from leukemia patients. A longer incubation time can be used to isolate T cells in any situation where T cells are fewer than other cell types, such as in isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immune-impaired individuals. Furthermore, using a longer incubation time can improve the efficiency of capturing CD8+ T cells. Therefore, by simply shortening or lengthening the time allowed for T cells to bind to CD3 / CD28 beads, and / or by increasing or decreasing the bead-to-T-cell ratio (as further described herein), a T-cell subset can be preferentially selected or excluded at the start of culture or at other points in the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, a T-cell subset can be preferentially selected or excluded at the start of culture or at other desired points in time. Those skilled in the art will recognize that multiple rounds of selection can also be used within the context of this invention. In some embodiments, a selection procedure may need to be performed and “unselected” cells may be used during activation and expansion. “Unselected” cells may also undergo further rounds of selection.

[0364] Enrichment of T cell populations by negative selection can be accomplished using an antibody combination targeting specific surface markers of the negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some implementations, enrichment or positive selection may be required for cells that typically express CD4+. + CD25 + CD62L hi GITR + and FoxP3 + Regulatory T cells. Alternatively, in some embodiments, regulatory T cells are consumed by anti-CD25 conjugated beads or other similar selection methods.

[0365] To separate the desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., particles, such as beads) can be varied. In some embodiments, it may be necessary to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In another embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a concentration greater than 100 million cells / ml is used. In a further embodiment, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 350 million, 40 million, 45 million, or 50 million cells / ml are used. In another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used. Using high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest (such as CD28-negative T cells) or cells from samples containing a large number of tumor cells (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and will be needed. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0366] In relevant embodiments, it may be necessary to use a lower concentration of cells. This is achieved by significantly diluting the mixture of T cells and the surface (e.g., particles, such as beads) to minimize the interaction between the particles and cells. This selects cells expressing high levels of the desired antigen to bind to the particles. For example, CD4+ T cells express higher levels of CD28 and are more effective at capture than CD8+ T cells at diluted concentrations. In one embodiment, the cell concentration used is 5 x 102 6 / ml. In other embodiments, the concentration used may be approximately 1x10⁻⁶. 5 / ml to 1x10 6 / ml and any integer values ​​in between.

[0367] In other implementations, cells can be incubated for varying durations at different rates on a rotator at 2-10°C or room temperature.

[0368] T cells used for stimulation can also be frozen after the washing step. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this case, one approach involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1°C per minute and storing them in the gas phase of a liquid nitrogen tank. Other controlled freezing methods can be used, as well as uncontrolled rapid freezing at -20°C or in liquid nitrogen. In some embodiments, as described herein, the cryopreserved cells are thawed and washed, and then allowed to stand at room temperature for 1 hour before activation using the method of the present invention.

[0369] In some embodiments, blood samples or apheresis products are also provided for collection from the subject at a time prior to the potential need for expanded cells as described herein. Therefore, the source of cells to be expanded can be collected at any necessary point in time, and the desired cells, such as T cells, can be isolated and frozen for subsequent use in T-cell therapy for any number of diseases or conditions that would benefit from T-cell therapy, such as those described herein. In one embodiment, the blood sample or apheresis product is taken from a generally healthy subject. In some embodiments, the blood sample or apheresis product is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed one, and the cells of interest are isolated and frozen for subsequent use. In some embodiments, T cells may be expanded, frozen, and subsequently used. In some embodiments, samples are collected from the patient shortly after the diagnosis of a specific disease as described herein, prior to any treatment. In a further embodiment, prior to any number of relevant treatments, cells are isolated from a blood sample or apheresis of the subject, including but not limited to treatment with agents such as nastatinumab, efazolinumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immune scavengers such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, and radiation. These drugs inhibit calcium-dependent phosphatases—calcium-dependent phosphatases (cyclosporine and FK506)—or inhibit p70S6 kinases important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, (1991); Henderson et al., Immun 73:316-321, (1991); Bierer et al., Curr. Opin. Immun 5:763-773, (1993)). In a further embodiment, cells are isolated from the patient and frozen for subsequent use in conjunction with bone marrow or stem cell transplantation, T-cell ablation therapy with chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (e.g., before, during, or after). In another embodiment, cells are pre-isolated and can be frozen for subsequent use after B-cell ablation therapy (such as agents that respond to CD20, such as rituximab).

[0370] In a further embodiment of the invention, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that the quality of T cells obtained shortly after certain cancer treatments, particularly with drugs that impair the immune system, and during the period when the patient typically recovers from treatment, may be optimal or improved due to their ability to expand in vitro. Similarly, after in vitro manipulation using the methods described herein, these cells can be in a preferred state for implantation and enhanced in vivo expansion. Therefore, in the context of the invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Further, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and modulation protocols may be used to create conditions in the subject in which the reproliferation, recirculation, regeneration, and / or expansion of particular cell types is advantageous, especially within a defined time window following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0371] T cell activation and expansion

[0372] Whether before or after T cells are engineered to express the CAR described herein, T cells can typically be activated and expanded using methods described, for example, in U.S. Patents 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0373] Typically, T cells, as described herein, expand by contact with a surface coated with reagents that stimulate CD3 / TCR complex-related signals and ligands that stimulate co-stimulatory molecules on the cell surface. Specifically, T cell populations can be stimulated as described herein, such as by contact with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies immobilized on the surface, or by contact with protein kinase C activators that bind calcium ionocarriers (e.g., lichenin). For co-stimulation of helper molecules on the T cell surface, ligands binding helper molecules are used. For example, T cell populations can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. For stimulating CD4... + T cells or CD8 +T cell proliferation was achieved using anti-CD3 and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used in the same manner as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp. Med. 190(9):1319-1328, (1999); Garland et al., J. Immunol Meth. 227(1-2):53-63, (1999)).

[0374] In some embodiments, the primary stimulatory signal and co-stimulatory signal of the T cells can be provided by different methods. For example, the agent providing each signal can be in solution or conjugated to a surface. When conjugated to a surface, the agent can be conjugated to the same surface (i.e., "cis" arrangement) or a different surface (i.e., "trans" arrangement). Alternatively, one agent can be conjugated to a surface while the other is in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution or conjugated to the surface. In some embodiments, both agents can be in solution. In another embodiment, the agent can be in a soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind to the agent. In this regard, see, for example, the artificial antigen-presenting cells (APCs) contemplated for activating and amplifying the T cells of the present invention in U.S. Patent Application Publications 20040101519 and 20060034810.

[0375] In one embodiment, two agents are immobilized on beads (immobilized on the same beads, i.e., "cis", or immobilized on different beads, i.e., "trans"). For example, the agent providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, while the agent providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment; and both agents are co-immobilized on the same beads with equal molecular weights. In one embodiment, each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used in a 1:1 ratio. In certain aspects of the invention, such an anti-CD3 antibody:anti-CD28 antibody ratio bound to the beads is used such that an increase in T cell expansion is observed compared to expansion observed using a 1:1 ratio. In a particular embodiment, an increase of about 1 to about 3 times is observed compared to expansion observed using a 1:1 ratio. In one embodiment, the CD3 antibody:CD28 antibody ratio bound to the beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies bind to the particles compared to anti-CD3 antibodies, i.e., the CD3:CD28 ratio is less than 1. In some embodiments of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies binding to the beads is greater than 2:1. In one particular embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:100. In another embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:75. In a further embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:50. In another embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:30. In an embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:10. In another embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 1:3. In yet another embodiment, the antibody-binding to the beads uses a CD3:CD28 ratio of 3:1.

[0376] A particle-to-cell ratio of 1:500 to 500:1 or any integer value therebetween can be used to stimulate T cells or other target cells. Those skilled in the art will readily understand that the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, smaller beads can bind only a small number of cells, while larger beads can bind many cells. In some embodiments, a cell-to-particle ratio ranging from 1:100 to 100:1 or any integer value therebetween, and in further embodiments, a ratio including 1:9 to 9:1 or any integer value therebetween, can also be used to stimulate T cells. As described above, the ratio of anti-CD3 and anti-CD28 conjugated granules to T cells leading to T cell stimulation can vary, but certain values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, where one of these ratios is a T cell / granule ratio of at least 1:1. In one embodiment, a granule:cell ratio of 1:1 or less is used. In a particular embodiment, the granule:cell ratio is 1:5. In a further embodiment, the granule:cell ratio can be varied depending on the number of days of stimulation. For example, in one embodiment, the particle:cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day for up to 10 days, resulting in a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In one particular embodiment, the particle:cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added daily or every other day until the final ratio on the first day is 1:1, and the final ratio on the third and fifth days of stimulation is 1:5. In another embodiment, the particle:cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In yet another embodiment, particles are added daily or every other day until the final ratio on the first day is 1:1, and the final ratio on the third and fifth days of stimulation is 1:10. Those skilled in the art will understand that a variety of other ratios are applicable to the present invention. In particular, the ratio will vary depending on particle size and cell size and type.

[0377] In a further embodiment described herein, immune effector cells such as T cells are combined with drug-coated beads, then the beads and cells are separated, followed by cell culture. In an alternative embodiment, the drug-coated beads and cells are not separated prior to culture, but are cultured together. In a further embodiment, the beads and cells are first concentrated by applying a force (such as magnetism), resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[0378] For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, the cells (e.g., 10 4 Up to 10 9 T cells) and beads (e.g., in a 1:1 ratio) M-450 CD3 / CD28 T paramagnetic beads, or from Miltenyi Biotec. MicroBeads are mixed in a buffer such as PBS (free of divalent cations, such as calcium and magnesium). Similarly, those skilled in the art will readily understand that any cell concentration can be used. For example, target cells may be very rare in a sample and constitute only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Therefore, any cell number is within the scope of this invention. In some embodiments, it may be necessary to significantly reduce the volume of particles and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / ml is used. In another embodiment, a concentration greater than 100 million cells / ml is used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 350 million, 40 million, 45 million, or 50 million cells / ml are used. In another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. In a further embodiment, concentrations of 125 million or 150 million cells / ml can be used. Using high concentrations can lead to increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are desirable in some embodiments. For example, using high cell concentrations allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0379] In one embodiment described herein, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any integer number of hours in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several stimulation cycles may also be required, so that the culture time of T cells may be 60 days or longer. Suitable conditions for T cell culture include appropriate culture media (e.g., essential medium or RPMI 1640 or X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives used for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate), and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, and Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with adequate amounts of serum (or plasma), or a defined set of hormones and / or cytokines in sufficient amounts to promote T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the cell culture to be injected into the subject. Target cells are maintained under conditions necessary for growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0380] T cells exposed to different stimulation times can exhibit different characteristics. For example, typical blood or apheresis peripheral blood mononuclear cell products have a larger population of cytotoxic T cells or suppressor T cells than T cells. C CD8 + helper T cell population (T) H CD4 + A T cell population is generated by stimulating CD3 and CD28 receptors to expand T cells in vitro. This T cell population is mainly composed of T cells before approximately day 8-9. H Cell composition, and after about 8-9 days, this T cell population contains more and more T cells. C Cell population. Therefore, depending on the therapeutic objective, the subject is infused with a mixture primarily composed of T cells. H A population of T cells may be advantageous. Similarly, if T cells have been isolated... CExpanding the cell's antigen-specific subgroup to a greater extent may be beneficial.

[0381] Furthermore, apart from CD4 and CD8 markers, other phenotypic markers showed significant changes, but these changes were largely reproducible during cell expansion. Therefore, this reproducibility allows for the customization of activated T cell products for specific purposes.

[0382] In some cases, immune effector cells (e.g., T cells) of the embodiments are co-cultured with activated and proliferating cells (AaPCs) to aid in cell expansion. AaPCs may also be referred to as artificial antigen-presenting cells (APCs). For example, antigen-presenting cells (APCs) can be used to prepare therapeutic compositions and cell therapy products of the embodiments. In one aspect, an AaPC ​​may be a genetically modified K562 cell. General guidance on the preparation and use of antigen-presenting systems can be found, for example, in U.S. Patents 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publications 2009 / 0017000 and 2009 / 0004142; and International Publication WO2007 / 103009, each of which is incorporated herein by reference. In yet another aspect of the embodiments, culturing genetically modified CAR cells comprises culturing the genetically modified CAR cells in the presence of dendritic cells or activated and proliferating cells (AaPCs) that stimulate the expansion of immune effector cells expressing CAR. In a further aspect, AaPC ​​contains a CAR-binding polypeptide or TCR-binding antibody or fragment thereof expressed on the surface of the AaPC. In some cases, AaPC ​​may include other molecules that activate or co-stimulate T cells. In some cases, other molecules may include membrane-bound Cγ cytokines. In a further aspect, AaPC ​​is inactivated or irradiated, or has been tested and confirmed to be free of infectious material. In a further aspect, culturing genetically modified CAR cells in the presence of AaPC ​​includes culturing genetically modified CAR cells in a medium containing soluble cytokines such as IL-15, IL-21, and / or IL-2. Cells can be cultured at ratios of about 10:1 to about 1:10, about 3:1 to about 1:5, about 1:1 to about 1:3 (immune effector cells to AaPC), or any range thereof. For example, a co-culture of T cells and AaPC ​​may be at a ratio of about 1:1, about 1:2, or about 1:3.

[0383] In one aspect, AaPCs may express CD137L. In some aspects, AaPCs may further express antigens targeted by CAR cells, such as ROR-1 (full-length, truncated, or any variant thereof). In other aspects, AaPCs may further express CD64, CD86, or mIL15. In some aspects, AaPCs may express at least one anti-CD3 antibody clone, such as OKT3 and / or UCHT1. In one aspect, AaPCs may be inactivated (e.g., by radiation). In one aspect, AaPCs may have been tested and confirmed to be free of infectious material. Methods for generating such AaPCs are known in the art. In one aspect, co-culturing a CAR-modified T cell population with AaPCs may include culturing cells at a ratio of about 10:1 to about 1:10, about 3:1 to about 1:5, about 1:1 to about 1:3 (T cells to AaPCs), or any range derived therefrom. For example, a co-culture of T cells and AaPCs may be at a ratio of about 1:1, about 1:2, or about 1:3. In one aspect, the culture step may further include culture with aminobisphosphonates (e.g., zoledronic acid).

[0384] In one aspect, the genetically modified CAR cell population is immediately infused into a subject or cryopreserved. In another aspect, the genetically modified CAR cell population is placed in a cytokine bath prior to infusion into a subject. In a further aspect, the genetically modified CAR cell population is cultured and / or stimulated for no more than 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, or 70 days. In one embodiment, stimulation includes co-culturing genetically modified CAR T cells with AaPC ​​to promote the growth of CAR-positive T cells. In another aspect, the genetically modified CAR cell population is stimulated for no more than: 1X stimulation, 2X stimulation, 3X stimulation, 4X stimulation, 5X stimulation, 6X stimulation, 7X stimulation, 8X stimulation, 9X stimulation, or 10X stimulation. In some cases, the genetically modified cells are cultured in vitro without the presence of AaPC. In some specific cases, the method of the embodiment further includes enriching the cell population with CAR-expressing immune effector cells (e.g., T cells) after the transfection and / or culture step. Enrichment may include fluorescence-activated cell sorting (FACS) to sort CAR-expressing cells. In a further aspect, sorting for CAR-expressing cells includes using a CAR-binding antibody. Enrichment may also include the consumption of CD56+ cells. In yet another aspect of the embodiment, the method further includes cryopreserving a sample of the genetically modified CAR cell population.

[0385] In some cases, AaPCs are incubated with peptides of optimal length, which allows the peptides to bind directly to MHC molecules without additional treatment. Alternatively, cells can express antigens of interest (i.e., in the case of MHC-independent antigen recognition). Furthermore, in some cases, APCs can express antibodies that bind to specific CAR peptides or to CAR peptides in general (e.g., universally activated and proliferating cells (uAPCs)). Such methods are disclosed in WO / 2014 / 190273, which is incorporated herein by reference. In addition to peptide-MHC molecules or antigens of interest, the AaPC ​​system may also contain at least one exogenous helper molecule. Any suitable number and combination of helper molecules can be used. Helper molecules can be selected from helper molecules such as co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD70 and B7.1 (B7.1 was formerly known as B7 and is also referred to as CD80), which, among other molecules, bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby influencing, for example, T cell proliferation, Th1 differentiation, short-term T cell survival, and cytokine secretion, such as interleukin (IL)-2. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Techniques, methods, and reagents for selecting, cloning, preparing, and expressing exemplary helper molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, for example, U.S. Patents 6,225,042, 6,355,479, and 6,362,001, which are incorporated herein by reference.

[0386] Cells selected to become AaPCs preferably have defects in intracellular antigen processing, intracellular peptide transport, and / or intracellular MHC class I or II molecule-peptide loading, or are thermophilic (i.e., less sensitive to temperature attack compared to mammalian cell lines), or have both defects and thermophilic properties. Preferably, cells selected to become AaPCs also lack the ability to express at least one endogenous counterpart of the exogenous MHC class I or II molecule and accessory molecule component introduced into the cell (e.g., endogenous MHC class I or II molecule and / or endogenous accessory molecules as described above). Furthermore, AaPCs preferably retain the defects and thermophilic properties that the cell possessed prior to its modification to generate an AaPC. Exemplary AaPCs are composed of or derived from transporters associated with antigen processing (TAP)-deficient cell lines such as insect cell lines. An exemplary poikilothermic insect cell line is a Drosophila cell line, such as the Schneider 2 cell line (see, for example, Schneider 1972, illustrative methods for the preparation, growth, and culture of Schneider 2 cells provided in U.S. Patents 6,225,042, 6,355,479, and 6,362,001).

[0387] In one embodiment, the AaPC ​​is also subjected to a freeze-thaw cycle. In an exemplary freeze-thaw cycle, the AaPC ​​can be frozen by contacting a suitable container holding the AaPC ​​with an appropriate amount of liquid nitrogen, solid carbon dioxide (i.e., dry ice), or a similar cryogenic material, causing the freezing to occur rapidly. The frozen AaPC ​​is then thawed by removing it from the cryogenic material and exposing it to room temperature conditions, or by accelerating the thawing process (where a warm water bath or warm hands are used to shorten the thawing time). Alternatively, the AaPC ​​can be frozen and stored for an extended period before thawing. The frozen AaPC ​​can also be thawed and subsequently freeze-dried before further use. Preferably, preservatives that may adversely affect the freeze-thaw procedure, such as dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and other preservatives, are not present in the culture medium containing the AaPC ​​that has undergone the freeze-thaw cycle, or are substantially removed, for example, by transferring the AaPC ​​to a culture medium that is substantially free of such preservatives.

[0388] In a further embodiment, the heterologous and endogenous nucleic acids relative to the AaPC ​​can be inactivated by cross-linking, such that cell growth, replication, or nucleic acid expression substantially does not occur after inactivation. In one embodiment, the AaPC ​​is inactivated at a point after the expression of exogenous MHC and helper molecules, the presentation of these molecules on the surface of the AaPC, and the loading of selected one or more peptides onto the presented MHC molecules. Thus, such inactivated and peptide-loaded AaPCs, while substantially unable to proliferate or replicate, retain the selected peptide presentation function. Preferably, cross-linking also produces AaPCs substantially free of contaminating microorganisms (such as bacteria and viruses) without substantially diminishing the antigen-presenting cell function of the AaPC. Thus, cross-linking preserves important AaPC ​​function while helping to alleviate safety concerns regarding cell therapy products developed using AaPCs. For methods related to cross-linking and AaPCs, see, for example, U.S. Patent Application Publication 20090017000, which is incorporated herein by reference.

[0389] In some embodiments, engineered antigen-presenting cells (APCs) are further provided. For example, as described above, such cells can be used to proliferate immune effector cells in vitro. In a further aspect, the engineered APCs themselves can be administered to a patient, thereby stimulating the expansion of immune effector cells in vivo. The engineered APCs of the embodiments can be used as therapeutic agents. In other embodiments, the engineered APCs can be used as therapeutic agents that can stimulate the activation of endogenous immune effector cells specific to the target antigen and / or increase the activity or persistence of adoptive immune effector cells specific to the target antigen.

[0390] As used herein, the term "engineered APC" refers to a cell containing at least a first transgene, wherein the first transgene encodes HLA. Such an engineered APC may further contain a second transgene for expressing an antigen, such that the antigen is complexed with HLA and presented on the surface of the APC. In some aspects, the engineered APC may be an antigen-presenting cell type (e.g., dendritic cells). In further aspects, the engineered APC may be generated from cell types that do not normally present antigens, such as T cells or T cell progenitor cells (referred to as "T-APC"). Thus, in some aspects, an engineered APC of an embodiment contains a first transgene encoding a target antigen and a second transgene encoding human leukocyte antigen (HLA), such that HLA is complexed with the epitope of the target antigen and expressed on the surface of the engineered APC. In some specific aspects, the HLA expressed in the engineered APC is HLA-A2.

[0391] In some aspects, the engineered APC of the implementation scheme may further include at least a third transgene encoding a co-stimulatory molecule. The co-stimulatory molecule may be a co-stimulatory cytokine, which may be a membrane-bound Cγ cytokine. In some aspects, the co-stimulatory cytokine is IL-15, such as membrane-bound IL-15. In some further aspects, the engineered APC may include an edited (deleted) gene. For example, repressive genes such as PD-1, LIM-3, CTLA-4, or TCR may be edited to reduce or eliminate the expression of that gene. The engineered APC of the implementation scheme may further include a transgene encoding any target antigen of interest.

[0392] care center

[0393] In one embodiment of this disclosure, the immune effector cells described herein are modified at a point of care. In some cases, the modified immune effector cells are also referred to as engineered T cells. In some cases, the point of care is located in a hospital or facility (e.g., a medical facility) near the subject requiring treatment. The subject undergoes apheresis, and peripheral blood mononuclear cells (PBMCs) or PBMC subsets can be enriched, for example, by washing or Ficoll separation. The enriched PBMCs or PBMC subsets can be cryopreserved in any suitable cryopreservation solution prior to further processing. In one case, the washing process is performed using a buffer solution containing human serum albumin. Immune effector cells, such as T cells, can be isolated by the selection methods described herein. In one case, the T cell selection method includes beads specific to CD3 on T cells or beads specific to CD4 and CD8 on T cells. In one case, the beads may be paramagnetic beads. The harvested immune effector cells can be cryopreserved in any suitable cryopreservation solution prior to modification. Immune effector cells can be thawed for up to 24, 36, 48, 72, or 96 hours before infusion. Thawed cells can be placed in cell culture buffers, such as RPMI supplemented with fetal bovine serum (FBS) or human serum AB, or in buffers containing cytokines such as IL-2 and IL-21, before modification. Alternatively, harvested immune effector cells can be modified immediately without cryopreservation.

[0394] In some cases, immune effector cells are modified by engineering / introducing a chimeric receptor, one or more cell tags, and / or cytokines into immune effector cells and then rapidly infusing them into a subject. In some cases, the source of immune effector cells may include allogeneic and autologous sources. In one case, the immune effector cells may be T cells or NK cells. In one case, the chimeric receptor may be a ROR-1 CAR. In another case, the cytokine may be mbIL-15. In one case, the mbIL-15 is SEQ ID NO:113, or a variant or fragment thereof. In yet another case, the expression of mbIL-15 is regulated by the ligand-inducible gene-switch expression system described herein. For example, a ligand such as veledimex may be delivered to the subject to regulate the expression of mbIL-15. In another case, the cytokine may be IL-12. In yet another case, the expression of IL-12 is regulated by the ligand-inducible gene-switch expression system described herein. For example, a ligand such as veledimex may be delivered to the subject to regulate the expression of IL-12.

[0395] On the other hand, veledimex is provided in doses of 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. Alternatively, lower doses of veledimex are provided, such as 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, or 20 mg. In one embodiment, veledimex is administered to the subject on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 prior to the infusion of modified immune effector cells. In another embodiment, veledimex is administered to the subject approximately every 12 hours, approximately every 24 hours, approximately every 36 hours, or approximately every 48 hours, continuing to be effective for a period of time after the infusion of modified immune effector cells. In one implementation, the effective duration of veledimex administration is approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 days. In other implementations, veledimex may be administered again after a rest period, after a withdrawal period, or when the subject experiences a relapse.

[0396] In certain circumstances, when adverse effects on the subject are observed or treatment is not required, cell tags can be activated, for example, via cetuximab, for conditional in vivo ablation of modified immune effector cells containing cell tags as described herein, such as truncated epidermal growth factor receptor tags.

[0397] In some implementations, these immune effector cells are modified via electroporation using the constructs described in Figures 1-2. In one case, an electroporator such as Lonza's Nucleofector is used. TM Electroporation is performed using an electroporator. In other embodiments, the vector containing the above-described construct is a non-viral or viral vector. In one case, the non-viral vector includes the Sleeping Beauty transposon-transposase system. In one case, specific sequences are used to electroporate immune effector cells. For example, immune effector cells can be electroporated with one transposon, then with DNA encoding the transposase, followed by electroporation with a second transposon. In another case, immune effector cells can be electroporated simultaneously with all transposons and the transposase. In yet another case, immune effector cells can be electroporated with the transposase, then with two transposons or one transposon at a time. During continuous electroporation, immune effector cells can rest for a period of time before the next electroporation step.

[0398] In some cases, modified immune effector cells do not undergo proliferation and activation steps. In others, they do not undergo incubation or culture steps (e.g., in vitro proliferation). In some cases, modified immune effector cells are placed in a buffer containing IL-2 and IL-21 prior to infusion. In other cases, they are placed or left to stand in a cell culture buffer, such as a cell culture buffer supplemented with fetal bovine serum (FBS) (e.g., RPMI), prior to infusion. Modified immune effector cells may be harvested, washed, and prepared in saline buffer for infusion into the recipient prior to infusion.

[0399] In one scenario, the subject has undergone lymphocyte consumption prior to infusion. In other scenarios, lymphocyte consumption is not required; instead, modified immune effector cells are rapidly infused into the subject. Exemplary lymphocyte consumption protocols are listed in Tables 2 and 3 below:

[0400] Table 2. Scheme 1

[0401] D-5 Fludarabine 25 mg / m2, cyclophosphamide 250 mg / m2 D-4 Fludarabine 25 mg / m2, cyclophosphamide 250 mg / m2 D-3 Fludarabine 25 mg / m2 IV, cyclophosphamide 250 mg / m2 D-2 REST D-1 REST D0 T-cell infusion

[0402] Table 3. Scheme 2

[0403]

[0404]

[0405] In another scenario, the subject experiences minimal lymphocyte depletion. Minimal lymphocyte depletion in this context refers to a reduced lymphocyte depletion regimen that allows the subject to receive infusions 1, 2, or 3 days after the lymphocyte depletion regimen. In one scenario, a reduced lymphocyte depletion regimen may include lower doses of fludarabine and / or cyclophosphamide. In another scenario, a reduced lymphocyte depletion regimen may include a shortened lymphocyte digestion period, such as 1 or 2 days.

[0406] In one implementation, immune effector cells are modified by engineering / introducing chimeric receptors and cytokines into the immune effector cells and then rapidly infusing them into a subject. In other cases, immune effector cells are modified by engineering / introducing chimeric receptors and cytokines into the cells and then infusing them into a subject within at least 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hours. In other cases, immune effector cells are modified by engineering / introducing chimeric receptors and cytokines into immune effector cells and then infusing them into subjects within 0, <1, <2, <3, <4, <5, <6, or <7 days.

[0407] In some embodiments, a specific amount of modified effector cells is administered to a subject in need, and this amount is determined based on efficacy and the potential to induce cytokine-related toxicity. In another embodiment, the modified effector cells are CARs. + and CD3 + Cells. In some cases, the amount of modified effector cells contains approximately 10. 4 To about 10 9 Modified effector cells / kg. In some cases, the amount of modified effector cells contains approximately 10 4 To about 10 5 Modified effector cells / kg. In some cases, the amount of modified effector cells contains approximately 10 5 To about 10 6 Modified effector cells / kg. In some cases, the amount of modified effector cells contains approximately 10 6 To about 10 7 Modified effector cells / kg. In some cases, the amount of modified effector cells may be >10. 4 But ≤10 5Modified effector cells / kg. In some cases, the amount of modified effector cells may be >10. 5 But ≤10 6 Modified effector cells / kg. In some cases, the amount of modified effector cells may be >10. 6 But ≤10 7 Modified effector cells / kg.

[0408] In one implementation, modified immune effector cells target cancer by direct delivery to a region of tumor tissue. For example, in ovarian cancer, modified immune effector cells can be delivered intraperitoneally (IP) to the abdomen or peritoneal cavity. This IP delivery can be performed by placing a port for delivering chemotherapy drugs or by using a pre-existing port. Other methods for regional delivery of modified immune effector cells may include catheter infusion into the resection cavity, ultrasound-guided intratumoral injection, hepatic artery infusion, or intrapleural delivery.

[0409] In one embodiment, the subject in need may begin treatment with a first dose of modified immune effector cells delivered via intravenous infusion (IP), followed by a second dose of modified immune effector cells delivered via intravenous infusion (IV). In another embodiment, the second dose of modified immune effector cells may be followed by subsequent doses that can be delivered via IV or IP. In one embodiment, the duration between the first and second or further subsequent doses may be approximately: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In one embodiment, the duration between the first and second or further subsequent doses may be about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. In another embodiment, the duration between the first and second or further subsequent doses may be about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0410] In another embodiment, the catheter can be placed at the tumor or metastatic site to further administer 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of modified immune effector cells. In some cases, the dose of modified effector cells may contain approximately 10... 2 To about 10 9 Modified effector cells / kg. In cases where toxicity is observed, the dose of modified effector cells may contain approximately 10 [units / kg]. 2 To about 105 Modified effector cells / kg. In some cases, the dose of modified effector cells can be started at approximately 10. 2 The modified effector cells / kg, and subsequent doses can be increased to approximately: 10 4 10 5 10 6 10 7 10 8 Or 10 9 Modified effector cells / kg.

[0411] In other embodiments, methods for stimulating the proliferation and / or survival of engineered cells include obtaining a cell sample from a subject and transfecting cells of that cell sample with one or more polynucleotides containing one or more transposons. In one embodiment, the transposon encodes a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, and a transposase that efficiently integrates the one or more polynucleotides into the genome of the cell to provide an engineered cell population. In one embodiment, the transposon encodes a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, a gene-switching polypeptide for ligand-inducible control of the cytokine, and a transposase that efficiently integrates the one or more polynucleotides into the human genome to provide an engineered cell population. In one embodiment, the gene-switching polypeptide comprises i) a first gene-switching polypeptide containing a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and ii) a second gene-switching polypeptide containing a transactivation domain fused to a second nuclear receptor ligand-binding domain. In some embodiments, the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker. In one case, lymphocyte consumption is not required before the engineered cells are administered to the subject.

[0412] In one embodiment, a method for in vivo proliferation of engineered cells includes obtaining a cell sample from a subject and transfecting cells of that cell sample with one or more polynucleotides containing one or more transposons. In one embodiment, the transposon encodes a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, and a transposase that efficiently integrates the one or more polynucleotides into the genome of the cell to provide an engineered cell population. In one embodiment, the transposon encodes a chimeric antigen receptor (CAR), a cytokine, one or more cell tags, a gene-switching polypeptide for ligand-inducible control of the cytokine, and a transposase that efficiently integrates the one or more polynucleotides into the human genome to provide an engineered cell population. In one embodiment, the gene-switching polypeptide comprises i) a first gene-switching polypeptide containing a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and ii) a second gene-switching polypeptide containing a transactivation domain fused to a second nuclear receptor ligand-binding domain. In some embodiments, the first and second gene-switching polypeptides are linked by a linker. In one embodiment, lymphocyte consumption is not required before the engineered cells are administered to the subject.

[0413] In another embodiment, a method for enhancing the in vivo persistence of engineered cells in a subject in need includes obtaining a cell sample from the subject and transfecting cells of that cell sample with one or more polynucleotides containing one or more transposons. In some cases, one or more transposons encode a chimeric antigen receptor (CAR), cytokines, one or more cell tags, and a transposase that efficiently integrates DNA into the genome of said cells to provide an engineered cell population. In some cases, one or more transposons encode a chimeric antigen receptor (CAR), cytokines, one or more cell tags, a gene-switching polypeptide for ligand-inducible control of cytokines, and a transposase that efficiently integrates DNA into the genome of said cells to provide an engineered cell population. In some cases, the gene-switching polypeptide comprises i) a first gene-switching polypeptide containing a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and ii) a second gene-switching polypeptide containing a transactivation domain fused to a second nuclear receptor ligand-binding domain, wherein the first and second gene-switching polypeptides are linked by a linker. In one case, lymphocyte consumption is not required before the engineered cells are administered to the subject.

[0414] In another embodiment, a method of treating a subject with a solid tumor includes obtaining a cell sample from the subject, transfecting the cells of the sample with one or more polynucleotides containing one or more transposons, and administering an engineered cell population to the subject. In one case, lymphocyte consumption is not required before administering the engineered cells to the subject. In some cases, one or more transposons encode a chimeric antigen receptor (CAR), cytokines, one or more cell tags, and a transposase for efficient integration of DNA into the genome of the cells. In some cases, the one or more transposons encode a chimeric antigen receptor (CAR), cytokines, one or more cell tags, a gene-switching polypeptide for ligand-inducible control of cytokines, and a transposase for efficient integration of DNA into the genome of the cells. In some cases, the gene-switching polypeptide comprises: i) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and ii) a second gene-switching polypeptide comprising a transactivation domain fused to a second nuclear receptor ligand-binding domain, wherein the first and second gene-switching polypeptides are linked by a linker. In some cases, cells are transfected by electroporation. In some cases, the polynucleotide encoding the gene-switching polypeptide is regulated by a promoter. In some cases, the promoter is a tissue-specific promoter or an EF1A promoter or a functional variant thereof. In some cases, the tissue-specific promoter contains a T cell-specific response element or an NFAT response element. In some cases, the cytokine includes at least one of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15 and IL-15R, or a variant of IL-15. In some cases, the cytokine is in a secreted form. In some cases, the cytokine is in a membrane-bound form. In some cases, the cell is an NK cell, NKT cell, T cell, or T cell progenitor cell. In some cases, the cell is administered to a subject (e.g., by infusing engineered cells into the subject). In some cases, the method further includes administering an effective amount of a ligand (e.g., veledimex) to induce cytokine expression. In some cases, the CAR is capable of binding at least ROR1. In some cases, the transposase is a salmon-type Tc1-like transposase. In some cases, the transposase is SB11 or SB100x. In other cases, the transposase is PiggyBac. In some cases, the cell tag contains at least one of a truncated HER1 variant or a truncated CD20 variant.

[0415] Therapeutic applications

[0416] In the embodiments described herein, immune effector cells (e.g., T cells) are transduced using Sleeping Beauty transposons and Sleeping Beauty transposases. For example, one or more Sleeping Beauty transposons may include a CAR that combines the antigen recognition domain of ROR-1 with a spacer region of the CD8α hinge and its variants, an intracellular domain of CD3-ζ, CD28, 4-1BB, or any combination thereof, and an intracellular domain of CD3ζ, one or more cell tags, one or more cytokines, and optionally components of the gene switch system described herein. Therefore, in some cases, the transduced T cells can elicit a CAR-mediated T cell response.

[0417] In the embodiments described herein, an application is provided for using CAR to specifically redirect primary T cells to the ROR-1 surface antigen. Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in mammals, comprising the step of administering CAR-expressing T cells to the mammal, wherein the CAR comprises a binding portion that specifically interacts with ROR-1, a spacer region, a ζ-chain portion comprising, for example, an intracellular domain of human CD3ζ, and a co-stimulatory signal transduction region.

[0418] In one embodiment, this disclosure includes a cell therapy in which T cells are genetically modified to express the ROR-1-specific CAR of the present invention, and the CAR T cells are infused into a recipient in need. The infused cells are able to kill cells in the recipient that overexpress ROR-1. Unlike antibody therapies, the CAR T cells described herein are capable of replicating in vivo, resulting in long-term persistence, which can lead to sustained action against tumor cells.

[0419] The present invention further provides a method for detecting a disease including ROR-1 overexpression in a subject, comprising: a) providing i) a sample from the subject, and ii) any one or more antibodies or antigen-binding fragments thereof described herein, b) contacting the sample with the antibody under conditions that allow the antibody to bind specifically to its antigen, and c) detecting an increase in the level of antibody binding to the sample compared to a control sample lacking the disease, thereby detecting the disease in the subject. In one embodiment, the disease is cancer. In a preferred embodiment, the cancer is selected from ovarian cancer and breast cancer. While not intended to limit the detection method, in one embodiment, the detection of antibody binding to the sample is performed by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS), Western blotting, immunoprecipitation, and / or radiographic imaging.

[0420] This article also provides a method for treating diseases including ROR-1 overexpression, comprising administering to a subject suffering from the disease a therapeutically effective amount of any one or more of the antibodies or antigen-binding fragments described herein. In one embodiment, the disease is cancer, such as ovarian cancer and breast cancer.

[0421] In one embodiment, the ROR-1 CAR T cells described herein can undergo stable in vivo T cell expansion for an extended period of time. In another embodiment, the CAR T cells described herein can evolve into specific memory T cells, which can be reactivated.

[0422] The CAR-modified T cells described herein can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. In the embodiments, the mammal is a human. Regarding in vitro immunization, at least one of the following events occurs in vitro prior to the administration of immune effector cells to the mammal: i) cell expansion, ii) introduction of nucleic acid encoding the CAR into the cells, and / or iii) cryopreservation of the cells.

[0423] In vitro procedures are well-known and will be discussed more fully below. In short, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0424] Procedures for in vitro expansion of hematopoietic stem cells and progenitor cells are described in U.S. Patent 5,199,942 (incorporated herein by reference) and can be applied to the cells of this invention. Other suitable methods are known in the art, and therefore this invention is not limited to any particular method of in vitro cell expansion. In summary, in vitro culture and expansion of T cells comprises: (1) collecting mammalian CD34+ hematopoietic stem cells and progenitor cells from peripheral blood harvests or bone marrow explants; and (2) in vitro expansion of such cells. In addition to the cell growth factors described in U.S. Patent 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligands may also be used for cell culture and expansion.

[0425] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0426] Generally, activated and expanded cells as described herein can be used to treat and prevent diseases occurring in immunocompromised individuals. In particular, the CAR-modified T cells of the present invention are used to treat ROR-1 malignancies, such as ROR-1. In some embodiments, the cells of the present invention are used to treat patients at risk of developing ROR-1. Therefore, methods for treating or preventing ROR-1 include administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need. In embodiments, activated and expanded cells as described herein can be used to treat ROR-1.

[0427] In short, the pharmaceutical compositions described herein may comprise the target cell populations described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In embodiments, the compositions of the present invention are formulated for intravenous administration.

[0428] The pharmaceutical compositions described herein may be administered in a manner suitable for the disease to be treated (or prevented). Dosage and frequency of administration will depend on factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosage may be determined based on clinical trials.

[0429] When an “immunologically effective dose” or “therapeutic dose” is indicated, the precise amount of the composition described herein to be administered may be determined by the physician taking into account individual differences in age, weight, and the patient’s (subject’s) condition. It may generally be specified that the pharmaceutical composition containing T cells described herein may be administered in doses of 10… 4 Up to 10 9 Cells / kg body weight, 10 5 Up to 10 6 The dosage is administered at 1 cell / kg body weight, including all integer values ​​within these ranges. The T-cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, (1988)). By monitoring the patient's disease indications and adjusting treatment accordingly, those skilled in the medical field can readily determine the optimal dosage and treatment regimen for a particular patient.

[0430] In some embodiments, it may be desirable to administer activated T cells to a subject, followed by a blood draw (or apheresis) to activate the T cells, and then re-infuse these activated and expanded T cells into the patient. This process can be performed multiple times every few weeks. In some embodiments, T cells can be activated from blood draws ranging from 10 cc to 400 cc. In some embodiments, T cells can be activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Without being bound by theory, this multiple blood draw / re-infusion protocol can be used to select certain T cell populations. In another embodiment, it may be desirable to administer activated T cells of the composition of the present invention to a patient after lymphatic depletion through radiation or chemotherapy.

[0431] The compositions described herein can be administered in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the ROR-1 CAR-T cell composition of the present invention is administered by intravenous injection. The T-cell components can be injected directly into lymph nodes or primary tumor or metastatic sites.

[0432] The dosage of the above-mentioned treatment to be administered to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosage increases or decreases for human administration can be made according to accepted practices in the art. For example, the dosage of the above-mentioned treatment may be 1 × 10⁻⁶. 4 CAR+ cells / kg up to 5×10 6 The range is CAR+ cells / kg. An exemplary dose could be 1×102 2 CAR+ cells / kg, 1×10 3 CAR+ cells / kg, 1×10 4 CAR+ cells / kg, 1×10 5 CAR+ cells / kg, 3×10 5 CAR+ cells / kg, 1×10 6 CAR+ cells / kg, 5×10 6 CAR+ cells / kg, 1×10 7 CAR+ cells / kg, 1×10 8 CAR+ cells / kg or 1×10 9 CAR+ cells / kg. The appropriate dose can be adjusted accordingly for adult or pediatric patients.

[0433] Alternatively, the typical amount of immune effector cells administered to mammals (e.g., humans) can range from one hundred, one thousand, ten thousand, one million to one hundred billion cells; however, amounts below or above this exemplary range are also within the scope of the invention. For example, the dose of host cells in the present invention can be from about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or any two of the values ​​defined above), or from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or about 100 million cells). The range is defined as approximately 10,000 cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or any two of the above values), approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, or any two of the above values).

[0434] The efficacy of treatment or prevention can be monitored by periodically assessing patients receiving treatment. For repeated administration over several days or longer, treatment may be repeated as needed until the desired suppression of disease symptoms is achieved. However, other dosing regimens may also be useful and are within the scope of this invention. The desired dose can be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion.

[0435] Compositions comprising immune effector cells expressing the disclosed nucleic acid sequences, or vectors comprising such nucleic acid sequences, may be administered co-administered with one or more other therapeutic agents that can be co-administered to mammals. "Co-administration" means administering one or more other therapeutic agents and compositions comprising the host cells or vectors of the present invention sufficiently close in time to enhance the effects of the one or more other therapeutic agents, or vice versa. In this regard, compositions comprising the immune effector cells or vectors described herein may be administered simultaneously with one or more other therapeutic agents, or the composition may be administered first, followed by one or more other therapeutic agents, or vice versa. Alternatively, compositions comprising the disclosed immune effector cells or vectors described herein and one or more other therapeutic agents may be administered simultaneously.

[0436] Examples of therapeutic agents that may be included in or co-administered with a composition comprising the host cell of the present invention and / or the vector of the present invention are interleukins, cytokines, interferons, adjuvants, and chemotherapeutic agents. In embodiments, the additional therapeutic agents are ligands of IFN-α, IFN-β, IFN-γ, GM-CSF, G-CSF, M-CSF, LT-β, ​​TNF-α, growth factors, and hGH, and ligands of human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

[0437] "Defoamers" reduce foaming during processing, which can cause agglomeration of aqueous dispersions, air bubbles in finished films, or otherwise impaired processing. Exemplary defoamers include silicone emulsions or sorbitan sesquoleate.

[0438] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite, and tocopherol. In some embodiments, antioxidants enhance chemical stability, if desired.

[0439] The formulations described herein may benefit from antioxidants, metal chelators, thiol-containing compounds, and other common stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol; (b) about 0.1% to about 1% w / v methionine; (c) about 0.1% to about 2% w / v monothioglycerol; (d) about 1 mM to about 10 mM EDTA; (e) about 0.01% to about 2% w / v ascorbic acid; (f) 0.003% to about 0.02% w / v polysorbate 80; (g) 0.001% to about 0.05% w / v polysorbate 20; (h) arginine; (i) heparin; (j) dextran sulfate; (k) cyclodextrin; (l) pentosan polysulfate esters and other heparin-like substances; (m) divalent cations, such as magnesium and zinc; or (n) combinations thereof.

[0440] "Adhesives" impart adhesive properties and include, for example, alginate and its salts; cellulose derivatives, such as carboxymethyl cellulose, methyl cellulose (e.g.) ), hydroxypropyl methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose (e.g.) ), ethyl cellulose (e.g.) ) and microcrystalline cellulose (e.g. Microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acid; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; polyvinylpyrrolidone; povidone; starch; pregelatinized starch; tragacanth gum; dextrin; sugars such as sucrose (e.g.) ), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g.) ) and lactose; natural or synthetic gums, such as gum arabic, gum tragali, solanum, psyllium husk gum, polyvinylpyrrolidone (e.g.) CL、 CL、 XL-10), larch arabinogalactan, Polyethylene glycol, wax, sodium alginate, etc.

[0441] "Carrier" or "carrier material" includes any excipient commonly used in pharmaceuticals and should be selected based on compatibility with compounds disclosed herein, such as ibrutinib and anticancer compounds, and the release profile properties of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials may include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurine, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium saccharide stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy, 19th edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L. (eds.), Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition (Lippincott Williams & Wilkins, 1999).

[0442] "Dispersants" and / or "viscosity modifiers" include materials that control the diffusion and homogeneity of a drug through a liquid medium, granulation method, or mixing method. In some embodiments, these agents also contribute to the effectiveness of coating or eroding a matrix. Exemplary diffusion promoters / dispersants include, for example, hydrophilic polymers, electrolytes, 60 or 80, PEG, polyvinylpyrrolidone (PVP; trade name) ) and carbohydrate-based dispersants such as hydroxypropyl cellulose (e.g., HPC, HPC-SL and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M and HPMC K100M), sodium carboxymethyl cellulose, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymers with ethylene oxide and formaldehyde (also known as teroxaprol), poloxamer (e.g., and These are block copolymers of ethylene oxide and propylene oxide; and poloxamine (e.g., Tetronic). Also known as Poloxamine It is a tetrafunctional block copolymer (BASF) obtained by sequentially adding propylene oxide and ethylene oxide to ethylenediamine. Corporation, Parsippany, NJ), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol, for example, having a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethyl cellulose, methylcellulose, polysorbate-80, sodium alginate, gums such as gum tragali and gum arabic, guar gum, xanthan gum (including xanthan gum), sugar, cellulose products such as sodium carboxymethyl cellulose, methylcellulose, sodium carboxymethyl cellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyvinylpyrrolidone monolaurate, polyvinylpyrrolidone, carbomer, polyvinyl alcohol (PVA), alginate, chitosan and combinations thereof. Plasticizers such as cellulose or triethylcellulose can also be used as dispersants. Dispersants that are particularly useful in liposome dispersions and self-emulsifying dispersions are myristoyl phosphatidylcholine, natural phosphatidylcholine derived from eggs, natural phosphatidylglycerol derived from eggs, cholesterol, and isopropyl myristate.

[0443] A combination of one or more etching promoters and one or more diffusion promoters may also be used in the compositions of the present invention.

[0444] The term "diluent" refers to a chemical compound used to dilute a compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they provide a more stable environment. Salts dissolved in buffered solutions (which may also provide pH control or maintenance) are used in the art as diluents, including but not limited to phosphate-buffered saline solutions. In some embodiments, the diluent increases the volume of the composition to facilitate compression or to produce sufficient volume for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, etc. Calcium hydrogen phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars, such as... (Amstar); Mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectionery; basic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, glucose dextrorate; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0445] "Filling agents" include compounds such as lactose, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, glucose binder, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0446] "Lubricant" and "flow aid" are compounds that prevent, reduce, or inhibit adhesion or friction between materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearoyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil. Higher fatty acids and their alkali metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, and zinc salts; stearic acid; sodium stearate; glycerol; talc; waxes; Boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxylated polyethylene glycol such as Carbowax TM Sodium oleate, sodium benzoate, glyceryl betaine, polyethylene glycol, magnesium dodecyl sulfate or sodium dodecyl sulfate, colloidal silica such as Syloid TM , Starch, such as corn starch, silicone oil, surfactants, etc.

[0447] "Plasticizer" is a compound used to soften microencapsulated materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. In some embodiments, plasticizers may also be used as dispersants or wetting agents.

[0448] "Solubilizers" include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium dodecyl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetrahydrofuran polyethylene glycol ether (glycofurol), diethylene glycol monoethyl ether (transcutol), propylene glycol, dimethyl isosorbide, etc.

[0449] "Stabilizers" include compounds such as any antioxidants, buffers, acids, preservatives, etc.

[0450] "Suspension agents" include polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol may have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, etc. Cellulose, hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums such as gum arabic and gum arabic, guar gum, xanthan gum (including xanthan gum), sugar, cellulose such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and other compounds.

[0451] "Surfactants" include substances such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin ETPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, etc. Compounds such as (BASF). Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40. In some embodiments, surfactants may be included to enhance physical stability or for other purposes.

[0452] "Viscosity enhancers" include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0453] "Wetting agents" include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E, TPGS, and ammonium salts.

[0454] reagent kits and compositions

[0455] One aspect of this disclosure relates to a kit and composition comprising a first vector including a coding region encoding the ROR-1-specific CAR of the present invention and optionally including genes for safety reasons, such as HER1t or HER1t-1 an...

Claims

1. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) A ROR-1 antigen-binding domain consisting of a single-stranded variable fragment (scFv), wherein the scFv is composed of the following: (i) a VL consisting of the amino acid sequence of SEQ ID NO: 36; (ii) a VH consisting of the amino acid sequence of SEQ ID NO: 17; and (iii) a flexible linker connecting the VL to the VH; (b) Transmembrane domain; and (c) Intracellular signal transduction domain.

2. The nucleic acid according to claim 1, wherein the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain.

3. The nucleic acid according to claim 2, wherein the co-stimulatory signal transduction domain comprises a 4-1BB signal transduction domain.

4. The nucleic acid according to claim 3, wherein the co-stimulatory signal transduction domain comprises the polypeptide shown in SEQ ID NO:

89.

5. The nucleic acid according to claim 2, wherein the co-stimulatory signal transduction domain comprises the CD28 signal transduction domain.

6. The nucleic acid according to claim 5, wherein the co-stimulatory signal transduction domain comprises the polypeptide shown in SEQ ID NO:

90.

7. The nucleic acid according to claim 1, wherein the intracellular signal transduction domain comprises the CD3ζ signal transduction domain.

8. The nucleic acid according to claim 7, wherein the CD3ζ signal transduction domain comprises the polypeptide shown in SEQ ID NO:

93.

9. The nucleic acid according to claim 1, further encoding a polypeptide shown in any one of SEQ ID NO: 94-108.

10. The nucleic acid according to claim 1, further encoding a cell tag.

11. The nucleic acid of claim 10, wherein the cell tag is a truncated epidermal growth factor receptor.

12. The nucleic acid of claim 10, wherein the cell tag comprises the polypeptide shown in SEQ ID NO:

109.

13. The nucleic acid of claim 11, wherein the cell tag comprises the polypeptide shown in SEQ ID NO:

110.

14. The nucleic acid according to claim 1, wherein the CAR further comprises a spacer region.

15. The nucleic acid according to claim 14, wherein the spacer region comprises a polypeptide shown in any one of SEQ ID NO: 83-86.

16. The nucleic acid of claim 15, wherein the spacer region comprises a stem region and a stem extension region.

17. The nucleic acid of claim 16, wherein the stem region comprises the hinge region of the fragment crystallizable region (Fc) of the antibody.

18. The nucleic acid of claim 17, wherein the stem region further comprises one or both constant domains of the Fc region of the antibody.

19. The nucleic acid of claim 16, wherein the stem region comprises a CD8α hinge region polypeptide.

20. The nucleic acid according to claim 19, wherein the stem region comprises the polypeptide shown in SEQ ID NO:

83.

21. The nucleic acid of claim 16, wherein the spacer region comprises 1 to 20 stem extension regions.

22. The nucleic acid of claim 16, wherein the spacer region comprises 1 to 5 stem extension regions.

23. The nucleic acid of claim 16, wherein each stem extension region comprises a polypeptide of the stem region.

24. The nucleic acid of claim 21, wherein each stem extension region has fewer dimerization sites than the stem region.

25. The nucleic acid according to claim 22, wherein each stem extension region lacks a dimerization site.

26. The nucleic acid according to claim 21, wherein each stem extension region differs from the stem region only in that each stem extension region lacks a dimerization site.

27. The nucleic acid of claim 26, wherein the stem region is located proximal to the transmembrane domain.

28. The nucleic acid of claim 16, wherein the spacer region comprises the polypeptide shown in SEQ ID NO:

85.

29. The nucleic acid according to claim 1, wherein the transmembrane domain comprises a polypeptide shown in any one of SEQ ID NO: 87 or 88.

30. The nucleic acid of claim 29, wherein the transmembrane domain comprises the polypeptide shown in SEQ ID NO:

87.

31. The nucleic acid of claim 1, further encoding: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain; and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker.

32. The nucleic acid according to claim 1, further encoding cytokines.

33. The nucleic acid according to claim 32, wherein the cytokine is IL-15 or IL-12.

34. The nucleic acid according to claim 1, further encoding a fusion protein, said fusion protein comprising: (a) IL-15; and (b) IL-15Rα.

35. The nucleic acid according to claim 34, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

113.

36. The nucleic acid according to claim 1, further encoding self-cleavage. Thosea asigna Viral (T2A) peptide.

37. The nucleic acid according to claim 1, wherein the VH and the VL are present in the scFv in an N-terminal to C-terminal orientation.

38. The nucleic acid according to claim 1, wherein: (a) The transmembrane domain comprises the polypeptide shown in SEQ ID NO: 87; and (b) The intracellular signal transduction domain comprises the polypeptide shown in SEQ ID NO: 90 and the polypeptide shown in SEQ ID NO: 93; and The CAR further includes a spacer region containing the polypeptide shown in SEQ ID NO:

85.

39. The nucleic acid according to claim 38, further encoding the polypeptide shown in SEQ ID NO:

108.

40. The nucleic acid of claim 38, further encoding a cell tag, said cell tag comprising the polypeptide shown in SEQ ID NO:

110.

41. The nucleic acid according to claim 38, further encoding a fusion protein, said fusion protein comprising the polypeptide shown in SEQ ID NO:

113.

42. The nucleic acid of claim 38, wherein the VH and the VL are present in the scFv in an N-terminal to C-terminal orientation.

43. The nucleic acid according to claim 38, further encoding: (a) A cell tag comprising the polypeptide shown in SEQ ID NO: 110; and (b) A fusion protein comprising the polypeptide shown in SEQ ID NO:

113.

44. The nucleic acid according to claim 1, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:

79.

45. A vector comprising a backbone and a nucleic acid according to any one of claims 1 to 44.

46. ​​The vector according to claim 45, wherein the vector is a lentiviral vector, a retroviral vector, or a non-viral vector.

47. The carrier according to claim 45, wherein the skeleton is Sleeping Beauty Transposon DNA plasmid.

48. A modified immune effector cell comprising the nucleic acid of any one of claims 1 to 44.

49. A modified immune effector cell comprising the vector of claim 45.

50. A modified immune effector cell comprising: (a) Cell tag; (b) Cytokines; and (c) A chimeric antigen receptor (CAR), wherein the CAR comprises: (i) A ROR-1 antigen-binding domain consisting of a single-stranded variable fragment (scFv), wherein the scFv is composed of the following: A VL consisting of the amino acid sequence of SEQ ID NO: 36; a VH consisting of the amino acid sequence of SEQ ID NO: 17; and a flexible linker connecting the VL and the VH; (ii) Transmembrane domain; and (iii) Intracellular signal transduction domain.

51. The modified immune effector cells of claim 50, wherein the cytokine comprises IL-15.

52. The modified immune effector cells of claim 51, wherein the IL-15 is contained in the fusion protein, and the fusion protein further contains IL-15Rα.

53. The modified immune effector cell of claim 50, wherein the cell tag comprises a truncated epidermal growth factor receptor.

54. The modified immune effector cell of claim 50, wherein the cell tag comprises the polypeptide shown in SEQ ID NO: 109 or 110.

55. The modified immune effector cell of claim 50, wherein the cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, or a regulatory T cell.

56. The modified immune effector cell of claim 50, further comprising: (a) a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain; and (b) a second gene-switching polypeptide comprising a trans-activation domain fused to a second nuclear receptor ligand-binding domain; wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker.

57. Use of the modified immune effector cells according to claim 50 in the preparation of a medicament for the treatment of non-Hodgkin's lymphoma.

58. A composition comprising the carrier of claim 45.

59. A kit comprising the vector of claim 45.

60. A composition comprising the modified immune effector cells of claim 48.

61. A method for expressing CAR in immune effector cells, the method comprising introducing the vector of claim 45 into the immune effector cells.

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