Methods, compositions, and components for CRISPR-CAS9 editing of TGFBR2 in T cells for immunotherapy

Abstract

Provided are CRISPR / CAS-related genome editing systems, compositions and methods for targeting the TGFBR2 locus, and cells edited using these systems, compositions and methods.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 580,320, filed November 1, 2017, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to CRISPR / Cas9-related methods and components for editing a target nucleic acid sequence, such as the transforming growth factor beta receptor II (TGFBR2) gene, or for regulating the expression of a target nucleic acid sequence, such as the TGFBR2 gene. [Background technology]

[0003] background Adoptive T cell transfer using genetically modified T cells has entered clinical trials as a treatment for solid and hematological malignancies. In Phase I and II trials targeting hematological malignancies (e.g., lymphoma, chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL)), many patients have shown at least partial responses, and some have shown complete responses (Kochenderfer, JN et al., 2012 Blood 119, 2709-2720 (Non-Patent Document 1)). However, the effects observed in solid tumor types (including melanoma, renal cell carcinoma, and colorectal cancer) have not always been robust (Johnson, LA et al., 2009 Blood 114, 535-546; Lamers, CH et al., 2013 Mol. Ther. 21, 904-912; Warren, RS et al., 1998 Cancer Gene Ther. 5, S1-S2 (Non-Patent Documents 2-4)).

[0004] While not wishing to be bound by any particular theory, the efficacy of adoptive T cell therapy for patients with solid tumors may be influenced by many factors, including (1) T cell proliferation, e.g., limited proliferation of T cells after adoptive transfer; (2) T cell survival, e.g., induction of T cell apoptosis by factors within the target cell environment, such as cancer cells; and (3) T cell function, e.g., inhibition of cytotoxic T cell function by inhibitory factors secreted by host immune cells and target cells, e.g., cancer cells. These factors may in turn be influenced by the activity of transforming growth factor β (TGF-β), a cytokine produced by a wide variety of tumor types that has been shown to directly suppress tumor-infiltrating lymphocytes as well as induce and promote the function of regulatory T cells (Tregs), which can suppress antitumor immunity.

[0005] TGFBR2 is a receptor for TGF-β, expressed on numerous cell types, including immune cells. TGF-β binding by TGFBR2 has been demonstrated to downregulate T cell activation, proliferation, and differentiation. The development of TGFBR2 inhibitors that can improve TGFBR2 activity against tumor-responsive T cells has been complicated by the lack of preclinical mouse models due to the severe autoimmune phenotype observed in mice containing T cells engineered to conditionally delete TGFBR2. Consequently, effective strategies to reduce or eliminate the T cell inhibitory effects of TGF-β are needed, particularly in the context of T cell-mediated immunotherapy.

[0006] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attacks. Upon exposure to a virus, a short segment of viral DNA is integrated into the CRISPR locus. RNA is transcribed from the portion of the CRISPR locus that contains the viral sequence. The RNA, which contains a sequence complementary to the viral genome, mediates the targeting of an RNA-guided nuclease to the target sequence in the viral genome. The RNA-guided nuclease then cleaves the viral target, thereby silencing it.

[0007] Recently, the CRISPR / Cas9 system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double-strand breaks (DSBs) allows targeted sequence modification via endogenous DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). CRISPR / Cas9 has shown promise for addressing TGF-β-mediated inhibition of T cells in the context of tumor therapy, but to date, no viable approach has been identified for addressing this issue in T cells for use in tumor therapy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Kochenderfer, JN et al., 2012 Blood 119, 2709-2720 [Non-patent document 2] Johnson, LA et al., 2009 Blood 114, 535-546 [Non-patent document 3] Lamers, CH et al., 2013 Mol. Ther. 21, 904-912 [Non-patent document 4] Warren, RS et al., 1998 Cancer Gene Ther. 5, S1-S2 Summary of the Invention

[0009] overview In certain aspects, provided herein is a genome editing system for targeted editing of the nucleic acid sequence of TGFBR2, and related compositions and methods.In certain embodiments, such targeted editing results in the alteration (e.g., downregulation) of TGFBR2 expression.In certain embodiments, such alteration of expression occurs in T cells.In certain embodiments, the alteration of TGFBR2 expression in T cells comprises the use of a ribonucleoprotein (RNP) complex as a genome editing system, comprising an RNA-guided nuclease protein complexed with a gRNA that targets the TGFBR2 gene.In certain embodiments, the alteration of TGFBR2 expression occurs as a result of RNP-induced double-strand breaks and subsequent imperfect repair, resulting in indels in the target TGFBR2 sequence and / or indels adjacent to the target TGFBR2 sequence.

[0010] In certain embodiments, the present disclosure relates to a genome editing system comprising a guide RNA having a targeting domain complementary to a target sequence of the TGFBR2 gene, and the RNA-guided nuclease is a Cas9 nuclease. The targeting domain can be 70%, 80%, 85%, 90%, 95%, or 100% complementary.

[0011] In certain embodiments, the targeting domain has a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides.

[0012] In certain embodiments, the targeting domain has at least 18 contiguous nucleotides complementary to the TGFBR2 gene.

[0013] In certain embodiments, the targeting domain comprises a nucleotide sequence identical to or differing by no more than 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs:5036-5096. In certain embodiments, the targeting domain is configured to form a double-stranded or single-stranded break within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position.

[0014] In certain embodiments disclosed herein, the genome editing system can modify the TGFBR2 gene by knocking out expression of the TGFBR2 gene or by knocking down expression of the TGFBR2 gene.

[0015] In certain aspects, the genome editing systems disclosed herein incorporate a gRNA comprising a targeting domain configured to target a coding or non-coding region of the TGFBR2 gene, wherein the non-coding region comprises a promoter region, an enhancer region, an intron, a 3' UTR, a 5' UTR, or a polyadenylation signal region of the TGFBR2 gene, and the coding region comprises, for example, the early coding region of the TGFBR2 gene.

[0016] In certain embodiments, the genome editing system disclosed herein has a target sequence of the TGFBR2 gene comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0017] In certain embodiments, the genome editing system disclosed herein has a target sequence of the TGFBR2 gene comprising a sequence selected from the group consisting of SEQ ID NOs: 4-10.

[0018] In certain embodiments, the genome editing systems disclosed herein incorporate a targeting domain comprising a nucleotide sequence identical to or differing by no more than 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5036-5096. In certain embodiments, the targeting domain comprises a nucleotide sequence identical to or differing by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 5041, (b) SEQ ID NO: 5042, (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050, (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO: 5093. In certain embodiments, the genome editing system incorporates a pair of gRNA molecules, including, for example, a gRNA pair having target sequences SEQ ID NOs:5042 and 5041, or 5042 and 5092, or SEQ ID NOs:5042 and 5093, or SEQ ID NOs:5093 and 5041.

[0019] In certain embodiments, the present disclosure relates to a composition comprising a gRNA molecule comprising a targeting domain complementary to a target sequence of the TGFBR2 gene. In certain embodiments, the composition comprises one, two, three, or four gRNA molecules. In certain embodiments, the composition further comprises an RNA-guided nuclease, e.g., a Cas9 molecule. In certain embodiments, the targeting domain incorporated into such a composition comprises a nucleotide sequence identical to or differing by no more than three nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5036-5096. In certain embodiments, the targeting domain comprises a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO:5041, (b) SEQ ID NO:5042, (c) SEQ ID NO:5047, (d) SEQ ID NO:5050, (e) SEQ ID NO:5052, (f) SEQ ID NO:5092, and (g) SEQ ID NO:5093. In certain embodiments, the composition incorporates a pair of gRNA molecules, including, for example, a gRNA pair having target sequences SEQ ID NOs:5042 and 5041, or 5042 and 5092, or SEQ ID NOs:5042 and 5093, or SEQ ID NOs:5093 and 5041.

[0020] In certain embodiments, the present disclosure relates to a vector encoding a gRNA molecule comprising a targeting domain complementary to a target sequence of the TGFBR2 gene. In certain embodiments, the vector further encodes an RNA-guided nuclease, e.g., a Cas9 molecule. In certain embodiments, the targeting domain of the gRNA encoded by the vector comprises a nucleotide sequence identical to or differing by no more than 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs:5036-5096. In certain embodiments, the targeting domain comprises a nucleotide sequence identical to or differing by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of (a) SEQ ID NO:5041, (b) SEQ ID NO:5042, (c) SEQ ID NO:5047, (d) SEQ ID NO:5050, (e) SEQ ID NO:5052, (f) SEQ ID NO:5092, and (g) SEQ ID NO:5093. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is an adeno-associated viral (AAV) vector or a lentiviral (LV) vector.

[0021] In certain aspects, the present disclosure relates to methods of modifying a TGFBR2 gene in a cell, comprising administering to the cell one of the following: (i) a genome editing system comprising a gRNA molecule comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and a Cas9 molecule; (ii) a vector comprising a polynucleotide encoding a gRNA molecule comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and a polynucleotide encoding a Cas9 molecule; or (iii) a composition comprising a gRNA molecule comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and a Cas9 molecule.

[0022] In certain embodiments, the present disclosure relates to a cell comprising the genome editing system described herein, the gRNA composition described herein, or the vector described herein.In certain embodiments, the cell expresses TGFBR2.In certain embodiments, the cell is a T cell.

[0023] In certain aspects, the present disclosure relates to gRNAs and RNA-guided nucleases that constitute a ribonucleoprotein (RNP) complex.

[0024] In certain aspects, the present disclosure relates to administering to a cell two or more RNP complexes comprising gRNAs with different targeting domains.

[0025] In certain aspects, the present disclosure relates to RNP complexes comprising an enzymatically active Cas9 (eaCas9) nuclease.

[0026] In certain aspects, the present disclosure relates to an RNP complex comprising an eaCas9 nuclease that forms a double-stranded break in a target nucleic acid or a single-stranded break in a target nucleic acid.

[0027] In certain aspects, the present disclosure relates to two RNP complexes comprising separate gRNAs that are used to create an offset single strand break in the TGFBR2 gene in a cell.

[0028] In certain aspects, the present disclosure relates to a cell that is a T cell or a natural killer (NK) cell. In certain aspects, the cell further comprises an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR).

[0029] In certain aspects, the present disclosure relates to an RNA-guided nuclease-mediated method for modifying TGFBR2 gene expression in a cell, comprising: (a) contacting a cell with a sufficient amount of a gRNA that targets TGFBR2 and an RNA-guided nuclease; and (b) forming a first DNA double-stranded break near the TGFBR2 target location within the TGFBR2 gene of the cell, wherein the first DNA double-stranded break is repaired by NHEJ, and the repair modifies expression of the TGFBR2 gene.

[0030] In certain embodiments, the present disclosure relates to forming a second DNA double-strand break near TGFBR2 target position.In certain embodiments, the first double-strand break is formed within about 500bp, about 450bp, about 400bp, about 350bp, about 300bp, about 250bp, about 200bp, about 150bp, about 100bp, about 50bp, about 25bp or about 10bp of TGFBR2 target position.In certain embodiments, the first and second double-strand breaks are formed within about 500bp, about 450bp, about 400bp, about 350bp, about 300bp, about 250bp, about 200bp, about 150bp, about 100bp, about 50bp, about 25bp or about 10bp of TGFBR2 target position. In certain embodiments, the first double-stranded break is formed in a coding or non-coding region of the TGFBR2 gene, and the non-coding region comprises a promoter region, an enhancer region, an intron, a 3'UTR, a 5'UTR, or a polyadenylation signal region of the TGFBR2 gene. In certain embodiments, the first and second double-stranded breaks are formed in a coding or non-coding region of the TGFBR2 gene, and the non-coding region comprises a promoter region, an enhancer region, an intron, a 3'UTR, a 5'UTR, or a polyadenylation signal region of the TGFBR2 gene.

[0031] In certain embodiments, the coding region is selected from exon 3, exon 4 and exon 5.

[0032] In certain embodiments, the targeting domain comprises a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5036-5096.

[0033] In certain embodiments, the RNA-guided nuclease is a Streptococcus pyogenes (S. pyogenes) Cas9 nuclease, and the targeting domain comprises a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO:5041, (b) SEQ ID NO:5042, (c) SEQ ID NO:5047, (d) SEQ ID NO:5050, (e) SEQ ID NO:5052, (f) SEQ ID NO:5092, and (g) SEQ ID NO:5093.

[0034] In certain embodiments, the RNA-guided nuclease is the Staphylococcus aureus (S. aureus) Cas9 nuclease.

[0035] In certain embodiments, the RNA-guided nuclease is a mutant Cas9 nuclease.

[0036] In certain embodiments, NHEJ repair results in insertions or deletions at a frequency of 20% or greater.

[0037] In particular embodiments, the frequency of insertions or deletions is 30% or more, 40% or more, or 50% or more.

[0038] In certain aspects, the present disclosure relates to genomically engineered cells comprising an insertion or deletion near or at a target location in the TGFBR2 gene, wherein the target location comprises a nucleotide sequence that is complementary to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs:5036-5096.

[0039] In certain embodiments, the insertion or deletion is within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp or about 10 bp of the TGFBR2 target position.

[0040] In certain embodiments, the cell is a T cell or an NK cell. In certain embodiments, the cell further comprises an eTCR or a CAR.

[0041] In certain aspects, the disclosure relates to a composition comprising: (a) a population of genomically engineered cells comprising an insertion or deletion near or at a target location in the TGFBR2 gene, wherein the target location comprises a nucleotide sequence that is complementary to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs:5036-5096; and (b) a pharmaceutically acceptable buffer.

[0042] In certain embodiments, the population of cells comprises T cells or NK cells. In certain embodiments, the cells further comprise an eTCR or a CAR.

[0043] In certain aspects, the present disclosure relates to methods of treating cancer in a subject comprising administering to the subject engineered immune cells, wherein the engineered immune cells have reduced expression of TGFBR2, and wherein the engineered immune cells optionally express an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR), and wherein the engineered immune cells have an insertion or deletion near or at a target location in the TGFBR2 gene.

[0044] In certain embodiments, the engineered immune cells comprise T cells or NK cells. In certain embodiments, the cells further comprise an eTCR or a CAR.

[0045] In certain embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, low-grade B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, mesothelioma, and / or any cancer type that expresses TGF-β.

[0046] In certain embodiments, the T cells are CD4+ T cells and / or CD8+ T cells.

[0047] In certain aspects, the engineered immune cells maintain or have enhanced lytic activity against target cancer cells compared to non-engineered immune cells.

[0048] In certain embodiments, the engineered immune cells maintain or have increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to non-engineered immune cells.

[0049] In certain aspects, the engineered immune cells maintain or have improved persistence to repeated antigen stimulation compared to non-engineered immune cells.

[0050] In certain embodiments, the engineered immune cells maintain or have increased expression of CD25 compared to non-engineered immune cells.

[0051] In certain embodiments, the engineered immune cells maintain or have reduced expression of PD-1 compared to non-engineered immune cells.

[0052] In certain aspects, the engineered immune cells maintain or have increased proliferation compared to non-engineered immune cells.

[0053] In certain aspects, the present disclosure relates to a composition comprising a plurality of engineered T cells, wherein the engineered T cells exhibit reduced TGFBR2 gene expression compared to non-engineered T cells.

[0054] In certain embodiments, the engineered T cells exhibit TGFBR2 gene expression levels that are about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the TGFBR2 expression levels in non-engineered T cells.

[0055] In certain embodiments, the engineered T cells further comprise expression of an eTCR or a CAR.

[0056] In certain embodiments, the T cells are CD4+ T cells and / or CD8+ T cells.

[0057] In certain embodiments, the engineered T cells are further characterized by having (a) enhanced lytic activity against target cancer cells compared to non-engineered T cells, (b) maintained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to non-engineered T cells, (c) maintained or increased persistence to repeated antigen stimulation compared to non-engineered T cells, (d) maintained or increased expression of CD25 compared to non-engineered T cells, (e) maintained or decreased expression of PD-1 compared to non-engineered T cells, and / or (f) maintained or increased proliferation compared to non-engineered T cells.

[0058] In certain aspects, the present disclosure relates to a composition comprising a plurality of engineered T cells, wherein the engineered T cells exhibit reduced TGFBR2 gene expression compared to non-engineered T cells, and the engineered T cells are generated by contacting non-engineered T cells with a genome editing system, wherein the genome editing system comprises a gRNA comprising a targeting domain complementary to a target sequence in the TGFBR2 gene and an RNA-guided nuclease.

[0059] In certain embodiments, the engineered T cells are further transduced with a vector that expresses eTCR or CAR. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentivirus (LV) vector.

[0060] In certain embodiments, the RNA-guided nuclease is a Streptococcus pyogenes (S. pyogenes) Cas9 nuclease, and the targeting domain comprises a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO:5041, (b) SEQ ID NO:5042, (c) SEQ ID NO:5047, (d) SEQ ID NO:5050, (e) SEQ ID NO:5052, (f) SEQ ID NO:5092, and (g) SEQ ID NO:5093.

[0061] In certain aspects, the present disclosure relates to a composition comprising a plurality of engineered T cells, wherein the engineered T cells are deficient in TGFBR2 signaling.

[0062] In certain embodiments, defective TGFBR2 signaling is mediated by expressing a dominant-negative (DN) form of TGFBR2 in engineered T cells.

[0063] In certain embodiments, the engineered T cells are further transduced with a vector that expresses eTCR or CAR. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector or a lentivirus (LV) vector.

[0064] In certain embodiments, the T cells are CD4+ T cells and / or CD8+ T cells.

[0065] In certain embodiments, the engineered T cells are further characterized by having (a) enhanced lytic activity against target cancer cells compared to non-engineered T cells, (b) maintained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to non-engineered T cells, (c) maintained or increased persistence to repeated antigen stimulation compared to non-engineered T cells, (d) maintained or increased expression of CD25 compared to non-engineered T cells, (e) maintained or decreased expression of PD-1 compared to non-engineered T cells, and / or (f) maintained or increased proliferation compared to non-engineered T cells.

[0066] In certain embodiments, the engineered immune cells further comprise reduced expression of wild-type TGFBR2.

[0067] In certain embodiments, wild-type TGFBR2 expression is reduced by contacting the engineered immune cells with a genome editing system, the genome editing system comprising a gRNA comprising a targeting domain complementary to a target sequence in the TGFBR2 gene and an RNA-guided nuclease.

[0068] In certain aspects, the present disclosure relates to a ribonucleoprotein (RNP) complex comprising a gRNA that includes a targeting domain complementary to a target sequence of the TGFBR2 gene and an RNA-guided nuclease.

[0069] 118. The RNP of claim 117, wherein in a particular embodiment, the RNA-guided nuclease is a Cas9 nuclease.

[0070] 118. The RNP of claim 117, wherein in certain embodiments, the RNP is electroporated into a cell.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.

[0072] Headings, including numerical and alphabetical headings and subheadings, are for organizational and presentation purposes and are not intended to be limiting.

[0073] [The present invention 1001] a gRNA comprising a targeting domain complementary to a target sequence of the transforming growth factor beta receptor II (TGFBR2) gene; RNA-guided nucleases and A genome editing system comprising: [The present invention 1002] The genome editing system of the present invention 1001, wherein the target sequence of the TGFBR2 gene comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2 and 3. [The present invention 1003] The genome editing system of the present invention 1001, wherein the target sequence of the TGFBR2 gene comprises a sequence selected from the group consisting of SEQ ID NOs: 4 to 10. [The present invention 1004] A genome editing system of the present invention 1001, wherein the targeting domain has at least 85% complementarity to the target sequence of the TGFBR2 gene. [The present invention 1005] 1001. A genome editing system of the present invention, wherein the targeting domain is configured to form a double-stranded break or a single-stranded break within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position, thereby modifying the TGFBR2 gene. [The present invention 1006] A genome editing system of the present invention 1005, in which TGFBR2 gene expression is knocked out or knocked down. [The present invention 1007] A genome editing system according to any one of claims 1001 to 1006, wherein the gRNA targets a coding region or non-coding region of the TGFBR2 gene, and the non-coding region includes a promoter region, an enhancer region, an intron, a 3'UTR, a 5'UTR, or a polyadenylation signal region of the TGFBR2 gene. [The present invention 1008] The genome editing system of the present invention 1007, wherein the coding region is selected from exon 3, exon 4, and exon 5. [The present invention 1009] The targeting domain is A nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5036 to 5096. A genome editing system according to any one of claims 1001 to 1008, comprising: [The present invention 1010] the RNA-guided nuclease is Streptococcus pyogenes (S. pyogenes) Cas9 nuclease; The targeting domain is (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO:5093 a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of: A genome editing system according to any one of claims 1001 to 1009 of the present invention, comprising: [The present invention 1011] Streptococcus pyogenes Cas9 nuclease recognizes the NGG protospacer adjacent motif (PAM), The genome editing system targets TGFBR2, The targeting domain is A nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5041-5042, 5047, 5050, 5052, and 5092-5093. Including, The genome editing system of the present invention 1010. [The present invention 1012] The genome editing system of any one of claims 1001 to 1009, wherein the RNA-guided nuclease is Staphylococcus aureus (S. aureus) Cas9 nuclease. [The present invention 1013] A genome editing system of the present invention, wherein the Staphylococcus aureus Cas9 nuclease recognizes either NNNRRT or NNNRRV PAM, and the genome editing system targets TGFBR2. [The present invention 1014] The genome editing system of any one of claims 1001 to 1013, wherein the RNA-guided nuclease is a mutant Cas9 nuclease. [The present invention 1015] A genome editing system of any of claims 1001 to 1014, wherein the gRNA is a modular gRNA or a chimeric gRNA. [The present invention 1016] Any of the genome editing systems of the present inventions 1001 to 1015, wherein the targeting domain has a length of about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides. [The present invention 1017] A genome editing system of the present invention, wherein the targeting domain comprises at least about 18 consecutive nucleotides complementary to the TGFBR2 gene. [The present invention 1018] A genome editing system of any of claims 1001 to 1017, comprising two, three or four gRNAs. [The present invention 1019] at least one Streptococcus pyogenes Cas9 nuclease; and gRNA comprising a combination of SEQ ID NOs: 5042 and 5041, or a combination of 5042 and 5092, or a combination of SEQ ID NOs: 5042 and 5093, or a combination of SEQ ID NOs: 5093 and 5041. The genome editing system of the present invention 1009, comprising: [The present invention 1020] A genome editing system according to any one of claims 1001 to 1019, for use in modifying a TGFBR2 gene in a cell. [The present invention 1021] The genome editing system of the present invention 1020, wherein the cells are derived from a subject suffering from cancer. [The present invention 1022] A genome editing system of the present invention 1001, in which the expression of TGFBR2 is reduced by 30% or more compared to baseline measurements. [The present invention 1023] A genome editing system of the present invention 1022, wherein the expression of TGFBR2 protein is determined by Western blot or indirect intracellular staining flow cytometry. [The present invention 1024] 1001. A genome editing system according to claim 10, wherein a frameshift mutation is introduced into the TGFBR2 gene. [The present invention 1025] A composition comprising a gRNA comprising a targeting domain complementary to a target sequence of a TGFBR2 gene. [The present invention 1026] The composition of the present invention 1025, wherein the target sequence of the TGRBR2 gene comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, and 3. [The present invention 1027] The composition of the present invention 1025, wherein the target sequence of the TGRBR2 gene comprises a sequence selected from the group consisting of SEQ ID NO: 4 to 10. [The present invention 1028] 1025. The composition of the present invention, wherein the targeting domain has at least 85% complementarity to the target sequence of the TGRBR2 gene. [The present invention 1029] The composition of the invention 1025, wherein the targeting domain has a length of about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides. [The present invention 1030] 1029. The composition of claim 1029, wherein the targeting domain comprises at least about 18 contiguous nucleotides complementary to the TGFBR2 gene. [The present invention 1031] The targeting domain is A nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5036 to 5096. 1025. The composition of the present invention comprising: [The present invention 1032] The composition of any one of claims 1025 to 1031, comprising one, two, three, or four gRNAs. [The present invention 1033] The composition of any one of claims 1025 to 1032, further comprising a Cas9 nuclease. [The present invention 1034] The composition of the present invention 1033, wherein the Cas9 nuclease is Streptococcus pyogenes Cas9 nuclease or Staphylococcus aureus Cas9 nuclease. [This invention 1035] The composition of the present invention 1033, further comprising one or both of a wild-type Cas9 nuclease and a mutant Cas9 nuclease. [The present invention 1036] the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule; The targeting domain is (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO:5093 a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: Including, Composition of the present invention 1033. [This invention 1037] Streptococcus pyogenes Cas9 nuclease recognizes the NGG protospacer adjacent motif (PAM), the composition targets TGFBR2; The targeting domain is A nucleotide sequence that is identical to or differs by no more than 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5041-5042, 5047, 5050, 5052, 5092, and 5093. Including, Composition of the present invention 1036. [The present invention 1038] 1034. The composition of claim 1034, wherein the Staphylococcus aureus Cas9 nuclease recognizes either an NNNRRT or NNNRRV PAM, and wherein the composition targets TGFBR2. [This invention 1039] at least one Streptococcus pyogenes Cas9 nuclease; and gRNA comprising a combination of SEQ ID NOs: 5042 and 5041, or a combination of 5042 and 5092, or a combination of SEQ ID NOs: 5042 and 5093, or a combination of SEQ ID NOs: 5093 and 5041. Any of the compositions of 1025 to 1038 of the present invention, comprising: [The present invention 1040] 1039. The composition of any of claims 1025 to 1039 for use in reducing or eliminating TGFBR2 gene expression in a cell. [This invention 1041] The composition of claim 1040, wherein the cell is derived from a subject suffering from cancer. [The present invention 1042] A vector comprising a polynucleotide encoding a gRNA comprising a targeting domain complementary to a target sequence of the TGFBR2 gene. [This invention 1043] The targeting domain is A nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5036 to 5096. 1042. The vector of the present invention, comprising: [This invention 1044] The vector of the present invention 1042 or 1043, further comprising a polynucleotide encoding a Cas9 nuclease. [This invention 1045] 1044. The vector of the present invention, wherein the Cas9 nuclease is Streptococcus pyogenes Cas9 nuclease or Staphylococcus aureus Cas9 nuclease. [The present invention 1046] The vector of the present invention 1044 or 1045, wherein the Cas9 nuclease further comprises one or both of a wild-type Cas9 nuclease and a mutant Cas9 nuclease. [This invention 1047] the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease; The targeting domain is (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO:5093 a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: Including, The vector of the present invention 1044. [This invention 1048] Streptococcus pyogenes Cas9 nuclease recognizes the NGG protospacer adjacent motif (PAM), the vector encodes a gRNA that targets TGFBR2; The targeting domain is A nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5041-5042, 5047, 5050, 5052, and 5092-5093. Including, The vector of the present invention 1047. [This invention 1049] 1045. The vector of the present invention, wherein the Staphylococcus aureus Cas9 nuclease recognizes either an NNNRRT or NNNRRV PAM, and the vector encodes a gRNA that targets TGFBR2. [The present invention 1050] The vector of any one of 1042 to 1049 of the present invention, wherein the vector is a viral vector. [This invention 1051] The vector of the present invention 1050, wherein the viral vector is an adeno-associated viral (AAV) vector or a lentiviral (LV) vector. [This invention 1052] The vector of any of claims 1042 to 1051 for use in reducing or eliminating TGFBR2 gene expression in a cell. [This invention 1053] The vector of the present invention 1052, wherein the cell is derived from a subject suffering from cancer. [This invention 1054] (i) a genome editing system comprising a gRNA comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and an RNA-guided nuclease; or (ii) a vector comprising a polynucleotide encoding a gRNA comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and a polynucleotide encoding an RNA-guided nuclease; A method for modifying expression of the TGFBR2 gene in a cell, comprising administering to the cell one of the following: [This invention 1055] The method of claim 1054, wherein the modification comprises knocking out TGFBR2 gene expression or knocking down TGFBR2 gene expression. [The present invention 1056] The method of any one of claims 1054 to 1055, wherein the cell is derived from a subject suffering from cancer. [This invention 1057] The method of claim 1054, wherein the gRNA and the RNA-guided nuclease comprise a ribonucleoprotein (RNP) complex. [This invention 1058] 1057. The method of claim 1057, comprising administering to a cell two or more RNP complexes comprising gRNAs with different targeting domains. [This invention 1059] The method of claim 1057, wherein the RNP complex comprises an enzymatically active Cas9 (eaCas9) nuclease. [The present invention 1060] The method of claim 1059, wherein the RNP complex comprises an eaCas9 nuclease that forms a double-stranded break in the target nucleic acid or a single-stranded break in the target nucleic acid. [The present invention 1061] 1058. The method of claim 1058, wherein two RNP complexes containing separate gRNAs are used to create offset single-stranded breaks in the TGFBR2 gene in the cell. [The present invention 1062] A cell comprising any one of the genome editing systems of the present inventions 1001 to 1024, any one of the compositions of the present inventions 1024 to 1041, or any one of the vectors of the present inventions 1042 to 1053. [The present invention 1063] The cell of the present invention 1062, which expresses TGFBR2. [The present invention 1064] The cell of the present invention 1062 or 1063, which is a T cell or a natural killer (NK) cell. [This invention 1065] The cell of claim 1064, further comprising an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR). [The present invention 1066] A cell modified according to any one of inventions 1054 to 1061. [This invention 1067] a) contacting a cell with a sufficient amount of a gRNA targeting TGFBR2 and an RNA-guided nuclease; and b) creating a first DNA double-strand break near a TGFBR2 target location within the TGFBR2 gene of the cell, wherein the first DNA double-strand break is repaired by NHEJ, and wherein the repair alters expression of the TGFBR2 gene. 1. An RNA-guided nuclease-mediated method for modifying TGFBR2 gene expression in a cell, comprising: [The present invention 1068] The method of claim 1068, further comprising forming a second DNA double-strand break near the TGFBR2 target location. [This invention 1069] 1067. The method of claim 1067, wherein the first double-stranded break is formed within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position. [The present invention 1070] The method of the present invention 1068, wherein the first and second double-stranded breaks are formed within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position. [This invention 1071] The method of claim 1067, wherein the first double-stranded break is formed in a coding or non-coding region of the TGFBR2 gene, and the non-coding region comprises a promoter region, an enhancer region, an intron, a 3' UTR, a 5' UTR, or a polyadenylation signal region of the TGFBR2 gene. [This invention 1072] The method of claim 1068, wherein the first and second double-stranded breaks are formed in a coding or non-coding region of the TGFBR2 gene, and the non-coding region comprises a promoter region, an enhancer region, an intron, a 3'UTR, a 5'UTR, or a polyadenylation signal region of the TGFBR2 gene. [This invention 1073] 1073. The method of any of claims 1067 to 1072, wherein the coding region is selected from exon 3, exon 4, and exon 5. [This invention 1074] The targeting domain is A nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5036 to 5096. Any of the methods of claims 1067 to 1073, comprising: [This invention 1075] the RNA-guided nuclease is Streptococcus pyogenes Cas9 nuclease; The targeting domain is (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO:5093 a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: Including, Any of the methods of the present invention 1067 to 1074. [This invention 1076] 1075. The method of any one of claims 1067 to 1074, wherein the RNA-guided nuclease is Staphylococcus aureus Cas9 nuclease. [This invention 1077] 1077. The method of claim 1075 or 1076, wherein the RNA-guided nuclease is a mutant Cas9 nuclease. [This invention 1078] 1067. The method of claim 1067, wherein the NHEJ repair results in an insertion or deletion at a frequency of 20% or more. [This invention 1079] The method of claim 1078, wherein the frequency of insertions or deletions is 30% or more, 40% or more, or 50% or more. [The present invention 1080] 1. A genomically engineered cell comprising an insertion or deletion near or at a target location in the TGFBR2 gene, The target location is A nucleotide sequence that is complementary to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5036 to 5096. Including, Genome-engineered cells. [This invention 1081] 1080. The cell of the invention, wherein the insertion or deletion is within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position. [This invention 1082] The cell of the present invention, 1080, which is a T cell or an NK cell. [This invention 1083] The cell of claim 1082, further comprising an eTCR or CAR. [This invention 1084] a. a population of genomically engineered cells comprising an insertion or deletion near or at a target location in the TGFBR2 gene, wherein the target location comprises a nucleotide sequence that is complementary to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from SEQ ID NOs: 5036-5096; b. A pharmaceutically acceptable buffer; A composition comprising: [This invention 1085] The composition of claim 1084, wherein the population of cells comprises T cells or NK cells. [The present invention 1086] The composition of the present invention 1085, wherein the T cell or NK cell further comprises an eTCR or CAR. [This invention 1087] 1. A method of treating cancer in a subject, comprising administering engineered immune cells to the subject, The engineered immune cells have reduced expression of TGFBR2, the engineered immune cells optionally express an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR), and the engineered immune cells have an insertion or deletion near or at a target location in the TGFBR2 gene. method. [This invention 1088] The method of claim 1087, wherein the engineered immune cells comprise T cells or NK cells. [This invention 1089] The method of claim 1087, wherein the eTCR or CAR has antigen specificity for the cancer cell. [The present invention 1090] 1087. The method of claim 1087, wherein the cancer is selected from the group consisting of leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, low-grade B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, mesothelioma, and / or any cancer type that expresses TGF-β. [This invention 1091] The method of claim 1088, wherein the T cells are CD4+ T cells and / or CD8+ T cells. [This invention 1092] The method of claim 1087, wherein the engineered immune cells maintain or have enhanced lytic activity against the target cancer cells compared to non-engineered immune cells. [This invention 1093] The method of claim 1087, wherein the engineered immune cells maintain or increase expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to non-engineered immune cells. [This invention 1094] 1087. The method of claim 1087, wherein the engineered immune cells maintain or have improved persistence to repeated antigen stimulation compared to non-engineered immune cells. [This invention 1095] 1087. The method of claim 1087, wherein the engineered immune cells maintain or have increased expression of CD25 compared to non-engineered immune cells. [This invention 1096] The method of claim 1087, wherein the engineered immune cells maintain or have reduced expression of PD-1 compared to non-engineered immune cells. [This invention 1097] The method of claim 1087, wherein the engineered immune cells maintain or increase proliferation compared to non-engineered immune cells. [This invention 1098] A composition comprising a plurality of engineered T cells, wherein the engineered T cells exhibit reduced TGFBR2 gene expression compared to non-engineered T cells. [This invention 1099] The composition of the present invention 1098, wherein the engineered T cells exhibit a TGFBR2 gene expression level that is about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of the TGFBR2 expression level in non-engineered T cells. [The present invention 1100] The composition of claim 1098, wherein the engineered T cell further comprises expression of an eTCR or CAR. [The present invention 1101] The composition of the present invention 1098, wherein the T cells are CD4+ T cells and / or CD8+ T cells. [The present invention 1102] a) enhanced lytic activity against target cancer cells compared to non-engineered T cells; b) sustained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to unmanipulated T cells; c) sustained or increased persistence to repeated antigen stimulation compared to unmanipulated T cells; d) maintained or increased expression of CD25 compared to unmanipulated T cells; e) maintained or reduced expression of PD-1 compared to unmanipulated T cells, and / or f) Sustained or increased proliferation compared to unmanipulated T cells The composition of the present invention 1098, wherein the engineered T cells are further characterized by having: [The present invention 1103] 1. A composition comprising a plurality of engineered T cells, engineered T cells exhibit reduced TGFBR2 gene expression compared to non-engineered T cells; The engineered T cells a gRNA containing a targeting domain complementary to a target sequence of the TGFBR2 gene; and RNA-guided nucleases by contacting unmanipulated T cells with a genome editing system comprising composition. [The present invention 1104] The composition of the present invention 1103, wherein the engineered T cells are further transduced with a vector expressing an eTCR or a CAR. [This invention 1105] The composition of claim 1104, wherein the vector is a viral vector. [The present invention 1106] The composition of the present invention 1105, wherein the viral vector is an adeno-associated viral (AAV) vector or a lentiviral (LV) vector. [This invention 1107] the RNA-guided nuclease is Streptococcus pyogenes Cas9 nuclease; The targeting domain is (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and (g) SEQ ID NO:5093 a nucleotide sequence that is identical to or differs by no more than about 3 nucleotides from a nucleotide sequence selected from the group consisting of: Including, Composition of the present invention 1103. [This invention 1108] A composition comprising a plurality of engineered T cells, wherein the engineered T cells are deficient in TGFBR2 signaling. [This invention 1109] The composition of the present invention 1108, wherein the defective TGFBR2 signaling is mediated by expressing a dominant negative (DN) form of TGFBR2 in the engineered T cells. [The present invention 1110] The composition of the present invention 1109, wherein the engineered T cells are further transduced with a vector expressing an eTCR or a CAR. [The present invention 1111] The composition of claim 1110, wherein the vector is a viral vector. [The present invention 1112] The composition of the present invention 1111, wherein the viral vector is an adeno-associated viral (AAV) vector or a lentiviral (LV) vector. [The present invention 1113] The composition of the present invention 1108, wherein the T cells are CD4+ T cells and / or CD8+ T cells. [This invention 1114] a) enhanced lytic activity against target cancer cells compared to non-engineered T cells; b) sustained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to unmanipulated T cells; c) sustained or increased persistence to repeated antigen stimulation compared to unmanipulated T cells; d) maintained or increased expression of CD25 compared to unmanipulated T cells; e) maintained or reduced expression of PD-1 compared to unmanipulated T cells, and / or f) Sustained or increased proliferation compared to unmanipulated T cells The composition of claim 1108, wherein the engineered T cells are further characterized by having: [This invention 1115] 1109. The composition of claim 1109, wherein the engineered immune cells further comprise reduced expression of wild-type TGFBR2. [The present invention 1116] Wild-type TGFBR2 expression a gRNA containing a targeting domain complementary to a target sequence of the TGFBR2 gene; and RNA-guided nucleases The composition of the present invention 1115, wherein the gene is reduced by contacting engineered immune cells with a genome editing system comprising: [This invention 1117] A ribonucleoprotein (RNP) complex containing a gRNA with a targeting domain complementary to the target sequence of the TGFBR2 gene and an RNA-guided nuclease. [This invention 1118] The RNP of the present invention 1117, wherein the RNA-guided nuclease is a Cas9 nuclease. [This invention 1119] The RNP of the present invention 1117, wherein the RNP is electroporated into a cell. Other features and advantages of the invention will become apparent from the detailed description, examples, drawings, and claims.

[0074] The accompanying drawings are intended to provide illustrative and schematic examples of certain aspects and embodiments of the present disclosure, rather than being comprehensive. The drawings are not intended to be limited or bound to any particular theory or model, and are not necessarily drawn to scale. Without limiting the above, nucleic acids and polypeptides may be shown as linear sequences or as schematic two-dimensional or three-dimensional structures. These depictions are intended to be illustrative, rather than limited or bound to any particular model or theory regarding their structure. [Brief explanation of the drawings]

[0075] [Figure 1] Figure 1 shows a list of exemplary gRNAs targeting exons 1A-5 of transforming growth factor beta receptor II (TGFBR2) and their associated non-homologous end joining (NHEJ) activity rates (%). [Figure 2] Figure 2 shows the genome editing efficiency of certain exemplary gRNA pairs. [Figure 3] Figure 3 shows the indel formation rate (%) determined by miSeq for the TGFBR2 gene. Seven gRNAs were tested at various concentrations of RNP to generate a dose-response curve. [Figure 4] Figure 4A-4B show the edited cell rate (%) (Figure 4A) and indel frequency rate (%) (Figure 4B) using several tested gRNAs targeting TGFBR2 and a control gRNA targeting the AAVS1 locus. [Figure 5] Figure 5 shows a comparison of two TGFBR2-targeting gRNAs in their ability to generate out-of-frame indel mutations. [Figure 6] Figure 6 shows the relative IFN-γ production of primary T cells transduced to express anti-BCMA CAR in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. Single or paired gRNAs were compared. [Figure 7] Figure 7 shows the relative cell proliferation of primary T cells transduced to express anti-BCMA CAR in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. Single or paired gRNAs were compared. [Figure 8] Figures 8A-8B show CD25 (Figure 8A) and PD-1 (Figure 8B) expression in primary T cells transduced to express anti-BCMA CAR in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. Single or paired gRNAs were compared. Cells were similarly stimulated with RPMI 8226 cells for 48 hours. [Figure 9]Figure 9 shows Smad 2 / 3 phosphorylation of T cells transduced to express different CARs in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. [Figure 10] Figure 10 shows relative Granzyme B expression in T cells transduced to express different CARs in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. [Figure 11] Figure 11 shows IFN-γ production of T cells transduced to express different CARs in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. [Figure 12] Figure 12 shows the relative T cell proliferation of T cells transduced to express different CARs in a TGFBR2 gene-edited background in the presence (10 ng / ml) or absence of TGFβ. [Figure 13] Figures 13A-B show granzyme B (Figure 13A) and interferon gamma (Figure 13B) production of primary T cells transduced to express an anti-BCMA CAR in a TGFBR2 gene-edited or TGFBR2 DN background in the presence (10 ng / ml) or absence of TGFβ. [Figure 14] Figure 14 shows the relative % lysis of RPMI 8226 cells by anti-BCMA CAR-expressing T cells in a TGFBR2 gene-edited or TGFBR2 DN background in the presence (10 ng / ml) or absence of TGFβ. [Figure 15A]Figures 15A-15F show anti-BCMA CAR-expressing T cells in a TGFBR2 unedited background, a TGFBR2 gene-edited background, or a TGFBR2 DN background using repeated TGFβ antigen stimulation. Figures 15A-15C show predicted cell numbers after stimulation with three different antigens. Figures 15D-15F show the % anti-BCMA CAR-expressing T cells over time using repeated TGFβ antigen stimulation. [Figure 15B] See Figure 15A for illustration. [Figure 15C] See Figure 15A for illustration. [Figure 15D] See Figure 15A for illustration. [Figure 15E] See Figure 15A for illustration. [Figure 15F] See Figure 15A for illustration. [Figure 16] Figures 16A-B show INF-γ production of primary T cells transduced to express an anti-BCMA CAR in a TGFBR2 gene-edited or TGFBR2 DN background in the presence or absence of TGFβ (10 ng / ml). T cells were co-incubated with either RPMI 8226 cells (Figure 16A) or OPM2 cells (Figure 16B). [Figure 17] Figures 17A-B show CD25 expression in primary T cells transduced to express an anti-BCMA CAR in a TGFBR2 gene-edited or TGFBR2 DN background in the presence or absence of TGFβ (10 ng / ml). T cells were co-incubated with either RPMI 8226 cells (Figure 17A) or OPM2 cells (Figure 17B). [Figure 18]Figures 18A-B show PD-1 expression in primary T cells transduced to express an anti-BCMA CAR in a TGFBR2 gene-edited or TGFBR2 DN background in the presence or absence of TGFβ (10 ng / ml). T cells were co-incubated with either RPMI 8226 cells (Figure 18A) or OPM2 cells (Figure 18B). [Figure 19] Figures 19A-19B show the relative cell proliferation (Figure 19A) and relative IFN-γ production (Figure 19B) of anti-BCMA CAR-expressing T cells in a TGFBR2 gene-edited or TGFBR2 DN background in the presence (10 ng / ml) or absence of TGFβ. Cells were similarly stimulated using RPMI 8226 cells. [Figure 20] Figures 20A-B show the PD-1% cell percentage in several CD4+ (Figure 20A) and CD8+ (Figure 20B) anti-BCMA CAR-expressing T cells in a TGFBR2 gene-edited or TGFBR2 DN background with increasing concentrations of TGFβ. Cells were also stimulated with RPMI 8226 cells for 48 hours. [Figure 21] Figure 21 shows the expression levels of the TGFβ signaling pathway genes PMEPA1, SKIL, SKI, and LDLRAD4 in anti-BCMA CAR-expressing T cells in a TGFBR2 gene-edited or TGFBR2 DN background. T cells were isolated from mouse spleens or RPMI 8226 cell-derived tumors. [Figure 22] Figure 22 shows Smad 2 / 3 phosphorylation levels, proliferation and granzyme B expression of anti-CD19 CAR-expressing T cells in a TGFBR2 DN background in the presence and absence of TGFβ. [Figure 23] Figure 23 shows a schematic for determining whether TGFBR2 gene editing of T cells confers a selective advantage over wild-type cells. [Figure 24]Figure 24 shows various ratios of anti-BCMA CAR cells, TGFBR2-KO cells, and WT cells (1, 0.75, 0.5, 0.25). Cells were co-cultured with RPMI8226 cells at a 1:1 effector:target ratio ±10 ng / ml TGFβ. Cells were harvested every 7 days and analyzed for indel rates (%) by high-throughput sequencing. Cells were restimulated with fresh RPMI8226 and readjusted weekly to a 1:1 effector:target ratio. DETAILED DESCRIPTION OF THE INVENTION

[0076] Detailed Description Definitions and Abbreviations Unless otherwise specified, each of the following terms has the meaning associated with it in this section.

[0077] The indefinite articles "a" and "an" refer to at least one of the associated noun and are used interchangeably with the terms "at least one" and "one or more." For example, "a module" means at least one module or one or more modules.

[0078] The conjunctions "or" and "and / or" are used interchangeably as non-exclusive disjunctions.

[0079] The phrase "consisting essentially of" means that the recited species are the predominant species, but that other species may be present in minor amounts or quantities that do not affect the structure, function, or behavior of the subject composition. For example, a composition consisting essentially of a particular species generally contains 90%, 95%, 96%, or more of that species.

[0080] "Domain" is used to describe a segment of a protein or nucleic acid. Unless otherwise specified, a domain does not necessarily have any particular functional properties.

[0081] "Indel" refers to an insertion and / or deletion in a nucleic acid sequence. Indels can be the product of repairing DNA double-strand breaks, such as those formed by the genome editing system of the present disclosure. Indels are most often formed when breaks are repaired by an "error-prone" repair pathway, such as the NHEJ pathway described below. Indels can result in insertions or deletions that cause in-frame or out-of-frame mutations in the target sequence.

[0082] "Gene conversion" refers to the modification of DNA sequence by the incorporation of endogenous homologous sequence (for example, homologous sequence in gene array). "Gene correction" refers to the modification of DNA sequence by the incorporation of exogenous homologous sequence, such as exogenous single-stranded or double-stranded donor template DNA.Gene conversion and gene correction are the products of DNA double-strand break repair by HDR pathway, as described below.

[0083] Indels, gene conversions, gene corrections, and other genome editing results are typically assessed by sequencing (most commonly by "next-generation" or "sequencing-by-synthesis" methods, although Sanger sequencing can still be used) and quantified by the relative frequency of numerical changes (e.g., ±1, ±2 or more bases) at the site of interest among all sequencing reads. DNA samples for sequencing may be prepared by a variety of methods known in the art, including amplifying the site of interest by polymerase chain reaction (PCR), capturing DNA ends generated by double-strand breaks, as in the GUIDEseq process described in Tsai et al. (Nat. Biotechnol. 34(5):483(2016)), which is incorporated herein by reference, or by other means known in the art. The results of genome editing may also be assessed by in situ hybridization methods, such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France), and by any other suitable method known in the art.

[0084] "Alt-HDR", "alternative homology-directed repair" or "alternative HDR" are used interchangeably to refer to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid). Alt-HDR differs from standard HDR in that this process utilizes a different pathway from standard HDR and can be inhibited by standard HDR mediators RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of single-stranded or nick homologous nucleic acid templates, while standard HDR generally involves double-stranded homologous templates.

[0085] "Standard HDR", "standard homology-directed repair", or "cHDR" refers to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid). Standard HDR typically operates when there is significant excision at double-strand break, forming at least one single-stranded portion of DNA. In normal cells, cHDR typically involves a series of steps, such as break recognition, break stabilization, excision, single-stranded DNA stabilization, DNA crossover intermediate formation, crossover intermediate division, and ligation. This process requires RAD51 and BRCA2, and the homologous nucleic acid is typically double-stranded.

[0086] Unless otherwise specified, the term "HDR" as used herein encompasses both standard HDR and alt-HDR.

[0087] "Non-homologous end joining" or "NHEJ" refers to ligation-mediated and / or non-template-mediated repair, including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), including microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0088] "Substitution" or "substituted," when used in reference to the modification of a molecule (e.g., a nucleic acid or protein), does not necessarily qualify the process, but merely indicates that a replacement entity is present.

[0089] "Subject" means a human or non-human animal. A human subject may be of any age (e.g., infant, child, young adult, or adult) and may be suffering from a disease or in need of genetic modification. Alternatively, a subject may be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, and more specifically, non-human primates, rodents (e.g., mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, etc. In certain aspects of the present disclosure, the subject is a livestock animal, such as a cow, horse, sheep, or goat. In certain aspects, the subject is poultry.

[0090] "Treat," "treating," and "treatment" refer to the treatment of a disease in a subject (e.g., a human subject), and include one or more of: inhibiting the disease, i.e., halting or preventing its onset or progression; alleviating the disease, i.e., causing regression of the pathological state; alleviating one or more symptoms of the disease; and curing the disease.

[0091] "Prevent," "preventing," and "prevention" refer to the prevention of disease in a mammal, e.g., a human, and include (a) avoiding or eliminating the disease, (b) affecting a predisposition to the disease, or (c) preventing or delaying the onset of at least one symptom of the disease.

[0092] A "kit" refers to any collection of two or more components that together constitute a functional unit that can be used for a specific purpose. By way of example (and not limitation), a kit according to the present disclosure may include a guide RNA that is or can be complexed with an RNA-guided nuclease and that is associated with a pharmaceutically acceptable carrier (e.g., suspended or suspendable). The kit can be used, for example, to introduce the complex into a cell or a subject to induce a desired genome modification in such a cell or subject. The components of the kit can be packaged together or separately. A kit according to the present disclosure may also include a instructions for use (DFU) that describes, for example, the use of the kit according to the methods of the present disclosure. The DFU can be physically packaged with the kit or can be made available to a user of the kit, for example, by electronic means.

[0093] The terms "polynucleotide," "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, and refer to any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc., can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including, but not limited to, information used by cellular machinery to make proteins and enzymes. These terms include double-stranded or single-stranded genomic DNA, RNA, synthetic and genetically engineered polynucleotides, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0094] Conventional IUPAC notation is used in the nucleotide sequences presented herein, as shown in Table 1 below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10;13(9):3021-30, which is incorporated herein by reference). Note, however, that in cases where the sequence can be encoded by either DNA or RNA, for example, in the gRNA targeting domain, "T" means "thymine or uracil."

[0095] (Table 1) IUPAC Nucleic Acid Notation TIFF0007785452000001.tif90128

[0096] The terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to a continuous chain of amino acids linked together through peptide bonds. The term includes individual proteins, groups or complexes of proteins associated with each other, as well as fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left and continuing to the carboxyl or C-terminus on the right. Standard one- or three-letter abbreviations can be used.

[0097] The term "variant" refers to an entity such as a polypeptide, polynucleotide, or small molecule that exhibits significant structural identity with a reference entity, but that is structurally different from the reference entity in terms of the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a variant is also functionally different from the reference entity. Generally, whether a particular entity is appropriately considered to be a "variant" of a reference entity is based on the degree of structural identity with the reference entity.

[0098] overview The genome editing systems described herein generally include one or more gRNAs that include a targeting domain complementary to one or more TGFBR2 target sequences, which in turn include or are adjacent to a protospacer adjacent motif (PAM) sequence recognized by one or more RNA-guided nucleases to which the one or more gRNAs can bind (e.g., complex). Thus, the genome editing systems of the present disclosure function to target one or more TGFBR2 target sequences in a site-specific manner and introduce modifications within or adjacent to those TGFBR2 target sequences.

[0099] The modifications introduced into or adjacent to the TGFBR2 target sites by the genome editing system of the present disclosure most commonly involve DNA single-strand breaks (SSBs or "nicks") and / or double-strand breaks (DSBs). The cells then repair the nicks and DSBs in such a way as to introduce small indels or relatively large insertions or deletions at one or more TGFBR2 target sites, delete sequences between the two TGFBR2 target sites, and / or insert sequences (particularly exogenous sequences introduced into the cells via donor template oligonucleotides) into the TGFBR2 sites, or replace the endogenous cellular DNA sequences between the two TGFBR2 target sites. However, in some cases, the genome editing system introduces one or more point mutations (e.g., via cysteine ​​deamination), changes in DNA marking (e.g., DNA methylation, histone acetylation or deacetylation, or other chromatin modifications), and / or recruit trans-acting factors such as transcription factors. Alternatively, the genome editing systems of the present disclosure may associate with one or more TGFBR2 target sequences in a sustained (e.g., over intervals of weeks, months, or longer) or transient (over intervals of seconds, minutes, hours, or days) manner, thereby preventing other factors (particularly RNA polymerases, but also DNA polymerases, transcription factors, and / or other cis- or trans-acting factors that affect gene expression) from associating with the TGFBR2 target sequences. These and other modes of action of genome editing systems and their components are described in more detail below under the headings "RNA-Guided Nucleases" and "Modifications of RNA-Guided Nucleases."

[0100] TGFBR2 target sequences and corresponding gRNA targeting domain sequences are generally, but not necessarily, located in exons, where the introduction of small indels or relatively large insertions or deletions can result in one or more mutations that reduce or eliminate TGFBR2 protein function (e.g., frameshift mutations, nonsense mutations, introduction of codons for amino acids that disrupt the structure of the surrounding protein, and / or removal of codons for amino acids required for protein activity). Figure 1 shows the mapping of the cleavage activity of various S. pyogenes guide RNAs to target locations within the exon structure of the TGFBR2 gene. These mutations are referred to throughout this specification as "knockout" mutations, and their functional effect is a "knockout" of TGFBR2 protein function.

[0101] Certain TGFBR2 target sequences can be considered "hotspot" target sites of gRNA targeting domain sequences.As used herein, "hotspot" refers to a site that is preferentially targeted to produce a high indel frequency rate (%) or to effectively knock down or knock out the TGFBR2 gene.The gRNA that targets one or more of these preferred sites can produce an indel frequency rate (%) of 30% or more.For example, preferred target sites in the TGFBR2 gene can have complementary gRNA targeting domains that produce an indel frequency rate (%) of 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more.Hotspot target sites in the TGFBR2 gene are described herein.

[0102] Preferred hotspot TGFBR2 regions are shown in Table x.

[0103] Table 2. Preferred TGFBR2 target sites TIFF0007785452000002.tif123155

[0104] Particularly preferred hotspot TGFBR2 regions are shown in Table 3.

[0105] (Table 3) TIFF0007785452000003.tif74150

[0106] The TGFBR2 target sequence can be located, for example, in exon 3, 4, or 5 of the TGFBR2 gene. A gRNA targeting domain sequence corresponding to a TGFBR2 target sequence present in exon 3, 4, or 5 of the TGFBR2 gene can comprise a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, a nucleotide sequence selected from SEQ ID NOs: 5036-5096. For example, and without limitation, exemplary targeting domains can comprise a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO:5041; (b) SEQ ID NO:5042; (c) SEQ ID NO: 5047, (d) SEQ ID NO: 5050; (e) SEQ ID NO: 5052, (f) SEQ ID NO: 5092, and g) SEQ ID NO:5093.

[0107] Table 4: Targeting sequences TIFF0007785452000004.tif45135

[0108] Instead of knocking out TGFBR2 expression, a transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region controlling the transcription of the TGFBR2 gene), can be targeted to alter (e.g., knockdown) gene expression. As described herein, the targeted knockdown approach can be mediated by a CRISPR / Cas system comprising an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcriptional repressor domain or a chromatin-modifying protein). For example, one or more gRNA molecules comprising a targeting domain can be configured to target an eiCas9 molecule or eiCas9 fusion protein sufficiently close to a transcriptional regulatory region, e.g., a promoter region (e.g., a promoter region controlling the transcription of the TGFBR2 gene), so that transcription of the TGFBR2 gene is reduced and / or eliminated. In certain embodiments, eiCas9 or an eiCas9 fusion protein can be used to knockdown TGFBR2 expression in T cells, e.g., human T cells.

[0109] TGFBR2 knockout and / or knockdown can be assessed by any suitable method, including, but not limited to, examining the sequence of the TGFBR2 gene, assessing TGFBR2 protein expression on the cell surface (e.g., by immunostaining and cell sorting, particularly by fluorescence-activated cell sorting, or FACS, including indirect intracellular staining flow cytometry), detecting cellular or molecular changes mediated by TGFBR2, assessing the effect of TGF-β on cell proliferation or survival, or by Western blot to detect TGFBR2 protein levels. Particularly with respect to T cells, TGFBR2 knockout can be confirmed by (a) sequencing the TGFBR2 locus or T7E1 primer extension assays, and / or (b) intracellular FACS assessment of SMAD2 / 3 phosphorylation. Sequencing and T7E1 are described in further detail below, and the intracellular SMAD2 / 3 phosphorylation assay is described, for example, in Chen et al., J. Experimental Med. Volume 198, Number 12, December 15, 2003 1875-1886 (this reference is incorporated by reference in its entirety for all purposes), in particular the Materials & Methods section on page 1877 and Supplemental Figure S2.

[0110] Knockout and / or knockdown of TGFBR2 can correspond to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% reduction in TGFBR2 expression compared to baseline measurements or wild-type cells.

[0111] In some aspects, the compositions and methods provided include those in which at least about or greater than 50%, at least about or greater than 60%, at least about or greater than 65%, at least about or greater than 70%, at least about or greater than 75%, at least about or greater than 80%, at least about or greater than 85%, at least about or greater than 90%, or at least about or greater than 95% of the cells in a composition of cells into which an agent (e.g., gRNA / Cas9) for knockout or gene disruption of the TGFBR2 gene has been introduced comprise a gene disruption, do not express an endogenous TGFBR2 polypeptide, and do not contain a contiguous TGFBR2 gene, a TGFBR2 gene, and / or a functional TGFBR2 gene. In some embodiments, methods, compositions, and cells according to the present disclosure include those in which at least about or greater than about 50%, at least about or greater than about 60%, at least about or greater than about 65%, at least about or greater than about 70%, at least about or greater than about 75%, at least about or greater than about 80%, at least about or greater than about 85%, at least about or greater than about 90%, or at least about or greater than about 95% of cells in a composition of cells into which an agent for knocking out or disrupting the TGFBR2 gene (e.g., gRNA / Cas9) has been introduced do not express TGFBR2 polypeptide, such as on the cell surface. In some embodiments, at least about or greater than about 50%, at least about or greater than about 60%, at least about or greater than about 65%, at least about or greater than about 70%, at least about or greater than about 75%, at least about or greater than about 80%, at least about or greater than about 85%, at least about or greater than about 90%, or at least about or greater than about 95% of the cells in a composition of cells into which an agent for knocking out or disrupting the TGFBR2 gene (e.g., gRNA / Cas9) has been introduced are knocked out in both alleles, i.e., contain a biallelic deletion within such percentage of cells.

[0112] Genome editing systems targeting TGFBR2 can be implemented in a variety of ways, and these implementations can be tailored to the setting in which the cells are edited. Certain embodiments of the present disclosure involve delivering a TGFBR2-targeting RNA-guided nuclease and guide RNA to cells ex vivo in the form of a ribonucleoprotein (RNP) complex by electroporation using an electroporator and cuvette available from commercial suppliers such as MaxCyte (Gaithersburg, MD) or Lonza (Basel, Switzerland). However, other embodiments may implement in vivo nucleic acid vectors, such as viral vectors or lipid nanoparticles, for either in vivo or ex vivo editing. Details of these implementations are described in more detail below under the heading "Implementation of the Genome Editing System."

[0113] Knockout and / or knockdown of TGFBR2 can be useful in a variety of contexts, including, but not limited to, adaptive T cell therapy. According to certain embodiments of the present disclosure, TGFBR2 is knocked out in immune cells, such as T cells, used for therapy. By way of example, the T cells may express an engineered receptor, such as a chimeric antigen receptor (CAR) or a heterologous T cell receptor (TCR), which can be configured to recognize an antigen on cells or tissues involved in a pathology, such as a tumor. TGFBR2 knockout T cells according to the present disclosure, whether they express an engineered receptor or not, can be used to target tissues or organs where TGF-β is present in an amount sufficient to reduce the proliferation or activity of TGFBR2-expressing T cells.

[0114] TGFBR2 knockout and / or knockdown cells may be used in "autologous" cell therapy, in which cells are harvested from a subject, modified to knock out or knock down TGFBR2 expression, and then returned to the same subject. Alternatively, these cells may be administered to a different subject in "allogeneic" cell therapy. In either approach, the TGFBR2 cells of the present disclosure may be manipulated in various ways between harvest and administration, such as by expansion, stimulation, purification or sorting, transgene transduction, freezing and / or thawing.

[0115] As described herein, knocking out or knocking down the presence of the TGFBR2 gene can (1) improve T cell proliferation, (2) improve T cell survival, and / or (3) improve T cell function. As described herein, knocking down the expression of the TGFBR2 gene can similarly (1) improve T cell proliferation, (2) improve T cell survival, and / or (3) improve T cell function.

[0116] Genome editing system The term "genome editing system" refers to any system with RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that can associate with a specific nucleic acid sequence and edit the DNA within or around the nucleic acid sequence, for example, by creating one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or point mutations. In certain embodiments, the double-strand break or single-strand break is within about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 100 bp, about 50 bp, about 25 bp, or about 10 bp of the TGFBR2 target position, thereby inducing modification of the expression of the TGFBR2 gene.

[0117] Naturally occurring CRISPR systems have been evolutionarily classified into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun;9(6):467-477 (Makarova), incorporated herein by reference), and although the genome editing system of the present disclosure can adapt components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from class 2 and type II or V CRISPR systems. Class 2 systems, including types II and V, are characterized by a relatively large, multi-domain RNA-guided nuclease protein (e.g., Cas9 or Cpfl) and one or more guide RNAs (e.g., crRNA and optionally tracrRNA) that form a ribonucleoprotein (RNP) complex that associates with (i.e., targets) and cleaves a specific genetic locus complementary to the targeting (or spacer) sequence of the crRNA. The genome editing system according to the present disclosure similarly targets and edits cellular DNA sequences, but differs significantly from naturally occurring CRISPR systems. For example, the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to the present disclosure may incorporate any number of non-natural modifications.

[0118] The genome editing system can be implemented (e.g., administered or delivered to a cell or subject) in a variety of ways, and the various implementations may be suitable for distinct applications. For example, in certain embodiments, the genome editing system is implemented as a protein / RNA complex (ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier and / or encapsulating agent, such as a lipid or polymer microparticle or nanoparticle, a micelle, a liposome, or the like. In certain embodiments, the genome editing system is implemented as one or more nucleic acids encoding the above-described RNA-guided nuclease and guide RNA components (optionally with one or more additional components). In certain embodiments, the genome editing system is implemented as one or more vectors, e.g., viral vectors such as adeno-associated viruses, containing such nucleic acids. In certain embodiments, the genome editing system is implemented as any combination of the above. Additional or modified implementations that function according to the principles described herein will be apparent to those skilled in the art and are within the scope of this disclosure.

[0119] It should be noted that the genome editing system of the present disclosure can target a single specific nucleotide sequence, or can target and simultaneously edit two or more specific nucleotide sequences by using two or more guide RNAs. The use of multiple gRNAs, referred to throughout this disclosure as "multiplexing," can be used to target multiple unrelated target sequences of interest or to create multiple SSBs or DSBs within a single target domain, and in some cases, to effect specific editing within such a target domain. For example, International Patent Publication No. WO2015 / 138510 by Maeder et al. (Maeder), incorporated herein by reference, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene, which generates a cryptic splice site and thereby reduces or eliminates the function of the gene. Maeder's genome editing system utilizes two guide RNAs that target sequences on either side (i.e., adjacent) of the point mutation, creating a DSB adjacent to the mutation. This then promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.

[0120] As another example, WO2016 / 073990 by Cotta-Ramusino et al. ("Cotta-Ramusino"), incorporated herein by reference, describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (Cas9 that creates single-stranded nicks, such as Streptococcus pyogenes D10A), a configuration called the "dual nickase system." The Cotta-Ramusino dual nickase system is configured to create two nicks on opposite strands of the target sequence, offset by one or more nucleotides, which then join to create a double-stranded break with an overhang (5' in the case of Cotta-Ramusino, but a 3' overhang is also possible). The overhang can then, in some circumstances, promote homologous recombination repair events. Also, as another example, WO2015 / 070083 by Palestrant et al. ("Palestrant," incorporated herein by reference) describes a gRNA that targets a nucleotide sequence encoding Cas9 (referred to as a "governing RNA"), which can be included in a genome editing system that includes one or more additional gRNAs, for example, to allow transient expression of Cas9, which may otherwise be constitutively expressed in some virally transduced cells. These multiplexing applications are intended to be illustrative rather than limiting, and one of skill in the art will recognize that other applications of multiplexing are generally compatible with the genome editing systems described herein.

[0121] Genome editing systems can sometimes form double-strand breaks that are repaired by cellular DNA double-strand break mechanisms, such as NHEJ or HDR.These mechanisms are described throughout the literature, for example, by Davis & Maizels, PNAS, 111(10): E924-932, March 11, 2014 (Davis) (description of Alt-HDR), Frit et al. DNA Repair 17(2014)81-97 (Frit) (description of Alt-NHEJ), and Iyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (Iyama) (general description of standard HDR and NHEJ pathways).

[0122] When genome editing system functions by forming DSB, such system can optionally include one or more components that promote specific double-strand break repair or specific repair results.For example, Cotta-Ramusino also describes a genome editing system that adds a single-stranded oligonucleotide "donor template".The donor template can be integrated into the target region of cellular DNA that is cut by genome editing system, resulting in the change of target sequence.

[0123] In certain embodiments, the genome editing system modifies the target sequence or modifies the expression of a gene within or near the target sequence without causing a single-strand or double-strand break. For example, the genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As an example, the RNA-guided nuclease may be connected (e.g., fused) to a cytidine deaminase functional domain and function by causing a targeted C to A substitution. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) ("Komor"), which is incorporated by reference. Alternatively, genome editing systems may utilize cleavage-inactivating (i.e., "dead") nucleases, such as dead Cas9 (dCas9), which function by forming a stable complex on one or more targeted regions of cellular DNA, thereby disrupting functions involving the targeted regions, including, but not limited to, mRNA transcription, chromatin remodeling, etc.

[0124] Guide RNA (gRNA) molecules The terms "guide RNA" and "gRNA" refer to any nucleic acid that facilitates the specific association (or "targeting") of an RNA-guided nuclease, such as Cas9 or Cpf1, with a target sequence, such as a genomic or episomal sequence within a cell. gRNAs can be unimolecular (comprising a single RNA molecule, or called a chimera) or modular (comprising multiple, typically two separate RNA molecules, such as crRNA and tracrRNA, that are usually linked to each other, e.g., by duplexing). gRNAs and their component parts are described throughout the literature, for example, in Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner)), and Cotta-Ramusino.

[0125] In bacteria and archaea, Type II CRISPR systems generally comprise an RNA-guided nuclease protein, such as Cas9, a CRISPR RNA (crRNA) that contains a 5' region complementary to an exogenous sequence, and a trans-activating crRNA (tracrRNA) that contains a 5' region complementary to and duplexed with the 3' region of the crRNA. Without intending to be bound by any theory, it is believed that this duplex promotes the formation of the Cas9 / gRNA complex and is required for its activity. As Type II CRISPR systems have been adapted for use in gene editing, it has been discovered that, in one non-limiting example, the crRNA and tracrRNA can be linked to a single, unimolecular, or chimeric guide RNA by a four-nucleotide (e.g., GAAA) "tetraloop" or "linker" sequence that bridges the complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end). (Mali et al. Science. 2013 Feb. 15; 339(6121): 823-826 (“Mali”); Jiang et al. Nat. Biotechnol. 2013 Mar. 31(3): 233-239 (“Jiang”); and Jinek et al., 2012 Science Aug. 17; 337(6096): 816-821 (“Jinek”), all of which are incorporated herein by reference.)

[0126] Guide RNAs, whether single-molecule or modular, contain a "targeting domain" that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence within the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including, but not limited to, "guide sequences" (Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832 ("Hsu"), incorporated herein by reference), "complementarity regions" (Cotta-Ramusino), "spacers" (Briner), and collectively, "crRNAs" (Jiang). Regardless of the name they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments, 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length), and are located at or near the 5' end in the case of Cas9 gRNAs and at or near the 3' end in the case of Cpf1 gRNAs.

[0127] In addition to the targeting domain, gRNAs typically (but not necessarily, as explained below) contain multiple domains that can affect the formation or activity of the gRNA / Cas9 complex. For example, as described above, the duplex structure formed by the first and second complementarity domains of the gRNA (also referred to as a "repeat:anti-repeat duplex") can interact with the recognition (REC) lobe of Cas9 and mediate the formation of the Cas9 / gRNA complex. (Nishimasu et al., Cell 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both of which are incorporated herein by reference.) Note that the first and / or second complementarity domains may contain one or more polyA tracts that can be recognized as termination signals by RNA polymerase. Thus, the sequences of the first and second complementarity domains are optionally modified to eliminate these tracts and facilitate complete in vitro transcription of the gRNA, for example, through the use of AG swaps, as described in Briner, or AU swaps. These and other similar modifications to the first and second complementarity domains are within the scope of this disclosure.

[0128] In addition to the first and second complementarity domains, Cas9 gRNAs typically contain two or more additional double-stranded regions that are involved in nuclease activity in vivo but not necessarily in vitro (Nishimasu 2015). The first stem-loop near the 3' portion of the second complementarity domain is variously referred to as the "proximal domain" (Cotta-Ramusino), "stem-loop 1" (Nishimasu 2014 and 2015), and "nexus" (Briner). One or more additional stem-loop structures are usually present near the 3' end of the gRNA, the number of which varies by species. Streptococcus pyogenes gRNAs typically contain two 3' stem-loops (for a total of four stem-loop structures including the repeat:anti-repeat duplex), while Staphylococcus aureus and other species have only one (for a total of three stem-loop structures). A description of conserved stem-loop structures (and gRNA structures more generally) grouped by species is provided in Briner.

[0129] While the foregoing description has focused on gRNAs for use with Cas9, it should be understood that other RNA-guided nucleases have been discovered or invented (or may be discovered or invented in the future) that utilize gRNAs that differ in some respects from those described thus far. For example, Cpf1 ("CRISPR from Prevotella and Francisella 1") is a recently discovered RNA-guided nuclease that does not require a tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I) is incorporated herein by reference.) gRNAs for use with the Cpf1 genome editing system generally include a targeting domain and a complementary domain (also called a "handle"). Also, note that in gRNAs for use with Cpf1, the targeting domain is typically located at or near the 3' end (the handle is at or near the 5' end of the Cpf1 gRNA) rather than the 5' end as described above for Cas9 gRNAs.

[0130] Those skilled in the art will understand that although there may be structural differences between gRNAs from various prokaryotic species, or between Cpfl and Cas9 gRNAs, the principles by which gRNAs function are generally consistent.Because of this consistency of function, gRNAs can be broadly defined by their targeting domain sequence, and those skilled in the art will understand that a given targeting domain sequence can be incorporated into any suitable gRNA, including single-molecule or chimeric gRNAs or gRNAs that contain one or more chemical modifications and / or sequence modifications (substitutions, additional nucleotides, truncations, etc.).Therefore, for the sake of simplicity in the present disclosure, gRNAs will only be described in terms of their targeting domain sequence.

[0131] More generally, those skilled in the art will understand that some aspects of the present disclosure relate to systems, methods, and compositions that can be implemented using multiple RNA-guided nucleases. Thus, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, not just a gRNA compatible with a particular species of Cas9 or Cpfl. For example, in certain embodiments, the term gRNA can include any RNA-guided nuclease that occurs in a class 2 CRISPR system, such as a type II or type V or CRISPR system, or a gRNA for use with an RNA-guided nuclease derived from or adapted thereto.

[0132] Table 2 below shows exemplary gRNAs for targeting TGFBR2 using Streptococcus pyogenes Cas9.

[0133] (Table 5) TIFF0007785452000005.tif127136

[0134] gRNA design Methods for target sequence selection and validation, as well as off-target analysis, have been previously described, for example, in Mali; Hsu; Fu et al., 2014 Nat Biotechnol 32(3): 279-84, Heigwer et al., 2014 Nat methods 11(2): 122-3; Bae et al. (2014) Bioinformatics 30(10): 1 473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. Each of these references is incorporated herein by reference. As a non-limiting example, gRNA design may involve the use of software tools to optimize the selection of potential target sequences corresponding to the user's target sequence, e.g., to minimize total off-target activity across the genome. Off-target activity is not limited to cleavage, but the cleavage efficiency at each off-target sequence can be predicted, for example, using an empirically derived weighting scheme. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

[0135] gRNA modification The activity, stability, or other properties of gRNAs can be modified by incorporating specific modifications. For example, transiently expressed or delivered nucleic acids may be susceptible to degradation by, for example, cellular nucleases. Therefore, the gRNAs described herein may contain one or more modified nucleosides or nucleotides that provide stability against nucleases. Without wishing to be bound by theory, it is also believed that certain modified gRNAs described herein may exhibit reduced innate immune responses when introduced into cells. Those skilled in the art will recognize specific cellular responses commonly observed in cells, such as mammalian cells, in response to exogenous nucleic acids, particularly exogenous nucleic acids of viral or bacterial origin. Such responses, which may include the induction and release of cytokine expression and cell death, can be completely reduced or eliminated by the modifications provided herein.

[0136] Certain exemplary modifications described in this section can be included anywhere within the gRNA sequence, including, but not limited to, at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end) and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some cases, modifications are located within functional motifs such as the repeat-antirepeat duplex of a Cas9 gRNA, the stem-loop structure of a Cas9 or Cpf1 gRNA, and / or the targeting domain of a gRNA.

[0137] As an example, the 5' end of the gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., G(5')ppp(5')G cap analog, mG(5')ppp(5')G cap analog, or 3'-O-Me-mG(5')ppp(5')G anti reverse cap analog (ARCA)), as shown below. TIFF0007785452000006.tif39128A cap or cap analog can be included either during chemical synthesis of the gRNA or during in vitro transcription.

[0138] Along similar lines, the 5' end of the gRNA may lack a 5' triphosphate group. For example, in vitro transcribed gRNAs can be treated with a phosphatase (e.g., using calf intestinal alkaline phosphatase) to remove the 5' triphosphate group.

[0139] Another common modification involves adding multiple (e.g., 1-10, 10-20, or 25-200) adenine (A) residues, called a polyA tract, to the 3' end of the gRNA. PolyA tracts can be added to gRNAs during chemical synthesis, after in vitro transcription using a polyadenosine polymerase (e.g., E. coli poly(A) polymerase), or in vivo using a polyadenylation sequence as described by Maeder.

[0140] It should be noted that the modifications described herein can be combined in any suitable manner, e.g., whether the gRNA is transcribed in vivo from a DNA vector or in vitro, the gRNA can include either or both a 5' cap structure or cap analog and a 3' polyA tract.

[0141] Guide RNAs can be modified with a 3'-terminal U-ribose, for example, by oxidizing the two terminal hydroxyl groups of U-ribose to aldehyde groups, concomitantly opening the ribose ring to yield modified nucleosides, such as those shown below. TIFF0007785452000007.tif22128In the formula, "U" can be unmodified or modified uridine.

[0142] The 3' terminal U ribose can be modified with a 2'3' cyclic phosphate as shown below. TIFF0007785452000008.tif30128In the formula, "U" can be unmodified or modified uridine.

[0143] The guide RNA may comprise a 3' nucleotide, which may be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridine may be replaced by modified uridine, for example, 5-(2-amino)propyluridine, 5-bromouridine, or any of the modified uridines described herein. Adenosine and guanosine may be replaced by modified adenosine and guanosine, for example, 8-bromoguanosine, which has a modification at the 8th position, or any of the modified adenosine or guanosine described herein.

[0144] In certain embodiments, gRNAs can incorporate sugar-modified ribonucleotides, e.g., the 2'OH-group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), amino (where amino can be, e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA may each independently be a modified or unmodified nucleotide containing, without limitation, a 2'-sugar modification, such as 2'-O-methyl, 2'-O-methoxyethyl, or a 2'-fluoro modification, including, for example, 2'-F or 2'-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2'-O-methyl, thymidine (T), 2'-F or 2'-O-methyl, guanosine (G), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0145] Guide RNAs can also include "locked" nucleic acids (LNAs) in which the 2'OH group can be connected to the 4' carbon of the same ribose sugar by, for example, a C1-6 alkylene or C1-6 heteroalkylene bridge. Any suitable moiety can be used, including, but not limited to, methylene, propylene, ether, or amino bridges; O-amino (amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino); and aminoalkoxy or O(CH2). n Such bridges can be provided comprising -amino (amino can be, for example, NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0146] In certain embodiments, gRNAs can include modified nucleotides that are polycyclic (e.g., tricyclic; and "unlocked" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs in which the ribose is replaced by a glycol unit attached to a phosphodiester bond), or threose nucleic acids (GNAs in which the ribose is replaced by α-L-threofuranosyl-(3'→2')).

[0147] Typically, gRNAs contain the sugar ribose, a five-membered ring containing oxygen. Exemplary modified gRNAs include, but are not limited to, substitution of oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylenes such as methylene or ethylene); double bond addition (e.g., replacing ribose with cyclopentenyl or cyclohexenyl); ribose ring contraction (e.g., forming a four-membered ring of cyclobutane or oxetane); and ribose ring expansion (e.g., forming a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also has a phosphoramidate backbone). While the majority of sugar analog modifications are located at the 2' position, other sites, including the 4' position, are also subject to modification. In certain embodiments, gRNAs contain 4'-S, 4'-Se, or 4'-C-aminomethyl-2'-O-Me modifications.

[0148] In certain embodiments, deazanucleotides, such as 7-deaza-adenosine, can be incorporated into gRNA. In certain embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into gRNA. In certain embodiments, one or more or all nucleotides in gRNA are deoxynucleotides.

[0149] RNA-guided nucleases RNA-guided nucleases according to the present disclosure include, but are not limited to, naturally occurring class 2 CRISPR nucleases, such as Cas9 and Cpf1, as well as other nucleases derived therefrom. In functional terms, an RNA-guided nuclease is defined as a nuclease that (a) interacts with (e.g., complexes with) a gRNA and (b) associates with (i) a sequence complementary to the targeting domain of the gRNA, and optionally cleaves or modifies a target region of DNA that contains (ii) an additional sequence called a "protospacer adjacent motif" or "PAM," which will be described in more detail below. As described in the examples below, RNA-guided nucleases can be broadly defined by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Those skilled in the art will understand that some aspects of the present disclosure relate to systems, methods, and compositions that can be implemented using any suitable RNA-guided nuclease with a particular PAM specificity and / or cleavage activity. Thus, unless otherwise specified, the term RNA-guided nuclease should be understood as a general term and should not be limited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., Streptococcus pyogenes vs. Staphylococcus aureus) or mutation (e.g., full-length vs. cleavage or split; naturally occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0150] The PAM sequence derives its name from its sequence relationship to a "protospacer" sequence that is complementary to the gRNA targeting domain (or "spacer"). Together with the protospacer sequence, the PAM sequence defines a target region or sequence for a specific RNA-guided nuclease / gRNA combination.

[0151] Different RNA-guided nucleases may require different sequence relationships between the PAM and the protospacer.

[0152] In addition to recognizing specific PAM and protospacer sequence orientations, RNA-guided nucleases can also recognize specific PAM sequences. For example, Staphylococcus aureus Cas9 recognizes the NNGRRT or NNGRRV PAM sequence, with the N residue immediately 3' to the region recognized by the gRNA targeting domain. Streptococcus pyogenes Cas9 recognizes the NGG PAM sequence. Furthermore, F. novicida Cpf1 recognizes the TTN PAM sequence. PAM sequences have been identified for various RNA-guided nucleases, and a strategy for identifying novel PAM sequences was described by Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that an engineered RNA-guided nuclease may have a PAM specificity that differs from that of the reference molecule (e.g., in the case of an engineered RNA-guided nuclease, the reference molecule may be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant with the greatest amino acid sequence homology to the engineered RNA-guided nuclease).

[0153] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity. Naturally occurring RNA-guided nucleases typically form DSBs within target nucleic acids, but engineered mutants have been created that generate only SSBs (as discussed above) or no cleavage at all (Ran & Hsu, et al., Cell 154(6), 1380-1389, September 12, 2013 (Ran)).

[0154] Cas9 Crystal structures have been determined for Streptococcus pyogenes Cas9 (Jinek 2014) and for Staphylococcus aureus Cas9 in complex with a single guide RNA and target DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

[0155] Naturally occurring Cas9 proteins contain two lobes, the recognition (REC) lobe and the nuclease (NUC) lobe, each containing specific structural and / or functional domains. The REC lobe contains an arginine-rich bridge helix (BH) domain and at least one REC domain (e.g., a REC1 domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analysis suggests specific functional roles for the BH and REC domains. The BH domain appears to play a role in gRNA:DNA recognition, while the REC domain is thought to interact with the repeat:antirepeat duplex of the gRNA and mediate the formation of the Cas9 / gRNA complex.

[0156] The NUC lobe contains a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. The RuvC domain can be formed from two or more split RuvC motifs (e.g., RuvC I, RuvC II, and RuvC III in Streptococcus pyogenes and Staphylococcus aureus). Meanwhile, the HNH domain is structurally similar to the HNN endonuclease motif and cleaves the complementary (i.e., top) strand of the target nucleic acid. As its name suggests, the PI domain contributes to PAM specificity.

[0157] While certain functions of Cas9 are related to (but not necessarily entirely determined by) the specific domains described above, these and other functions can be mediated or influenced by other Cas9 domains or by multiple domains on either lobe. For example, in Streptococcus pyogenes Cas9, as described in Nishimasu 2014, the repeat:antirepeat duplex of the gRNA fits into the groove between the REC and NUC lobes, and nucleotides of the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem-loop structure interact with amino acids in multiple domains (PI, BH, and REC1), as well as some nucleotides in the second and third stem-loops (RuvC and PI domains).

[0158] Cpf1 The crystal structure of Acidaminococcus sp. Cpf1 in complex with crRNA and a double-stranded (ds) DNA target containing a TTTN PAM sequence has been solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949-962 (Yamano)), which is incorporated herein by reference. Similar to Cas9, Cpf1 has two lobes: the REC (recognition) lobe and the NUC (nuclease) lobe. The REC lobe contains the REC1 and REC2 domains, which lack similarity to known protein structures. Meanwhile, the NUC lobe contains three RuvC domains (RuvC-I, RuvC-II, and RuvC-III) and a BH domain. However, in contrast to Cas9, the Cpf1 REC lobe lacks the HNH domain and contains other domains that also lack similarity to known protein structures, namely, a structurally unique PI domain, three Wedge (WED) domains (WED-I, WED-II, and WED-III), and a nuclease (Nuc) domain.

[0159] Although Cas9 and Cpf1 share structural and functional similarities, it should be understood that certain Cpf1 activities are mediated by structural domains that are not similar to either Cas9 domain. For example, cleavage of the complementary strand of target DNA appears to be mediated by the Nuc domain, which is sequentially and spatially distinct from the HNH domain of Cas9. Furthermore, the non-targeting portion (handle) of the Cpf1 gRNA adopts a pseudoknot structure rather than the stem-loop structure formed by the repeat:antirepeat duplex of the Cas9 gRNA.

[0160] Modification of RNA-guided nucleases While the RNA-guided nucleases described above have activities and properties that may be useful for a variety of applications, those skilled in the art will understand that RNA-guided nucleases may be modified in certain cases to alter cleavage activity, PAM specificity, or other structural or functional characteristics.

[0161] First, turning to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe are described above. Exemplary mutations that can be made to the RuvC domain, the Cas9 HNH domain, or the Cpf1 Nuc domain are described in Ran and Yamano and Cotta-Ramusino. Typically, mutations that reduce or eliminate the activity of one of the two nuclease domains result in an RNA-guided nuclease with nickase activity, but it should be noted that the type of nickase activity differs depending on which domain is inactivated. For example, inactivating the RuvC domain of Cas9 results in a nickase that cleaves the complementary or upper strand. On the other hand, inactivating the Cas9 HNH domain results in a nickase that cleaves the lower or non-complementary strand.

[0162] Modifications of PAM specificity relative to the naturally occurring Cas9 reference molecule have been described by Kleinstiver et al. for both Streptococcus pyogenes (Kleinstiver et al., Nature. 2015 Jul 23; 523(7561): 481-5 (Kleinstiver I) and Staphylococcus aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec; 33(12): 1293-1298 (Kleinstiver II)). Kleinstiver et al. also describe modifications that improve the targeting fidelity of Cas9 (Nature, 2016 January 28; 529, 490-495 (Kleinstiver III)). Each of these references is incorporated herein by reference.

[0163] RNA-guided nucleases have been split into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 Feb; 33(2): 139-42 (Zetsche II) incorporated by reference) and Fine et al. (Sci Rep. 2015 Jul 1; 5: 10777 (Fine) incorporated by reference).

[0164] In certain embodiments, the RNA-guided nuclease can be size-optimized or truncated, for example, through one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activity. In certain embodiments, the RNA-guided nuclease is covalently or non-covalently linked to another polypeptide, nucleotide, or other structure, optionally via a linker. Exemplary linked nucleases and linkers are described in Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated herein by reference for all purposes.

[0165] The RNA-guided nuclease may also comprise a tag, such as, but not limited to, a nuclear localization signal, to facilitate the transport of the RNA-guided nuclease protein to the nucleus. In certain embodiments, the RNA-guided nuclease may incorporate a C-terminal and / or N-terminal nuclear localization signal. Nuclear localization sequences are known in the art and are described in Maeder et al.

[0166] The foregoing list of modifications is intended to be exemplary in nature, and those skilled in the art will understand, in light of the present disclosure, that other modifications may be possible or desirable for particular applications. Thus, for the sake of brevity, the exemplary systems, methods, and compositions of the present disclosure are presented with reference to specific RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used may be modified in ways that do not alter their functional principles. Such modifications are within the scope of the present disclosure.

[0167] Nucleic acid encoding an RNA-guided nuclease The nucleic acid provided herein encodes RNA-guided nuclease, for example, Cas9, Cpf1, or functional fragment thereof.The exemplary nucleic acid encoding RNA-guided nuclease has been previously described (see, for example, Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0168] In some cases, the nucleic acid encoding the RNA-guided nuclease may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. In certain embodiments, the mRNA encoding the RNA-guided nuclease has one or more (for example, all) of the following properties: it may be capped, polyadenylated, and substituted with 5-methylcytidine and / or pseudouridine.

[0169] Synthetic nucleic acid sequences can also be codon-optimized, for example, at least one non-common codon or at least one low-frequency codon is replaced by a common codon.For example, synthetic nucleic acid can lead to the synthesis of optimized messenger mRNA, for example, optimized for expression in mammalian expression system as described herein.An example of codon-optimized Cas9 coding sequence is provided in Cotta-Ramusino.

[0170] Additionally or alternatively, the nucleic acid encoding the RNA-guided nuclease may contain a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0171] Functional analysis of candidate molecules Candidate RNA-guided nuclease, gRNA, and their complexes can be evaluated by standard methods known in the art.See, for example, Cotta-Ramusino.The stability of RNP complexes can be evaluated by differential scanning fluorimetry, as described below.

[0172] Differential Scanning Fluorometry (DSF) The thermal stability of ribonucleoprotein (RNP) complexes containing gRNA and RNA-guided nucleases can be measured via DSF. The DSF technique measures the thermal stability of proteins, which can be increased under favorable conditions, such as the addition of a binding RNA molecule such as gRNA.

[0173] The DSF assay can be performed according to any suitable protocol and can be used in any suitable setting, including, but not limited to, (a) testing different conditions (e.g., different stoichiometric ratios of gRNA:RNA-guided nuclease protein, different buffers, etc.) to identify optimal conditions for RNP formation, and (b) testing modifications of the RNA-guided nuclease and / or gRNA (e.g., chemical modifications, sequence alterations, etc.) to identify modifications that improve RNP formation or stability. One readout of the DSF assay is the shift in the melting temperature of the RNP complex. A relatively high shift suggests that the RNP complex is more stable than a reference RNP complex characterized by a relatively low shift (and therefore may have higher activity or advantageous formation, disassembly, or other functional characteristics than the reference RNP complex). When the DSF assay is deployed as a screening tool, a threshold melting temperature shift can be specified, resulting in an output of one or more RNPs with a melting temperature shift equal to or greater than the threshold. For example, the threshold can be 5-10°C (e.g., 5°, 6°, 7°, 8°, 9°, 10°) or greater, and the output can be one or more RNPs characterized by a melting temperature shift above the threshold.

[0174] Two non-limiting examples of DSF assay conditions are described below.

[0175] To determine the best solution for RNP complex formation, a fixed concentration (e.g., 2 μM) of Cas9 aqueous solution plus 10x SYPRO Orange® (Life Technologies catalog number S-6650) was dispensed into a 384-well plate. Equimolar amounts of gRNA diluted in solutions of various pH levels and salt were then added. After 10 minutes of incubation at room temperature and brief centrifugation to remove air bubbles, a gradient from 20°C to 90°C was run using a Bio-Rad CFX384™ Real-Time System C1000 Touch™ thermal cycler with Bio-Rad CFX Manager software, increasing the temperature by 1°C every 10 seconds.

[0176] The second assay consisted of mixing various concentrations of gRNA with a fixed concentration (e.g., 2 μM) of Cas9 in the optimal buffer from assay 1 above and incubating in a 384-well plate (e.g., at room temperature for 10 minutes). An equal volume of optimal buffer plus 10x SYPRO Orange® (Life Technologies catalog number S-6650) was added, and the plate was sealed with Microseal® B adhesive (MSB-1001). After a brief centrifugation to remove air bubbles, a gradient from 20°C to 90°C was run using a Bio-Rad CFX384™ Real-Time System C1000 Touch™ thermal cycler with Bio-Rad CFX Manager software, increasing the temperature by 1°C every 10 seconds.

[0177] Genome editing strategies In various embodiments of the present disclosure, the above-mentioned genome editing system is used to edit (i.e., modify) the target region of DNA in a cell or DNA obtained from a cell (e.g., TGFBR2 DNA).Various strategies for creating specific edits are described herein, and these strategies are generally described in terms of the desired repair result, the number and location of individual edits (e.g., SSB or DSB), and the target site of such edits.

[0178] Genome editing strategies involving the formation of SSBs or DSBs are characterized by repair outcomes including (a) deletion of all or part of the target region, (b) insertion or substitution of all or part of the target region, or (c) disruption of all or part of the target region. This grouping is not intended to limit or bind to any particular theory or model, but is provided solely for simplicity of presentation. Those skilled in the art will understand that the listed outcomes are not mutually exclusive, and that some repairs may lead to other outcomes. Description of a particular editing strategy or editing method should not be understood to require a particular repair outcome unless otherwise specified.

[0179] Replacement of a target region typically involves replacing all or part of the existing sequence in the target region with a homologous sequence, for example, through gene correction or gene conversion, two repair outcomes mediated by the HDR pathway. HDR is facilitated by the use of a donor template, which can be single-stranded or double-stranded, as described in more detail below. The single-stranded or double-stranded template can be exogenous, in which case they facilitate gene correction to facilitate gene conversion, or endogenous (e.g., a homologous sequence within the cellular genome). The exogenous template can have an asymmetric overhang (i.e., the portion of the template complementary to the site of the DSB can be offset in the 3' or 5' direction rather than centered within the donor template), as described, for example, by Richardson et al. (Nature Biotechnology 34, 339-344 (2016), (Richardson)). If the template is single-stranded, it can correspond to either the complementary (top) strand or the non-complementary (bottom) strand of the target region.

[0180] Gene conversion and gene correction are sometimes facilitated by creating one or more nicks within or around the target region, as described by Ran and Cotta-Ramusino. In some cases, a double-nickase strategy is used to create two offset SSBs that sequentially form a single DSB with an overhang (e.g., a 5' overhang).

[0181] Various repair results can achieve the interruption and / or deletion of all or part of the target sequence.For example, as described by Maeder for LCA10 mutation, the sequence can be deleted by simultaneously generating two or more DSBs adjacent to the target region, and then excised when the DSB is repaired.For another example, the sequence can be interrupted by the deletion generated by forming a double-strand break with a single-strand overhang, and then the overhang is subjected to exonuclease treatment before repair.

[0182] The formation of indels within a target sequence mediates one specific subset of target sequence interruptions, and repair outcomes are typically mediated by the NHEJ pathway (including Alt-NHEJ). NHEJ is referred to as an "error-prone" repair pathway due to its association with indel mutations. However, in some cases, DSBs are repaired by NHEJ without altering the surrounding sequence (so-called "complete" or "intact" repair). This generally requires complete ligation of both ends of the DSB. In contrast, indels are thought to result from enzymatic treatment of free DNA ends prior to ligation, which adds and / or removes nucleotides from one or both strands of the free ends.

[0183] Because enzymatic processing of free DSB ends can be stochastic in nature, indel mutations tend to be variable and occur along a distribution, potentially influenced by a variety of factors, including the specific target site, the cell type used, and the genome editing strategy employed. Nevertheless, it is possible to draw limited generalizations regarding indel formation. Deletions formed by repair of a single DSB are most often in the 1-50 bp range, but can also exceed 100-200 bp. Insertions formed by repair of a single DSB tend to be short and often contain short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence can often be traced to other regions of the genome or to plasmid DNA present in the cell.

[0184] Indel mutations and genome editing systems configured to generate indels are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or frameshift mutations are acceptable. They can also be useful in situations where a specific sequence is preferred, as long as the desired specific sequence tends to preferentially result from repair of an SSB or DSB at a given site. Indel mutations are also useful tools for evaluating or screening the activity of specific genome editing systems and their components. In these and other situations, indels can be characterized by (a) their relative and absolute frequency in the genome of cells contacted with the genome editing system and (b) the distribution of numerical differences relative to the unedited sequence, e.g., ±1, ±2, ±3, etc. As an example, in a lead discovery setting, multiple gRNAs can be screened to identify the gRNA that most efficiently promotes cleavage at the target site based on indel readouts under controlled conditions. Guides that generate indels at or above a threshold frequency, or that generate a specific distribution of indels, can be selected for subsequent testing and development. The frequency and distribution of indels can also be useful as a readout to evaluate various genome editing system implementations or formulations and delivery methods, for example, by keeping the gRNA constant and varying certain other reaction conditions or delivery methods.

[0185] Multiplexing Strategy Although the exemplary strategies described above have focused on repair outcomes mediated by a single DSB, the genome editing system disclosed herein can also be used to generate two or more DSBs, either at the same locus or at different loci. Strategies for editing involving the formation of multiple DSBs or SSBs are described, for example, in Cotta-Ramusino.

[0186] Donor Template Design Donor template design is described in detail in Cotta-Ramusino et al. DNA oligomer donor templates (oligodeoxynucleotides, or ODNs), which can be single-stranded (ssODN) or double-stranded (dsODN), can be used to facilitate HDR-based repair of DSBs and are particularly useful for introducing modifications into target DNA sequences, inserting novel sequences into target sequences, or replacing the entire target sequence.

[0187] Whether single-stranded or double-stranded, the donor template typically contains regions of homology to regions of DNA within or near (e.g., adjacent or abutting) the target sequence to be cleaved. These regions of homology are referred to herein as "homology arms" and are shown schematically below. [5' homology arm]-[replacement sequence]-[3' homology arm].

[0188] The homology arms can have any suitable length (including 0 nucleotides if only one homology arm is used), and the 3' and 5' homology arms can have the same length or different lengths. The selection of an appropriate homology arm length can be influenced by various factors, such as the desire to avoid homology or microhomology with specific sequences, such as Alu repeats or other highly common elements. For example, the 5' homology arm can be shortened to avoid sequence repeat elements. In other embodiments, the 3' homology arm can be shortened to avoid sequence repeat elements. In some embodiments, both the 5' and 3' homology arms can be shortened to avoid the inclusion of specific sequence repeat elements. Furthermore, some homology arm designs can improve editing efficiency or increase the frequency of desired repair outcomes. For example, Richardson et al. Nature Biotechnology 34, 339-344(2016) (Richardson), incorporated by reference, found that the relative asymmetry of the 3' and 5' homology arms of a single-stranded donor template affects the repair rate and / or repair outcome.

[0189] The replacement sequence of the donor template has been described elsewhere, including Cotta-Ramusino et al. The replacement sequence can be of any suitable length (including 0 nucleotides if the desired repair outcome is a deletion) and typically contains one, two, three, or more sequence modifications relative to a naturally occurring sequence in the cell for which editing is desired. One common sequence modification involves modifying a naturally occurring sequence to repair a mutation associated with a disease or condition for which treatment is desired. Another common sequence modification involves modifying one or more sequences complementary to or encoding the PAM sequence of an RNA-guided nuclease or the targeting domain of a gRNA used to generate an SSB or DSB, to reduce or eliminate recurrent cleavage of the target site after the replacement sequence is integrated into the target site.

[0190] When a linear ssODN is used, it can be configured to (i) anneal to a nicked strand of a target nucleic acid, (ii) anneal to an intact strand of a target nucleic acid, (iii) anneal to a plus strand of a target nucleic acid, and / or (iv) anneal to a minus strand of a target nucleic acid. The ssODN can have any suitable length, for example, about, at least, or up to 150-200 nucleotides (e.g., 150, 160, 170, 180, 190, or 200 nucleotides).

[0191] It should be noted that the template nucleic acid can also be a nucleic acid vector, such as a viral genome or circular double-stranded DNA, e.g., a plasmid. The nucleic acid vector containing the donor template can contain other coding or non-coding elements. For example, the template nucleic acid can be delivered as part of a viral genome (e.g., an AAV or lentiviral genome) that contains specific genomic backbone elements (e.g., inverted terminal repeats in the case of an AAV genome) and optionally contains additional sequences encoding gRNAs and / or RNA-guided nucleases. In certain embodiments, the donor template can promote the formation of free DSBs on one or both ends of the donor template adjacent to target sites recognized by one or more gRNAs, which can participate in the repair of corresponding SSBs or DSBs formed in cellular DNA using the same gRNAs. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino.

[0192] Using either format, the template nucleic acid can be designed to avoid undesired sequences, in certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.

[0193] target cell The genome editing system according to the present disclosure can be used to manipulate or modify cells, for example, to edit or modify target nucleic acids. Manipulation can, in various aspects, occur in vivo or ex vivo.

[0194] Various cell types can be engineered or modified according to embodiments of the present disclosure, and in some cases, such as in vivo applications, multiple cell types are engineered or modified, for example, by delivering a genome editing system according to the present disclosure to multiple cell types. However, in other cases, it may be desirable to limit the engineering or modification to one or more specific cell types. For example, in some instances, it may be desirable to edit cells with limited differentiation potential, such as photoreceptor cells in the case of Maeder, or terminally differentiated cells, where genotypic modification is predicted to result in a change in cell phenotype. However, in other cases, it may be desirable to edit less differentiated, pluripotent stem or progenitor cells. By way of example, the cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or other stem or progenitor cell types that differentiate into cell types relevant to a given use or indication. In certain embodiments, the cells are T cells.

[0195] As a corollary, the cells that are modified or engineered may be variously dividing or non-dividing cells, depending on the cell type targeted and / or the desired editing outcome.

[0196] Once the cells have been manipulated or modified ex vivo, the cells can be used immediately (e.g., administered to a subject), or the cells can be maintained or stored for later use. Those skilled in the art will understand that cells can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art.

[0197] Implementing genome editing systems: delivery, formulation, and administration routes As explained above, the genome editing system of the present disclosure can be implemented in any suitable manner, meaning that the components of such a system, including, without limitation, an RNA-guided nuclease, a gRNA, and optionally a donor template nucleic acid, can be delivered, formulated, or administered in any suitable form or combination of forms that result in the transduction, expression, or introduction of the genome editing system and / or cause the desired repair result in a cell, tissue, or subject. Tables 3 and 4 list some non-limiting examples of genome editing system implementations. However, those skilled in the art will understand that these lists are not comprehensive and that other implementations are possible. With particular reference to Table 3, the table lists some exemplary implementations of a genome editing system that include a single gRNA and, optionally, a donor template. However, a genome editing system according to the present disclosure can incorporate other components, such as multiple gRNAs, multiple RNA-guided nucleases, and proteins, and various implementations will be apparent to those skilled in the art based on the principles set forth in the table. In the tables, [N / A] indicates that the genome editing system does not include the indicated component.

[0198] (Table 6) TIFF0007785452000009.tif216131

[0199] Table 7 summarizes various delivery methods for the components of the genome editing systems described herein. Again, the list is intended to be illustrative rather than limiting.

[0200] (Table 7) TIFF0007785452000010.tif220150

[0201] Nucleic acid-based delivery of genome editing systems The nucleic acids encoding various elements of the genome editing system according to the present disclosure can be administered to a subject or delivered to a cell by methods known in the art or as described herein.For example, the DNA encoding the RNA-guided nuclease and / or the DNA encoding the gRNA, and the donor template nucleic acid can be delivered by, for example, a vector (e.g., a viral vector or a non-viral vector), a non-vector-based method (e.g., using naked DNA or DNA complex), or a combination thereof.

[0202] Nucleic acids encoding elements of a genome editing system can include sequences encoding one, two, three, four, or more gRNAs. For example, the nucleic acid can encode both a first and a second gRNA molecule, e.g., the second gRNA has a second targeting domain complementary to a second target sequence of the TGFBR2 gene. The nucleic acids disclosed herein can further include a nucleotide sequence encoding a third gRNA molecule having a third targeting domain complementary to a third target sequence of the TGFBR2 gene. The nucleic acid compositions disclosed herein can further include a nucleotide sequence encoding a fourth gRNA molecule described herein having a fourth targeting domain complementary to a fourth target sequence of the TGFBR2 gene. In certain embodiments, the second, third, and / or fourth gRNA molecule comprises a targeting domain comprising a nucleotide sequence selected from SEQ ID NOs:5036-5096.

[0203] Nucleic acids encoding the genome editing system or its components can be delivered directly to cells as naked DNA or RNA, for example, by transfection or electroporation, or can be conjugated to a molecule (e.g., N-acetylgalactosamine) that facilitates uptake by target cells (e.g., red blood cells, HSCs). Nucleic acid vectors, such as those summarized in Table 4, can also be used.

[0204] The nucleic acid vector can include one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA, and / or a donor template. The vector can also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted into or fused to) the protein-encoding sequence. In one example, the nucleic acid vector can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40).

[0205] The nucleic acid vector can also include any suitable number of regulatory / control elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES), etc. These elements are well known in the art and described in Cotta-Ramusino.

[0206] Nucleic acid vectors according to the present disclosure include recombinant viral vectors. Exemplary viral vectors are listed in Table 7, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. In certain embodiments, the vector is a viral vector, such as an adeno-associated viral (AAV) vector or a lentiviral (LV) vector. Other viral vectors known in the art can also be used. Furthermore, viral particles can be used to deliver genome editing system components in the form of nucleic acids and / or peptides. For example, "empty" viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to modify target tissue specificity.

[0207] In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding the genome editing system of the present disclosure. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For example, organic (e.g., lipid and / or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of the present disclosure. Table 8 lists exemplary lipids for use in nanoparticle formulations and / or gene transfer, and Table 9 lists exemplary polymers for use in gene transfer and / or nanoparticle formulations.

[0208] Table 8. Lipids used for gene transfer TIFF0007785452000011.tif76155TIFF0007785452000012.tif204155

[0209] Table 9. Polymers used for gene transfer TIFF0007785452000013.tif222150

[0210] Non-viral vectors optionally include targeting modifications to improve uptake and / or selectively target specific cell types. These targeting modifications can include, for example, cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell-penetrating peptides. Such vectors also optionally use fusogenic and endosome-destabilizing peptides / polymers, undergo acid-induced conformational changes (e.g., to accelerate endosomal escape of the cargo), and / or incorporate stimulus-cleavable polymers, for example, for release within cellular compartments. For example, disulfide-based cationic polymers that are cleaved in a reducing cellular environment can be used.

[0211] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than components of the genome editing system, such as the RNA-guided nuclease component and / or gRNA component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered simultaneously with one or more components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered before or after one or more components of the genome editing system are delivered (e.g., less than about 30 minutes, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 6 hours, less than about 9 hours, less than about 12 hours, less than about 1 day, less than about 2 days, less than about 3 days, less than about 1 week, less than about 2 weeks, or less than about 4 weeks). In certain embodiments, the nucleic acid molecule is delivered by a different means than that by which one or more components of the genome editing system, such as the RNA-guided nuclease component and / or gRNA component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, such as an integration-defective lentivirus, and the RNA-guided nuclease molecule component and / or gRNA component can be delivered by electroporation, for example, so that the toxicity caused by the nucleic acid (e.g., DNA) can be reduced. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, such as a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, such as an RNA molecule described herein.

[0212] Delivery of RNPs and / or RNAs encoding genome editing system components RNPs (complexes of gRNA and RNA-guided nuclease, i.e., ribonucleoprotein complexes) and / or RNA encoding the RNA-guided nuclease and gRNA can be delivered into cells or administered to a subject by methods known in the art, some of which are described in Cotta-Ramusino. In vitro, RNA encoding the RNA-guided nuclease and / or RNA encoding the gRNA can be delivered by, for example, microinjection, electroporation, transient cell compaction, or squeezing (see, e.g., Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc or other conjugate-mediated delivery, and combinations thereof can also be used for in vitro and in vivo delivery.

[0213] In vitro, delivery via electroporation involves mixing cells with RNA encoding an RNA-guided nuclease and / or gRNA in a cartridge, chamber, or cuvette, with or without a donor template nucleic acid molecule, and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tools and / or protocols can be used in connection with various aspects of the present disclosure.

[0214] In certain embodiments, the RNP complexes of the present disclosure, including, for example, RNP pharmaceutical compositions, can be used to (1) improve T cell proliferation, (2) improve T cell survival, and / or (3) improve T cell function. For example, without limitation, two or more RNP complexes containing separate gRNAs can be used simultaneously or sequentially to modify TGFBR2 gene expression in cells, e.g., T cells. Such RNP complexes can contain separate gRNAs targeting separate TGFBR2 gene sequences. The RNP complexes can, in certain instances, induce cleavage events, e.g., double-stranded or single-stranded breaks. For example, the RNP complexes can contain an enzymatically active Cas9 (eaCas9) molecule that forms a double-stranded break within a target nucleic acid, or an eaCas9 molecule (e.g., a nickase molecule) that forms a single-stranded break within a target nucleic acid. In certain embodiments, a dual-nickase RNP strategy can be used to form two offset single-stranded breaks, thereby forming a single double-stranded break with an overhang (e.g., a 5' overhang).

[0215] Route of administration Genome editing systems, or cells modified or manipulated using such systems, can be administered to subjects by suitable methods or routes, either locally or systemically. Systemic administration methods include oral and parenteral routes. Parenteral routes include, for example, intravenous, intramedullary, intraarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Systemically administered components can be modified or formulated to target, for example, HSCs, hematopoietic stem / progenitor cells, or erythroid progenitor cells.

[0216] Examples of local administration modes include intramedullary injection into the trabecular bone, or intrafemoral injection into the bone marrow space, and injection into the portal vein. In certain embodiments, significantly smaller amounts of components can be effective when administered locally (e.g., directly to the bone marrow) compared to when administered systemically (e.g., intravenously) (compared to systemic approaches). Local administration modes can reduce or eliminate the occurrence of toxic side effects that may occur when a therapeutically effective amount of a component is administered systemically.

[0217] Administration can be provided as periodic boluses (e.g., intravenously) or as continuous infusion from an internal reservoir or from an external reservoir (e.g., from an intravenous bag or implantable pump). The components can be administered locally, for example, by continuous release from a sustained-release drug delivery device.

[0218] Furthermore, the components can be formulated to allow release over an extended period of time. The release system can include a matrix of biodegradable material or a matrix of material that releases incorporated components by diffusion. The components can be uniformly or non-uniformly distributed within the release system. A variety of release systems can be useful, with the selection of an appropriate system dependent on the release rate required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents, such as, but not limited to, calcium carbonate and sugars (e.g., trehalose). The release system can be natural or synthetic. However, synthetic release systems are preferred because they generally provide more reliable, reproducible, and well-defined release profiles. The materials of the release system can be selected so that components of various molecular weights are released by diffusion through the material or by degradation of the material.

[0219] Representative synthetic biodegradable polymers include, for example, polyamides, such as poly(amino acids) and poly(peptides); polyesters, such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitution, addition, hydroxylation, oxidation of chemical groups, e.g., alkyl, alkylene, and other modifications routinely performed by one of ordinary skill in the art), copolymers, and mixtures thereof. Representative synthetic non-degradable polymers include, for example, polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic acid, and methacrylic acid—polyacrylates and polymethacrylates, and others such as poly(vinyl alcohol), poly(vinylpyrrolidone), and poly(vinyl acetate); poly(urethanes); celluloses and derivatives thereof, such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitution, addition, hydroxylation, oxidation of chemical groups, e.g., alkyl, alkylene, and other modifications routinely performed by one of ordinary skill in the art), copolymers, and mixtures thereof.

[0220] Poly(lactide-co-glycolide) microspheres can also be used. Typically, the microspheres are composed of polymers of lactic acid and glycolic acid and are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with the components described herein.

[0221] Multimodal or differential delivery of components

[00023] Those skilled in the art will understand, in light of the present disclosure, that the various components of the genome editing systems disclosed herein can be delivered together or separately, and simultaneously or non-simultaneously. Separate and / or asynchronous delivery of genome editing system components may be particularly desirable to provide temporal or spatial control over the function of the genome editing system and limit specific effects caused by their activity.

[0222] As used herein, different modes or differential modes refer to delivery modes that give different pharmacodynamic or pharmacokinetic properties to target component molecules, such as RNA guided nuclease molecules, gRNA, template nucleic acid or payload.For example, delivery modes can result in different tissue distribution, different half-life or different temporal distribution in selected compartments, tissues or organs.

[0223] Some delivery methods, such as delivery by nucleic acid vectors that persist within the cell or its progeny, for example by autonomous replication or insertion into cellular nucleic acid, enhance expression of the component and persistence of the component. Examples include delivery by viruses, such as AAV or lentivirus.

[0224] For example, components of genome editing system, such as RNA-guided nuclease and gRNA, can be delivered by different modes in terms of the resulting half-life or persistence of delivered components in the body or specific compartments, tissues or organs.In certain embodiments, gRNA can be delivered by such modes.RNA-guided nuclease molecular components can be delivered by modes that result in less persistence or exposure to the body or specific compartments, tissues or organs.

[0225] More generally, in certain embodiments, a first component is delivered using a first delivery mode and a second component is delivered using a second delivery mode. The first delivery mode confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, within the body, compartment, tissue, or organ. The second delivery mode confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, within the body, compartment, tissue, or organ.

[0226] In certain embodiments, the first pharmacodynamic or pharmacokinetic property, eg, distribution, duration, or exposure, is more restricted than the second pharmacodynamic or pharmacokinetic property.

[0227] In certain embodiments, the first delivery modality is selected to optimize, eg, minimize, a pharmacodynamic or pharmacokinetic property, eg, distribution, duration, or exposure.

[0228] In certain embodiments, the second delivery modality is selected to optimize, eg, maximize, a pharmacodynamic or pharmacokinetic property, eg, distribution, duration, or exposure.

[0229] In certain embodiments, the first delivery modality involves the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or a viral vector, e.g., an AAV or lentivirus. Because such vectors are relatively persistent, the products transcribed from them are also relatively persistent.

[0230] In certain embodiments, the second delivery modality comprises a relatively transient element, such as RNA or a protein.

[0231] In certain embodiments, the first component comprises gRNA, and the delivery method is relatively continuous, for example, gRNA is transcribed from a plasmid or a viral vector, for example, AAV or lentivirus.The transcription of these genes has little physiological effect, because the gene does not encode a protein product, and gRNA cannot act alone.The second component, the RNA-guided nuclease molecule, is delivered transiently, for example, as mRNA or protein, ensuring that the complete RNA-guided nuclease molecule / gRNA complex exists and is active for only a short period of time.

[0232] Furthermore, the components can be delivered in different molecular forms or using different delivery vectors that complement each other to enhance safety and tissue specificity.

[0233] The use of differential delivery modes can improve performance, safety, and / or efficacy, for example, reducing the potential for eventual off-target modification. Because peptides derived from bacterial Cas enzymes are presented on the cell surface by MHC molecules, delivery of immunogenic components, such as Cas9 molecules, in a less sustained manner may result in reduced immunogenicity. A two-part delivery system can mitigate these drawbacks.

[0234] Differential delivery modes can be used to deliver components to different but overlapping target regions. The formation of active complexes is minimized outside the overlapping target regions. Thus, in certain embodiments, a first component, such as gRNA, is delivered by a first delivery mode that results in a first spatial, e.g., tissue distribution. A second component, such as an RNA-guided nuclease molecule, is delivered by a second delivery mode that results in a second spatial, e.g., tissue distribution. In certain embodiments, the first mode comprises a first component selected from liposomes, nanoparticles, e.g., polymer nanoparticles, and nucleic acids, e.g., viral vectors. The second mode comprises a second component selected from the above group. In certain embodiments, the first delivery mode comprises a first targeting component, e.g., a cell-specific receptor or antibody, while the second delivery mode does not comprise that component. In certain embodiments, the second delivery mode comprises a second targeting component, e.g., a second cell-specific receptor or a second antibody.

[0235] When RNA-guided nuclease molecules are delivered via viral delivery vectors, liposomes, or polymer nanoparticles, they may be delivered to multiple tissues, potentially resulting in therapeutic activity in multiple tissues, when targeting only a single tissue would be desirable. A two-part delivery system can address this issue and enhance tissue specificity. When gRNA and RNA-guided nuclease molecules are packaged within separate delivery vehicles with distinct but overlapping tissue tropisms, only fully functional complexes are formed in tissues targeted by both vectors.

[0236] Genetically engineered cells and methods for producing cells expressing recombinant receptors Provided herein are cells for adoptive cell therapy, such as adoptive immunotherapy, and methods for producing or generating cells.Cells include immune cells, such as T cells.Cells are usually engineered by introducing one or more engineered nucleic acids or their products.Among such products are engineered antigen receptors, including engineered T cell receptors (TCRs), and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), including activating, stimulating, and costimulatory CARs, and combinations thereof.In some embodiments, cells are also simultaneously or sequentially introduced with the nucleic acid encoding the engineered antigen receptor and with an agent (e.g., Cas9 / gRNA RNP) that can disrupt the gene encoding TGFBR2.

[0237] In some aspects, cells (e.g., T cells) can be incubated or cultured before, during, and / or after introducing a nucleic acid molecule encoding a recombinant receptor and / or an agent (e.g., Cas9 / gRNA RNP). In some aspects, cells (e.g., T cells) can be incubated or cultured before, during, or after introducing a nucleic acid molecule encoding a recombinant receptor, e.g., before, during, or after transducing the cells with a viral vector (e.g., a lentiviral vector) encoding the recombinant receptor. In some embodiments, cells (e.g., T cells) can be incubated or cultured before, during, or after contacting the cells with an agent, or before, during, or after introducing an agent (e.g., Cas9 / gRNA RNP), e.g., before, during, or after delivering an agent to the cells via electroporation. In some embodiments, incubation can occur both when introducing a nucleic acid molecule encoding a recombinant receptor and when introducing an agent, e.g., Cas9 / gRNA RNP. In some embodiments, incubation may be in the presence of cytokines such as IL-2, IL-7, or IL-15, or in the presence of stimulatory or activating agents that induce cell proliferation or activation, such as anti-CD3 / anti-CD28 antibodies.

[0238] In some embodiments, the methods include activating or stimulating the cells with a stimulatory or activating agent (e.g., anti-CD3 / anti-CD28 antibody) prior to introducing the nucleic acid molecule encoding the recombinant receptor and an agent such as, for example, a Cas9 / gRNA RNP. In some embodiments, the incubation may also be carried out in the presence of a cytokine such as IL-2 (e.g., 1 U / mL to 500 U / mL, 10 U / mL to 200 U / mL, etc., e.g., at least 50 U / mL or at least 100 U / mL, or about 50 U / mL or about 100 U / mL), IL-7 (e.g., 0.5 ng / mL to 50 ng / mL, 1 ng / mL to 20 ng / mL, etc., e.g., at least 5 ng / mL or at least 10 ng / mL, or about 5 ng / mL or about 10 ng / mL), or IL-15 (e.g., 0.1 ng / mL to 50 ng / mL, 0.5 ng / mL to 25 ng / mL, etc., e.g., at least 1 ng / mL or at least 5 ng / mL, or about 1 ng / mL or about 5 ng / mL). In some embodiments, the cells are incubated for 6 to 96 hours, such as 24 to 48 hours or 24 to 36 hours, before introducing the nucleic acid molecule encoding the recombinant receptor (e.g., via transduction).

[0239] Cells and cell preparation for genetic manipulation The recombinant receptor that binds to the specific antigen and agent (for example, Cas9 / gRNA RNP) for gene editing of the TGFBR2 gene encoding TGFBR2 polypeptide can be introduced into a variety of cells.In some embodiments, the recombinant receptor is manipulated and / or the TGFBR2 target gene is manipulated ex vivo, and the resulting genetically engineered cells are administered to a subject.The source of the target cell for ex vivo manipulation can be, for example, the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow.The source of the target cell for ex vivo manipulation can also be, for example, xenogeneic donor blood, umbilical cord blood, or bone marrow.

[0240] In some embodiments, the cells, e.g., engineered cells, are eukaryotic cells, such as mammalian cells, e.g., human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs and are immune system cells, e.g., innate or adaptive immune cells, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. The cells may be allogeneic and / or autologous with respect to the subject being treated. The cells are typically primary cells, e.g., isolated directly from the subject and / or isolated and frozen from the subject.

[0241] In some embodiments, the target cell is a T cell, such as, for example, a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or granulocyte, such as, for example, a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In one embodiment, the target cell is an iPS cell, or a cell derived from an iPS cell, e.g., an induced pluripotent stem (iPS) cell, that has been generated from a subject and engineered to be modified (e.g., mutagenized) or have had the expression of one or more target genes manipulated to differentiate into, e.g., a T cell, such as a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0242] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total T cell population, CD4+ cells, CD8+ cells, etc., and subpopulations thereof defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation.

[0243] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells; effector T cells (TEFF); memory T cells and their subtypes such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells; tumor infiltrating lymphocytes (TIL); immature T cells; mature T cells; helper T cells; cytotoxic T cells; mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells; helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells; α / β T cells, and δ / γ T cells.

[0244] In some embodiments, the methods involve isolating cells from a subject and preparing, treating, culturing, and / or manipulating them. In some embodiments, preparing the manipulated cells involves one or more culturing and / or preparation steps. Cells for manipulation as described may be isolated from a sample, such as a biological sample, obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is one that has a disease or condition or is in need of, or for which, cell therapy will be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated.

[0245] Thus, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples obtained after one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, including processed samples derived therefrom.

[0246] In some embodiments, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestinal tract, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples of autologous and allogeneic origin, for example, in the context of cell therapy, such as adoptive cell therapy.

[0247] In some embodiments, the cells are derived from a cell line, such as, for example, a T cell line. In some embodiments, the cells are obtained from a heterologous source, such as, for example, mouse, rat, non-human primate, or pig.

[0248] In some embodiments, cell isolation involves one or more preparative and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesired components, enrich for desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0249] In some examples, cells from a subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some embodiments, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some embodiments, cells other than red blood cells and platelets.

[0250] In some embodiments, blood cells collected from a subject are washed, e.g., the plasma fraction is removed, and the cells are placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or most or all divalent cations. In some aspects, the washing step is accomplished in a semi-automated "through-flow" centrifuge (e.g., a Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, such as, for example, Ca++ / Mg++-free PBS. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0251] In some embodiments, the method involves density-based cell separation methods such as preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0252] In some embodiments, the isolation method involves separating different cell types based on the expression or presence in the cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, isolation in some aspects involves separating cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, on the cells, e.g., by incubation with an antibody or binding partner that specifically binds to such marker, generally followed by a washing step and separation of cells that have binding to the antibody or binding partner from cells that have not bound to the antibody or binding partner.

[0253] Such separation steps can be based on positive selection, in which cells that bind to the reagent are retained for further use, and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, and as a result, separation based on markers expressed by cells other than the desired population is best.

[0254] Separation does not necessarily result in 100% enrichment or removal of a specific cell population or the cells that express a specific marker.For example, positive selection or enrichment of a specific type of cell, such as one that expresses a marker, means increasing the number or percentage of such cells, but does not necessarily mean that the cells that do not express the marker are completely absent.Similarly, negative selection, removal or depletion of a specific type of cell, such as one that expresses a marker, means reducing the number or percentage of such cells, but does not necessarily mean that all such cells are completely removed.

[0255] In some cases, multiple separation steps are performed, and the positive or negative selection fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.In some cases, a single separation step can simultaneously deplete cells that express multiple markers, such as by incubating cells with multiple antibodies or binding partners that are specific to the markers that are the targets of negative selection.Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners that are expressed in various cell types.

[0256] In some embodiments, one or more T cell populations, such as surface markers, are positive for or express high levels of one or more particular markers (marker+). high ) cells, or cells that are negative for one or more markers (marker-) or express them at relatively low levels (marker low) cells are enriched or depleted. For example, in some embodiments, specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, such as, for example, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells), but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (such as CD8+ cells or T cells, e.g., CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L (i.e., positively selected) and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected). In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO and CD62L (or negative for CD45RA).

[0257] For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0258] In some embodiments, T cells are separated from peripheral blood mononuclear cell (PBMC) samples by negative selection markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further separated into subpopulations by positive or negative selection for markers expressed, or expressed to a relatively high degree, on one or more naive, memory, and / or effector T cell subpopulations.

[0259] In some embodiments, the CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to enhance efficacy, such as improving long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such subpopulations. (See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701.) In some embodiments, efficacy is further enhanced by combining TCM-enriched CD8+ T cells with CD4+ T cells.

[0260] In some embodiments, memory T cells reside in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, such as with anti-CD8 and anti-CD62L antibodies.

[0261] In some embodiments, the CD4+ T cell population and the CD8+ T cell subpopulation are enriched for central memory (TCM) cells, e.g., a subpopulation enriched for TCM cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of the TCM cell-enriched CD8+ population is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some embodiments, the same CD4 expression-based selection step used to prepare the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0262] In a particular example, a sample of PBMCs or other white blood cells is subjected to selection of CD4+ cells, and both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers of central memory T cells, such as CD62L or CCR7, with the positive and negative selection being performed in either order.

[0263] CD4+ T helper cells are classified into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO.

[0264] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are attached to a solid support or matrix, such as magnetic or paramagnetic beads, allowing for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (Copyright) Humana Press Inc., Totowa, NJ).

[0265] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culturing, culturing, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of stimulatory agents. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prepare cells for genetic manipulation, such as for the introduction of recombinant antigen receptors.

[0266] Conditions may include one or more particular media; temperature; oxygen content; carbon dioxide content; time; agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines; chemokines; antigens; binding partners; fusion proteins; recombinant soluble receptors; and any other agents designed to activate cells.

[0267] In some embodiments, the stimulatory conditions or agents include one or more agents, such as, for example, a ligand, capable of activating an intracellular signaling domain of the TCR complex. In some aspects, the agent activates or initiates the TCR / CD3 intracellular signaling cascade in the T cell. Such agents may include, for example, antibodies, such as those specific for TCR components and / or costimulatory receptors, such as anti-CD3 or anti-CD28, bound to a solid support, such as beads, and / or one or more cytokines. Optionally, the expansion method may further comprise adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, such as, for example, IL-2 at a concentration of at least about 10 units / ml.

[0268] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0269] In some embodiments, T cells are expanded by adding culture starting composition feeder cells, such as non-dividing PBMCs (e.g., so that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the medium prior to the addition of the T cell population.

[0270] In some embodiments, the stimulatory conditions include a temperature suitable for human T lymphocyte proliferation, such as, for example, at least about 25°C, typically at least about 30°C, and typically about 37°C. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma radiation in the range of about 6000 to 10,000 rads. The LCL feeder cells are provided in any suitable amount, such as, in some aspects, at a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0271] In some embodiments, the preparation method includes freezing, e.g., cryopreserving, the cells either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters can be used in certain aspects. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with medium to achieve final DMSO and HSA concentrations of 10% and 4%, respectively. The cells are then frozen to -80°C, typically at a rate of 1°C per minute, and stored in the vapor phase of a liquid nitrogen storage tank.

[0272] In some embodiments, the methods involve reintroducing the engineered cells into the same patient, either before or after cryopreservation.

[0273] Recombinant receptor In some embodiments, the cells comprise one or more nucleic acids encoding a recombinant receptor that have been introduced through genetic engineering, and the genetically engineered product of such nucleic acids. In some embodiments, the cells can be generated or produced by introducing a nucleic acid molecule encoding the recombinant receptor into the cell (e.g., through transduction of a viral vector such as a retroviral or lentiviral vector). In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or in a sample obtained from the cell, such as being obtained from another organism or cell that is not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature, including one comprising a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0274] In some embodiments, the target cells are engineered to bind to one or more target antigens, such as one or more tumor antigens. In some embodiments, the target antigens are selected from the group consisting of ROR1, B-cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFRvIII, Folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, melanoma preferentially expressed antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, bispecific antigen, cancer-testis antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, pathogen-specific antigens, and antigens associated with universal markers.In some embodiments, the target cells are engineered to bind, for example, by a TCR or CAR, to one or more of the following tumor antigens: Tumor antigens include AD034, AKT1, BRAP, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, fibulin-1, HAGE, HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / galectin-9, HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HUB1, KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGE-1, MAGE-1, MAGE-4a, MPP11, MSLN, NNP-1, NY-BR-1, and NY-BR-1. NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, OGFr, PLU-1, Rab38, RBPJκ, RHAMM, SCP1, SCP-1, SSX3, SSX4, SSX5, TOP2A, TOP2B, or tyrosinase.

[0275] antigen receptor Chimeric antigen receptor (CAR) The cells generally express recombinant receptors, such as antigen receptors, including functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), and other antigen-binding receptors, such as recombinant T cell receptors (TCRs), including other chimeric receptors.

[0276] Exemplary antigen receptors, such as CARs, and methods for engineering and introducing such receptors into cells are described in, for example, International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US2013014933 No. 7, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some embodiments, antigen receptors include CARs such as those described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.Examples of CARs include those disclosed in any of the aforementioned documents, such as WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9):689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US7,446,190, and US8,389,282. Chimeric receptors such as CARs generally comprise an extracellular antigen-binding domain, such as a portion of an antibody molecule, such as the variable heavy (VH) and / or variable light (VL) chain regions of an antibody, e.g., an scFv antibody fragment.

[0277] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or pathological cells, e.g., tumor or pathogenic cells, compared to normal cells or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0278] Antigens that may be targeted by the receptor include αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, C D22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III mutant (EGFRvIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor α, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erb B dimer, human high-molecular-weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A) , Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), and pathogen-specific antigens;

[0279] In some embodiments, antigens targeted by the receptors of some embodiments include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen (oncofetal antigen), ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, and cyclins such as MAGE A3, CE7, Wilms' tumor 1 (WT-1), and cyclin A1 (CCNA1), and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0280] In some embodiments, the CAR has binding specificity for a tumor-associated antigen such as, for example, CD19, CD20, carbonic anhydrase IX (CAIX), CD171, CEA, ERBB2, GD2, alpha-folate receptor, Lewis Y antigen, prostate-specific membrane antigen (PSMA), or tumor-associated glycoprotein 72 (TAG72).

[0281] In some embodiments, the CAR binds to a pathogen-specific antigen, hi some embodiments, the CAR is specific for a viral antigen (such as HIV, HCV, HBV), a bacterial antigen, and / or a parasitic antigen.

[0282] Chimeric receptors include chimeric antigen receptors (CARs). Chimeric receptors, such as CARs, generally contain the variable heavy (V) fragment of an antibody, e.g., an scFv antibody fragment. H ) chain region and / or variable light (V L The antibody generally comprises an extracellular antigen-binding domain, such as a portion of an antibody molecule, such as a nucleotide sequence (e.g., a nucleotide sequence), ... or a portion of an antibody molecule, such as an extracellular antigen-binding domain (e.g., a nucleotide sequence).

[0283] In some embodiments, the antibody portion of a recombinant receptor, e.g., a CAR, further comprises at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., an scFv, and the transmembrane domain. The spacer can be of a length that results in increased cellular responsiveness following antigen binding compared to the absence of the spacer. Exemplary spacers, e.g., hinge regions, include those described in International Publication No. WO2014031687. In some examples, the spacer is 12 amino acids in length, or about 12 amino acids in length, or less than 12 amino acids in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 or fewer amino acids, about 119 or fewer amino acids, or about 229 or fewer amino acids. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the CH2 and CH3 domains, or an IgG4 hinge linked to the CH3 domain.

[0284] Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication No. WO2014031687.

[0285] This antigen recognition domain is generally linked to one or more intracellular signaling components, such as a signaling component that mimics activation through an antigen receptor complex, such as a TCR complex in the case of a CAR, and / or a signal through another cell surface receptor. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally binds to a domain in a receptor, such as a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such a domain to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0286] In some embodiments, the transmembrane domain is derived from either natural or synthetic sources. If natural in origin, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprise at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains are primarily comprised of hydrophobic residues such as leucine and valine. In some aspects, a phenylalanine, tryptophan, and valine triplet will be found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain.

[0287] Intracellular signaling domains include those that mimic or approximate signals through natural antigen receptors, signals through combinations of such receptors with costimulatory receptors, and / or signals through costimulatory receptors alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2-10 amino acids in length, e.g., one containing glycine and serine, e.g., a glycine-serine duo, is present between the transmembrane domain and the cytoplasmic signaling domain of the CAR to form the linkage.

[0288] Receptors, such as CARs, generally comprise at least one intracellular signaling component. In some embodiments, receptors comprise an intracellular component of the TCR complex, such as a TCR CD3 chain that mediates T cell activation and cytotoxicity, such as the CD3ζ chain. Thus, in some aspects, the antigen-binding moiety is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, receptors, such as CARs, further comprise portions of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, a CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0289] In some embodiments, upon ligation with a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell, engineered to express the CAR. For example, in some situations, the CAR induces a T cell function, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or a costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., when it transmits an effector function signal. In some embodiments, the intracellular signaling domain comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also the cytoplasmic sequence of a co-receptor that acts in concert with such receptors in their natural context to initiate signal transduction following antigen receptor binding, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capability.

[0290] In the context of native TCRs, full activation generally requires not only signal transduction through the TCR but also a costimulatory signal. Thus, in some embodiments, the CAR also includes a component for generating a secondary or costimulatory signal to promote full activation. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell to provide a component for generating a secondary or costimulatory signal.

[0291] T cell activation has been described in some embodiments as mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of these signaling components.

[0292] In some embodiments, the CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that contain primary cytoplasmic signaling sequences include those derived from the CD3ζ chain, FcRγ, CD3γ, CD3δ, and CD3ε. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0293] In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR comprises both an activating component and a costimulatory component.

[0294] In some embodiments, the activation domain is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises both an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO 2014 / 055668). In some aspects, the cell comprises one or more stimulatory or activating CARs and / or costimulatory CARs. In some embodiments, the cell further comprises an inhibitory CAR (iCAR; see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR that recognizes an antigen other than that associated with and / or specific for the disease or condition, whereby the activation signal delivered through the disease-targeting CAR is reduced or inhibited by binding of the inhibitory CAR to its ligand, e.g., reducing off-target effects.

[0295] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3zeta intracellular domain.

[0296] In some embodiments, the CAR includes one or more costimulatory domains, e.g., two or more, and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components CD3-zeta, CD28, and 4-1BB.

[0297] In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, that may be used to confirm transduction or manipulation of cells expressing the receptor, such as a truncated cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding a CAR and EGFRt separated by a T2A ribosomal switch can express the two proteins from the same construct, such that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker, and optionally the linker sequence, can be any of those disclosed in published application no. WO2014 / 031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, such as a T2A cleavable linker sequence.

[0298] In some embodiments, the marker is a molecule that is not naturally found on or at the surface of a T cell, such as, for example, a cell surface protein or portion thereof.

[0299] In some embodiments, the marker is a molecule, such as a cell surface protein, that is, one that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0300] In some embodiments, the marker serves no therapeutic function and / or effect other than being used as a marker of genetic engineering, for example, to select successfully engineered cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some desired effect, such as a ligand for cells encountered in vivo, such as a costimulatory or immune checkpoint molecule, to enhance and / or suppress the cell's response upon adoptive transfer and encounter with the ligand.

[0301] In some cases, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some embodiments, first-generation CARs provide only a CD3 chain-induced signal upon antigen binding; in some embodiments, second-generation CARs provide such a signal and a costimulatory signal, such as those that include intracellular signaling domains derived from costimulatory receptors such as CD28 or CD137; in some embodiments, third-generation CARs include multiple costimulatory domains from different costimulatory receptors.

[0302] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv and an intracellular domain containing an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane can be connected directly or indirectly. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer as described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0303] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment; a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof; an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., an antibody fragment; a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof; an intracellular signaling domain that contains the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer that contains a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer.

[0304] In some embodiments, the transmembrane domain of a receptor, e.g., a CAR, is the transmembrane domain of human CD28, e.g., the 27 amino acid transmembrane domain of human CD28 (accession number P10747.1), or a variant thereof.

[0305] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0306] In some embodiments, the intracellular signaling domain comprises the intracellular costimulatory signaling domain of human CD28, or a functional variant or portion thereof, such as the 41 amino acid domain thereof, and / or a domain with an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular domain comprises the intracellular costimulatory signaling domain of 41BB, or a functional variant or portion thereof, such as the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1), or a functional variant or portion thereof.

[0307] In some embodiments, the intracellular signaling domain comprises a human CD3ζ stimulatory signaling domain, such as the 112 AA cytoplasmic domain of human CD3ζ (Accession No. P20963.2) or isoform 3 of the CD3ζ signaling domain, as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993, or a functional variant thereof.

[0308] In some aspects, the spacer contains only the hinge region of an IgG, such as only an IgG4 or IgG1 hinge. In other embodiments, the spacer is, for example, an Ig hinge, such as an IgG4 hinge linked to the CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge linked to the CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge linked to the CH3 domain only. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as a known flexible linker.

[0309] For example, in some embodiments, a CAR comprises an antibody or fragment that specifically binds to an antigen, a spacer such as any of the Ig hinge-containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, a CAR comprises an antibody or fragment that specifically binds to an antigen, a spacer such as any of the Ig hinge-containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, such CAR constructs further comprise a T2A ribosomal skip element and / or a tEGFR sequence, for example, downstream of the CAR.

[0310] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the receptor and other polypeptides provided, such as linkers or peptides, may contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, a polypeptide may contain modifications to the native or naturally occurring sequence, so long as the protein retains the desired activity. These modifications may be deliberate, such as site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors resulting from PCR amplification.

[0311] T cell receptor In some embodiments, the engineered antigen receptor comprises a recombinant T cell receptor (TCR) and / or a TCR cloned from a naturally occurring T cell. Thus, in some embodiments, the target cell is modified to contain specific T cell receptor (TCR) genes (e.g., TRAC and TRBC genes). TCRs or antigen-binding portions thereof include those that recognize peptide epitopes or T cell epitopes of targeting polypeptides, such as antigens of tumor, viral, or autoimmune proteins. In some embodiments, the TCR has binding specificity for a tumor-associated antigen, such as carcinoembryonic antigen (CEA), GP100, melanoma antigen recognized by T cell 1 (MART1), melanoma antigen A3 (MAGE A3), NYESO1, or p53.

[0312] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively), or antigen-binding portions thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in the αβ and γδ forms are generally structurally similar, although the T cells that express them may have distinct anatomical locations or functions. Generally, TCRs are expressed, or can be expressed, on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0313] In some embodiments, the TCR is a complete TCR or an antigen-binding portion or fragment thereof. In some embodiments, the TCR is an intact or full-length TCR, such as an αβ or γδ TCR. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but binds to a specific peptide bond in an MHC molecule, such as binding to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR may contain only a portion of the structural domain of a full-length or intact TCR, but can still bind to a peptide epitope, such as an MHC-peptide complex, to which the complete TCR binds. In some cases, the antigen-binding portion contains variable domains of the TCR, such as the variable α and β chains of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions (CDRs) involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0314] In some embodiments, the variable domain of a TCR contains hypervariable loops or CDRs, which are major contributors to antigen recognition and binding ability and specificity. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within the variable region of a TCR chain are generally separated by framework regions (FRs), which generally exhibit less variability between TCR molecules than CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR involved in antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and / or for interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the α chain may interact with the N-terminal portion of a particular antigenic peptide. In some circumstances, CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some circumstances, CDR2 contributes most significantly to, or is the primary CDR involved in interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the β chain may contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0315] In some embodiments, the TCR comprises a variable alpha domain (V α ) and / or variable β domain (V β), or an antigen-binding fragment thereof. In some embodiments, the α chain and / or β chain of the TCR may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some embodiments, the α chain constant domain is encoded by the TRAC gene (IMGT nomenclature) or a variant thereof. In some embodiments, the β chain constant region is encoded by the TRBC1 or TRBC2 gene (IMGT nomenclature) or a variant thereof. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains a CDR.

[0316] Determining or identifying the various domains or regions of a TCR is within the level of one of ordinary skill in the art. In some embodiments, residues of a TCR are known or can be identified by the International Immunogenetics Information System (IMGT) numbering system (see, e.g., www.imgt.org; see also, Lefranc et al. (2003) Developmental and Comparative Immunology, 2:55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequence within the TCR Vα and / or Vβ chain corresponds to the amino acids located at residues 27-38, the CDR2 sequence within the TCR Vα and / or Vβ chain corresponds to the amino acids located at residues 56-65, and the CDR3 sequence within the TCR Vα and / or Vβ chain corresponds to the amino acids located at residues 105-117.

[0317] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), linked by, for example, a disulfide bond. In some embodiments, the constant domain of the TCR may contain a short linking sequence in which cysteine ​​residues form disulfide bonds, thereby linking the two TCR chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain. In some embodiments, the constant domain and the variable domain each contain a disulfide bond formed by cysteine ​​residues.

[0318] In some embodiments, TCRs for engineering cells as described are generated from known TCR sequences, such as Vα and β chain sequences, for which substantially full-length coding sequences are readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cellular sources are well known. In some embodiments, nucleic acids encoding TCRs can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of nucleic acids encoding the TCR within or isolated from a given cell, or by synthesis of publicly available TCR DNA sequences. In some embodiments, TCRs can be obtained from biological sources, such as T cells (e.g., cytotoxic T cells), cells derived from T cell hybridomas, or other publicly available sources. In some embodiments, T cells can be obtained from in vivo isolated cells. In some embodiments, T cells can be cultured T cell hybridomas or clones. In some embodiments, TCRs or antigen-binding portions thereof can be generated synthetically from knowledge of the TCR sequence.

[0319] In some embodiments, high-affinity T cell clones against a target antigen (e.g., a cancer antigen) are identified, isolated from a patient, and introduced into the patient. In some embodiments, TCR clones for the target antigen are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs against the target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0320] In some embodiments, the TCR or antigen-binding portion thereof is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is achieved by display methods, including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves engineering or modifying a known parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to generate a mutagenized TCR, in which one or more residues in the CDRs are mutated and a variant with a desired altered property, such as higher affinity for a desired target antigen, is selected.

[0321] In some embodiments as described, the TCR may contain an introduced disulfide bond. In some embodiments, no native disulfide bond is present. In some embodiments, one or more native cysteines (e.g., in the constant domains of the α and β chains) that form native interchain disulfide bonds are substituted with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond may be formed by mutating non-cysteine ​​residues on the α and β chains, such as in the constant domains of the α and β chains, to cysteine. Exemplary non-native disulfide bonds of TCRs are described in International Publication Nos. WO2006 / 000830 and WO2006 / 037960. In some embodiments, cysteines may be introduced at residue Thr48 of the α chain and Ser57 of the β chain, at residue Thr45 of the α chain and Ser77 of the β chain, at residue Tyr10 of the α chain and Ser17 of the β chain, at residue Thr45 of the α chain and Asp59 of the β chain, and / or at residue Ser15 of the α chain and Glu15 of the β chain. In some embodiments, the presence of non-native cysteine ​​residues in a recombinant TCR (e.g., resulting in one or more non-native disulfide bonds) may assist in the production of a desired recombinant TCR in cells introduced with overexpression of a mismatched TCR pair containing native TCR chains.

[0322] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. Optionally, the TCR chain contains a cytoplasmic tail. In some aspects, each chain of a TCR (e.g., α or β) can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail. In some embodiments, the TCR binds, for example, through the cytoplasmic tail, to the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Optionally, this structure allows the TCR to bind to another molecule, such as CD3 and its subunits. For example, a TCR containing a constant domain with a transmembrane region may anchor the protein to the cell membrane and bind to the invariant subunit of the CD3 signaling apparatus or complex. The intracellular tail of the CD3 signaling subunit (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs, which are involved in the signaling ability of the TCR complex.

[0323] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). The TCR may be in a cell-associated form or a soluble form. In some embodiments, for purposes of the provided methods, the cell-associated form of the TCR is expressed on the surface of a cell.

[0324] In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to a TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR α chain constant region extracellular sequence, and a second polypeptide in which a sequence corresponding to a TCR β chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR β chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in a native dimeric αβ TCR. In some embodiments, the interchain disulfide bond is not present in native TCRs. For example, in some embodiments, one or more cysteines may be incorporated into the constant region extracellular sequences of the dTCR polypeptide pair. In some cases, both native and non-native disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence to anchor it to the membrane.

[0325] In some embodiments, the dTCR contains a TCR alpha chain containing a variable alpha domain, a constant alpha domain, and a first dimerization motif attached to the C-terminus of the constant alpha domain, and a TCR beta chain comprising a variable beta domain, a constant beta domain, and a first dimerization motif attached to the C-terminus of the constant beta domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between amino acids in the first dimerization motif and amino acids in the second dimerization motif, linking the TCR alpha chain and the TCR beta chain together.

[0326] In some embodiments, TCR is a single amino acid chain that contains an α chain and a β chain that can bind MHC-peptide complexes, scTCR.Typically, scTCR can be produced using methods known to those skilled in the art, see, for example, International Publication Nos. WO1996 / 13593, WO1996 / 18105, WO1999 / 18129, WO2004 / 033685, WO2006 / 037960, WO2011 / 044186; U.S. Patent No. 7,569,664; and Schlueter, CJ et al. J.Mol.Biol.256,859(1996).

[0327] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to a TCR alpha chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR beta chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0328] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to a TCR β chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR α chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0329] In some embodiments, the scTCR contains a first segment comprised of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, a second segment comprised of a beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant and transmembrane sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0330] In some embodiments, the scTCR contains a first segment comprised of a TCR beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant domain sequence, a second segment comprised of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant and transmembrane sequence, and optionally a linker sequence joining the C-terminus of the first segment to the N-terminus of the second segment.

[0331] In some embodiments, for an scTCR to bind to an MHC-peptide complex, the α and β chains must pair such that their variable region sequences are oriented for such binding. Various methods for promoting α and β pairing in scTCRs are well known in the art. In some embodiments, a linker sequence is included that links the α and β chains to form a single polypeptide chain. In some embodiments, the linker should be long enough to span the distance between the C-terminus of the α chain and the N-terminus of the β chain, or vice versa, while ensuring that the linker length is not so long as to prevent or reduce binding of the scTCR to the target peptide-MHC complex.

[0332] In some embodiments, the linker of the scTCR linking the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence can have the formula, for example, -P-AA-P-, where P is proline and AA represents an amino acid sequence containing glycine and serine amino acids. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. In some cases, the linker has sufficient length to span the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but is not so long as to prevent or reduce binding of the scTCR to its target ligand. In some embodiments, the linker can contain approximately 10-45 amino acids, such as 10-30 amino acids or 26-41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P- or -PGGG-(SGGGG)6-P-, where P is proline, G is glycine, and S is serine. In some embodiments, the linker has the sequence I have TIFF0007785452000014.tif3128.

[0333] In some embodiments, the scTCR contains disulfide bonds between residues of a single amino acid chain, which in some cases may promote stability of the pairing between the α and β regions of the single-chain molecule (see, e.g., U.S. Pat. No. 7,569,664). In some embodiments, the scTCR contains a covalent disulfide bond linking residues of the immunoglobulin region of the constant domain of the α chain with residues of the immunoglobulin region of the constant domain of the β chain of the single-chain molecule. In some embodiments, the disulfide bond corresponds to a native disulfide bond present in a native dTCR. In some embodiments, no disulfide bond is present in a native TCR. In some embodiments, the disulfide bond is introduced into a non-native disulfide bond, for example, by incorporating one or more cysteines into the constant regions of the extracellular sequences of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine ​​mutations include any of those described above. In some cases, both native and non-native disulfide bonds may be desirable.

[0334] An scTCR is a non-disulfide-linked truncated TCR with a heterologous leucine zipper fused to its C-terminus to facilitate chain association (see, e.g., International Publication No. WO 1999 / 60120). In some embodiments, an scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., International Publication No. WO 1999 / 18129).

[0335] In some embodiments, any TCR, including dTCR or scTCR, can be linked to a signaling domain that provides an active TCR on the T cell surface. In some embodiments, the TCR is expressed on the T cell surface. In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the transmembrane domain can be a Cα or Cβ transmembrane domain. In some embodiments, the transmembrane domain can be derived from a non-TCR source, such as a transmembrane region from CD3z, CD28, or B7.1. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR contains a CD3z signaling domain. In some embodiments, the TCR can form a TCR complex with CD3.

[0336] In some embodiments, the TCR or antigen-binding fragment thereof is about 10 -5 ~10 -12 It represents an affinity with an equilibrium binding constant for the target antigen, M, and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or a ligand.

[0337] In some embodiments, the TCR or antigen-binding portion thereof may be a recombinantly produced native protein or a mutant version thereof, in which one or more properties, such as binding characteristics, have been modified. In some embodiments, the TCR may be derived from one of a variety of animal species, such as human, mouse, rat, or other mammals. In some embodiments, to generate a vector encoding the TCR, the α and β chains may be PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains may be synthetically generated.

[0338] In some embodiments, the TCR α and β chains are isolated and cloned into a gene expression vector. In some embodiments, the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), allowing simultaneous expression of gene products (e.g., encoding the α and β chains) by messages from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA containing multiple genes (e.g., encoding the α and β chains) in a single open reading frame (ORF), separated from each other by sequences encoding a self-cleaving peptide (e.g., T2A) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polyprotein, which is cleaved into individual proteins either during translation (in the case of T2A) or post-translation. In some cases, peptides such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), resulting in separation between the end of the 2A sequence and the next downstream peptide. Examples of 2A cleavage peptides, including peptides that can induce ribosome skipping, are T2A, P2A, E2A, and F2A. In some embodiments, α and β chains are cloned into different vectors. In some embodiments, the α and β chains produced are incorporated into retroviral vectors, such as lentivirus.

[0339] In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skipping peptide so that both chains are co-expressed. In some embodiments, gene transfer of the TCR is achieved through a retroviral or lentiviral vector or through a transposon (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:1748-1757; and Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683).

[0340] Vectors and methods of manipulation The provided methods include expressing a recombinant receptor, such as a CAR or TCR, to generate genetically engineered cells that express such binding molecules. Genetic engineering generally involves introducing a nucleic acid encoding the recombinant or engineered component into a cell, such as by retroviral transduction, transfection, or transformation.

[0341] In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining them with a stimulus that induces a response such as proliferation, survival, and / or activation, as measured by, for example, expression of cytokines or activation markers, followed by transduction of the activated cells and expansion of the culture to numbers sufficient for clinical application.

[0342] For example, various methods for introducing genetically engineered components, such as antigen receptors (e.g., CAR), are well known and can be used with the provided methods and compositions.Exemplary methods include the method for transferring the nucleic acid encoding the receptor through viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.

[0343] In some embodiments, the nucleic acid encoding the recombinant receptor may be cloned into a suitable expression vector. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include vectors such as those designed for propagation and expansion, or for expression, or both, such as plasmids and viruses.

[0344] In some embodiments, the vector may be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. In some embodiments, plant expression vectors may be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors, such as retroviral vectors, are used.

[0345] In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors, whether DNA-based or RNA-based, can contain regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the host type (e.g., bacteria, fungi, plants, or animals) into which the vector will be introduced, as appropriate. In some embodiments, vectors can contain a non-native promoter operably linked to the nucleotide sequence encoding the recombinant receptor. In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and promoters found in the long terminal repeat of murine stem cell virus. Other promoters known to those skilled in the art are also contemplated.

[0346] In some embodiments, the recombinant nucleic acid is transferred into cells using a recombinant infectious viral particle, such as a vector derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using a recombinant lentiviral or retroviral vector, such as a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2,e93; Park et al., Trends Biotechnol. 2011 November 29(11):550-557).

[0347] In some embodiments, the retroviral vector has long terminal repeats (LTRs), such as those derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV). The majority of retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Several exemplary retroviral systems are described (e.g., in U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0348] Lentiviral transduction methods are known in the art. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother.35(9):689-701; Cooper et al. (2003) Blood.101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol.506:97-114; and Cavalieri et al. (2003) Blood.102(2):497-505.

[0349] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via gene transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2,e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0350] Other approaches and vectors for transferring nucleic acids encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014 / 055668 and U.S. Patent No. 7,446,190.

[0351] In some situations, overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) may be toxic to the subject. Thus, in some situations, the engineered cells contain gene segments that render the cells susceptible to negative selection in vivo, such as in the case of administration in adoptive immunotherapy. For example, in some embodiments, the cells are engineered so that they can be eliminated as a result of changes in the in vivo conditions of the patient to whom they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, such as a compound. Negative selection genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell II:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0352] In some embodiments, the cells are further engineered to promote the expression of cytokines or other factors.

[0353] For example, additional nucleic acids, such as genes, that can be introduced include those that improve the effectiveness of therapeutic methods, such as by promoting the survival and / or function of the transferred cells; genes that provide genetic markers for cell selection and / or evaluation, such as for evaluating in vivo survival or localization; genes that improve safety by making cells susceptible to negative selection in vivo, as described, for example, by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992). See also documents such as PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from the fusion of dominant positive and negative selectable markers. See, for example, U.S. Patent No. 6,040,177, columns 14-17, issued to Riddell et al.

[0354] Compositions and Formulations Populations of such cells, compositions containing such cells, and / or enriched in such cells are also provided, in which cells expressing the recombinant receptor constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells in the composition, or of a particular type, such as T cells or CD8+ or CD4+ cells. Compositions include pharmaceutical compositions and formulations for administration, such as adoptive cell therapy. Methods of treatment are also provided in which the cells and compositions are administered to a subject, e.g., a patient.

[0355] Also provided are compositions containing cells for administration, including pharmaceutical compositions and formulations, such as compositions in unit dose form containing a predetermined number of cells for administration of a dose or a fraction thereof. Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0356] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0357] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0358] In some embodiments, the choice of carrier will depend in part on the particular cell and / or administration method. Accordingly, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and may include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0359] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0360] The formulation may comprise an aqueous solution. The formulation or composition may also contain two or more active ingredients useful for the particular indication, disease, or condition being treated with the cells, preferably with complementary activities on the cells that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0361] In some embodiments, the pharmaceutical composition contains the cells in an amount effective to treat or prevent a disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, multiple boluses of cells, or continuous infusion of cells.

[0362] Cells and compositions may be administered using standard administration techniques, formulations, and / or devices. Cell administration can be autologous or xenogeneic. For example, immunoresponsive cells or progenitor cells can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood derived from immunoresponsive cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immune response cells) is administered, it is generally formulated in a unit-dose injectable form (solution, suspension, emulsion).

[0363] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0364] In some embodiments, the compositions are provided as sterile liquid preparations or viscous compositions, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, etc., which may be buffered to a selected pH in some embodiments. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact time with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0365] Sterile injectable solutions can be prepared by incorporating the cells in a solvent such as a mixture of suitable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring and / or coloring agents, depending on the route of administration and the desired preparation. In some embodiments, standard texts can be consulted to make appropriate preparations.

[0366] Various additives can be added to enhance the stability and sterility of composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers.Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid.For example, the use of absorption-delaying agents, such as aluminum monostearate and gelatin, can bring about the sustained absorption of injectable pharmaceutical forms.

[0367] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by passage through sterile filtration membranes.

[0368] Methods of Administration and Use in Adoptive Cell Therapy Provided herein are methods for administering the cells, populations, and compositions described herein, as well as uses of such cells, populations, and compositions described herein to treat or prevent diseases, conditions, and disorders, including cancer. In some embodiments, the cells, populations, and compositions are administered to a subject or patient with a particular disease or condition to be treated, e.g., through adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, following incubation and / or other processing steps, the cells and compositions prepared by the provided methods, such as engineered compositions and final product compositions, are administered to a subject, such as a subject with or at risk for a disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptoms of a disease or condition, such as by reducing tumor burden in cancers that express an antigen recognized by the engineered T cells.

[0369] The administration method of cells for adoptive cell therapy is known and can be used in connection with the provided methods and compositions.For example, adoptive T cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol.8(10):577-85).See, for example, Themeli et al. (2013) Nat Biotechnol.31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0370] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human. In some embodiments, a subject, e.g., a patient to whom a cell, cell population, or composition is administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0371] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a complete or partial improvement or reduction in a disease or condition or disorder, or its associated symptoms, adverse effects or outcomes, or phenotype. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The term does not imply complete cure of a disease, or complete elimination of all symptoms, or an effect on all symptoms or outcomes.

[0372] As used herein, "delaying the onset of disease" means to postpone, prevent, slow down, retard, stabilize, inhibit, and / or delay the onset of a disease (such as cancer). This delay can be of varying duration, depending on the disease being treated and / or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, may be delayed.

[0373] As used herein, "preventing" includes providing protection against the onset or recurrence of a disease in a subject who is susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the onset of the disease or slow the progression of the disease.

[0374] As used herein, to "inhibit" a function or activity is to decrease the function or activity when compared to conditions that are otherwise the same under the condition or parameter of interest, or when compared to another condition. For example, a cell that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the cell.

[0375] An "effective amount" of an agent, e.g., a pharmaceutical formulation, cell, or composition, in the context of administration, refers to an amount effective, at dosages / amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0376] For example, a "therapeutically effective amount" of an agent, such as a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the cell population being administered. In some embodiments, the methods provided involve administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount.

[0377] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some embodiments will be higher than the therapeutically effective amount.

[0378] In some embodiments, the subject has persistent or recurrent disease following treatment with another therapeutic intervention, such as chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having become refractory to another treatment.

[0379] The administration method of cells for adoptive cell therapy is known and can be used in connection with the provided methods and compositions.For example, adoptive T cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol.8(10):577-85).See, for example, Themeli et al. (2013) Nat Biotechnol.31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0380] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and / or otherwise prepared from the subject receiving the cell therapy or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject in need of treatment, e.g., a patient, and the cells are administered to the same subject following isolation and processing.

[0381] In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject receiving or ultimately receiving cell therapy, such as a first subject. In such embodiments, the cells are then administered to a different subject, such as a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0382] In some embodiments, the subject has been treated with a therapeutic agent targeting a disease or condition, such as a tumor, prior to administration of the cells or a composition containing the cells. In some aspects, the subject is refractory or non-responsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease following treatment with another therapeutic intervention, such as chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject becoming resistant to another treatment.

[0383] In some embodiments, the subject is responsive to another therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a recurrence of the disease or condition over time. In some embodiments, the subject is not relapsing. In some such embodiments, the subject is determined to be at high risk of recurrence, e.g., at high risk of recurrence, and therefore the cells are administered prophylactically, e.g., to reduce the likelihood of recurrence or prevent recurrence.

[0384] In some embodiments, the subject has not undergone previous treatment with another therapeutic agent.

[0385] Diseases, conditions, and disorders treated by the provided compositions, cells, methods, and uses include tumors such as solid tumors, hematological malignancies, and melanoma; infectious diseases, such as infections with viruses or other pathogens, e.g., HIV, HCV, HBV, CMV, etc.; and parasitic diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include, but are not limited to, leukemias, lymphomas such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[0386] In some embodiments, the disease or condition is an infectious disease or condition, including, but not limited to, viral, retroviral, bacterial, and protozoan infections, immunodeficiency disorders, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), type 1 diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or a transplant-related disease or condition.

[0387] In some embodiments, the antigen associated with the disease, disorder, or condition is ROR1, B-cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, melanoma preferentially expressed antigen (PRAME), survivin, TAG72, B7-H6, IL-13 receptor α2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, vb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, bispecific antigen, cancer-testis antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen antigen (CEA), Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms' tumor 1 (WT-1), cyclin, cyclin A2, CCL-1, CD138, and pathogen-specific antigens.

[0388] In some embodiments, the antigen associated with the disease or disorder is orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen (oncofetal antigen), ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen, PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3 and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0389] In some embodiments, the cells are administered at a desired dosage, and in some aspects, include a desired dose or cell number or cell type, and / or a desired cell type ratio. Thus, the dosage of cells is, in some embodiments, based on the total cell number (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on the desired total number (or number per kg body weight) of cells of each population or each cell type. In some embodiments, the dosage is based on a combination of characteristics, such as the desired total cell number, the desired ratio, and the desired total cell number of each population.

[0390] In some embodiments, CD8 + and CD4 +A population or subtype of cells, such as T cells, is administered at a desired total cell amount, e.g., a desired dose of T cells, or within a tolerance thereof. In some embodiments, the desired dose is a desired number of cells, or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some embodiments, the desired dose is equal to or greater than a minimum number of cells, or equal to or greater than a minimum number of cells per unit of body weight. In some embodiments, of the total total cells administered at the desired dose, individual populations or subtypes are administered at a desired output ratio (CD4 + Against CD8 + ratio, etc.), e.g., within a certain acceptable variation or error of such ratio.

[0391] In some embodiments, cells are administered at one or more desired doses of cells of individual populations or subtypes, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells, or within a tolerance thereof. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is equal to or greater than the minimum number of cells of the population or subtype, or the minimum number of cells of the population or subtype per unit of body weight.

[0392] Thus, in some embodiments, the dosage is based on a desired fixed dose and desired ratio of total cells, and / or based on one or more desired fixed doses of each of, for example, individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and CD4 + Against CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on desired fixation or minimum dose of cells.

[0393] In certain embodiments, individual populations of cells or subtypes of cells are, e.g., between 1 million and 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 a range defined by any two of the foregoing values), e.g., between about 10 million and 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, about 90 million cells, or a range defined by any two of the foregoing values). cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0394] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is, for example, 1 x 10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg, or 1 x 10 6 cells / kg body weight, or approximately 1 x 10 5 cells / kg, approximately 1.5×10 5 cells / kg, approximately 2×10 5 cells / kg, or approximately 1 x 10 6 cells / kg body weight, 10 5 ~10 6 cells / kg body weight, 10 4 ~10 9 cells / kilogram (kg) body weight or approximately 10 4 ~about 10 9 For example, in some embodiments, the cells are in the range of, for example, 1 x 10 cells / kilogram (kg) body weight. 5 T cells / kg, 1.5×105 T cells / kg, 2×10 5 T cells / kg, or 1 × 10 6 T cells / kg body weight, or approximately 1 × 10 5 T cells / kg, approximately 1.5×10 5 T cells / kg, approximately 2×10 5 T cells / kg, or approximately 1 × 10 6 T cells / kg body weight, 10 5 ~10 6 T cells / kg body weight, 10 4 ~10 9 T cells / kilogram (kg) body weight or approximately 10 4 ~about 10 9 T cells / kilogram (kg) body weight or within a specified margin of error thereof.

[0395] In some embodiments, the cells are, for example, 1 x 10 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 x 10 6 CD4 + and / or CD8 + cells / kg body weight, or approximately 1 x 10 5 CD4 + and / or CD8 + cells / kg, approximately 1.5×10 5 CD4 + and / or CD8 + cells / kg, approximately 2×10 5 CD4 + and / or CD8 + cells / kg, or approximately 1 x 10 6 CD4 + and / or CD8 + cells / kg body weight, 10 5 ~10 6 CD4 + and / or CD8 +cells / kg body weight, 10 4 ~ or 10 9 or about 10 4 ~ or about 10 9 CD4 + and / or CD8 + Dosed in cells / kilogram (kg) body weight or within a specified margin of error.

[0396] In some embodiments, the cells are at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 CD4 + cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 CD8+ cells, and / or at least about 1 × 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 In some embodiments, the cells are administered at about 10 T cells, or more than 10 T cells, or within a specified margin of error. 8 ~10 12 or about 10 10 ~10 11 T cells, about 10 8 ~10 12 or about 10 10 ~10 11 CD4 + cells, and / or approximately 10 8 ~10 12 or about 10 10 ~10 11 CD8 + It is administered in cells or within a specified margin of error.

[0397] In some embodiments, cells are administered at a desired output ratio of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes, or within a tolerated range thereof. In some aspects, the desired ratio can be a specific ratio or can be a ratio range, e.g., in some embodiments, a desired ratio (e.g., CD4 + Against CD8 + The cell ratio) may be 5:1 to 5:1 or about 5:1 to about 5:1 (or greater than about 1:5 but less than about 5:1), or 1:3 to 3:1 or about 1:3 to about 3:1 (or greater than about 1:3 but less than about 3:1), for example, 2:1 to 1:5 or about 2:1 to about 1:5 (or 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2. 5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5, or about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1. (greater than about 1:5 and less than about 2:1, such as 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5.) In some embodiments, the acceptable variance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0398] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the cells, and the discretion of the attending physician. The compositions and cells, in some embodiments, are suitably administered to the subject at one time or over a series of treatments.

[0399] The cells can be administered by any suitable means, such as by bolus injection, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and, if desired, by topical treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus of cells. In some embodiments, it is administered by multiple bolus administration of cells over a period of, for example, three days or less, or by continuous infusion of cells.

[0400] In some embodiments, the cells are administered as part of a combination therapy, such as with another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic drug or therapeutic agent, performed simultaneously or sequentially in any order. In some embodiments, the cells are co-administered with one or more additional therapeutic agents, or administered simultaneously or sequentially in any order in conjunction with another therapeutic intervention. In some situations, the cells are co-administered with another therapy close enough in time so that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, to enhance persistence. In some embodiments, the method comprises administering a chemotherapeutic agent.

[0401] Following administration of the cells, the biological activity of the engineered cell population in some embodiments is measured, for example, by any of several known methods. Parameters evaluated include specific binding of engineered or natural T cells or other immune cells to antigens in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays, as described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al. J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or burden.

[0402] In certain embodiments, engineered cells are further modified in many ways to enhance their therapeutic or preventive effects.For example, the engineered CAR or TCR expressed by the population can be directly or indirectly conjugated to targeting moiety via linker.For example, the practice of conjugating compounds such as CAR or TCR to targeting moiety is known in the art.For example, see Wadwa et al., J.Drug Targeting 3:111(1995) and U.S. Patent No. 5,087,616. [Example]

[0403] The following examples are illustrative only and are not intended to limit the scope or content of the present invention in any way.

[0404] Example 1 - Initial screening of gRNAs Guide RNAs were screened by complexing commercially synthesized gRNAs with Cas9 in vitro and delivering the gRNA / Cas9 ribonucleoprotein (RNP) into cells via electroporation.

[0405] Figure 1 shows the indel frequency rate (%) of the tested gRNAs. Figure 2 shows the genome editing efficiency (%) of certain exemplary gRNA pairs.

[0406] Example 2 - Analysis of gRNA candidates against TGFBR2 in T cells The goal of cellular CRISPR-Cas9 editing is to achieve the highest percentage of target gene knockout using the lowest possible concentration of gRNA / Cas9 complex and the fewest off-target cleavage events. To determine the optimal gRNA candidate for TGFBR2 gene editing, we tested seven potential gRNAs. T cells were transfected with various concentrations of gRNA / Cas9 RNP. Illumina miSeq analysis was then used to determine the indel frequency. The results show that the indel frequency percentages for the various gRNAs ranged from 20% to 80%, with all gRNAs achieving the highest indel frequency percentages at an RNP concentration of 2 μM (Figure 3). We subsequently used a 2 μM RNP concentration for all experiments.

[0407] Selected gRNAs were chosen from the miSeq analysis for subsequent analysis on BCMA CAR T cells. The editing rate (%) of the TGFBR2 gene was determined using gRNAs with SEQ ID NOs: 5050, 5052, 5093, and 5043 in an RNP re-cleavage assay. A dual gRNA approach was also tested using the SEQ ID NO: 5043 / 5093 combination. While individual gRNAs with SEQ ID NOs: 5043 and 5093 only achieved 20–40% editing, the combination was found to be more effective, yielding editing rates of approximately 80% (Figure 4A).

[0408] High-throughput sequencing analysis was performed using the selected gRNAs to determine the indel frequency rate (%). gRNAs of SEQ ID NOs: a and b, as well as the dual gRNA combination c / d, were tested. Surprisingly, a high indel frequency rate (%) of 95% was achieved from the selected gRNAs (Figure 4B).

[0409] Although the indel frequency rate (%) is a useful indicator of gRNA efficacy, some of the resulting indels may be in-frame insertions or deletions. Such in-frame indels may generate modified genes whose protein products retain some activity. When attempting to create gene knockouts, out-of-frame indels are preferred. To confirm that the tested gRNAs were indeed generating the desired out-of-frame indels, the sequencing results were analyzed for the specific types of indels that occurred. The results show that SEQ ID NO:5052 produced the highest indel frequency rate (%), while SEQ ID NO:5050 produced a higher out-of-frame indel frequency rate (%) (Figure 5).

[0410] Example 3 - In vitro efficacy of gene editing in primary and engineered T cells We analyzed the inhibitory effect of TGFβ on primary T cells modified to express a BCMA-targeting CAR. Anti-BCMA CAR T cells were further modified to harbor an edited TGFBR2 gene via the CRISPR-Cas9 system using selected gRNAs. The AAVS1 control for gene editing and anti-BCMA CAR-expressing T cells with an unedited TGFBR2 gene were used as controls. This cell line was co-cultured with the RPMI 8226 multiple myeloma cell line in the presence or absence of 10 ng / ml TGFβ. In the presence of excess TGFβ, primary T cells and anti-BCMA CAR T cells exhibited the expected inhibitory effect of reduced interferon-γ (IFNγ) production. However, the gene-edited anti-BCMA CAR T cells rescued the inhibitory effect of TGFβ, restoring IFNγ production to levels observed without TGFβ (Figure 6).

[0411] Inhibition of T cell proliferation is one of the effects caused by TGFβ signaling (Tiemessen et al. Int.I mmunol. 15: 1495-1504. 2003). To address the effect of TGFβ signaling on cell proliferation, we monitored anti-BCMA CAR T cell proliferation in a TGFBR2 gene-edited background using several gRNAs. Editing the TGFBR2 gene allowed anti-BCMA CAR T cells to proliferate in the presence of excess TGFβ (Figure 7).

[0412] T cell activity is affected by the expression of various stimulatory and inhibitory receptors. CD25 expression was assessed for TGFBR2 gene-edited anti-BCMA CAR T cells. TGFBR2 gene editing results in higher levels of CD25 expression than control cells upon exposure to TGFβ (Figure 8A). We also assessed the expression of the inhibitory receptor PD-1 by measuring the percentage of PD-1+ cells in the presence and absence of excess TGFβ. TGFBR2 gene-edited anti-BCMA CAR T cells showed a lower increase in PD-1 than control cells. Surprisingly, the gene-edited cells also gave rise to relatively fewer PD-1+ cells, even without the addition of TGFβ (Figure 8B).

[0413] Activation of the TGFβ signaling pathway leads to phosphorylation of the Smad2 / 3 complex, which then regulates many downstream processes in the TGFβ signaling pathway. For this reason, phosphorylated Smad2 / 3 is often used as an indicator of TGFβ signaling in cells. Phosphorylated Smad2 / 3 was detected in T cells transduced to express various CARs, with or without TGFBR2 gene editing. CAR-expressing T cells, including the AAVS1 gene-edited control, in the presence (10 ng / ml) or absence of TGFβ were used as controls. While excess TGFβ resulted in the expected increase in Smad2 / 3 phosphorylation in unedited cells, TGFBR2 gene-edited cells maintained the same level of Smad2 / 3 phosphorylation, regardless of the addition of TGFβ (Figure 9). These results demonstrate that the TGFBR2 CRISPR gene editing approach is effective in silencing the TGFβ signaling pathway.

[0414] To further analyze the effects of TGFBR2 gene editing in various CAR T cell backgrounds, we detected GzmB levels in the presence and absence of TGFβ. CAR-expressing T cells containing AAVS1 gene-edited control TGFβ (10 ng / ml) were used as controls. GzmB intracellular staining results reveal that TGFBR2 gene editing maintains GzmB expression in the presence of TGFβ (Figure 10).

[0415] A cytokine detection assay was used to analyze IFNγ production in experiments similar to those described in Figure 10. Consistent with previous results with GzmB expression, IFNγ production was maintained and even increased in the TGFBR2 gene-edited background compared to controls in conditions of excess TGFβ (Figure 11).

[0416] The Edu Click-It assay from ThermoFisher was used to analyze cell proliferation in experiments similar to those described in Figures 10 and 11. Under conditions of excess TGFβ, cell proliferation was maintained in the TGFBR2 gene-edited background compared to controls (Figure 12).

[0417] The overall results in Example 3 demonstrate the benefits of using the CRISPR-Cas9 gene editing system to edit the TGFBR2 gene. Using single and dual gRNA approaches, the TGFβ signaling pathway can be effectively suppressed in the context of CAR T cells.

[0418] Example 4 - Comparison of TGFBR2 dominant-negative approach with CRISPR-Cas9 gene editing approach Another approach to abrogating TGFβ signaling is to express a dominant-negative (DN) form of TGFBR2, which competes with wild-type TGFBR2 for TGFβ binding, thereby minimizing the effective signaling response.

[0419] The inhibitory effect of TGFβ on primary T cells transduced to express anti-BCMA CAR was analyzed. Anti-BCMA CAR-expressing T cells were further modified via the CRISPR-Cas9 system to express DNA or have an edited TGFBR2 gene. Dual gRNAs of SEQ ID NOs: 5043 and 5093 were used to edit the CRISPR-Cas9-edited cells. AAVS1 control for gene editing, anti-BCMA CAR-expressing T cells with an unedited TGFBR2 gene, and anti-BCMA CAR-expressing T cells with the AAVS1 gene editing control were used as controls. This cell line was co-cultured with RPMI 8226 multiple myeloma cell line in the presence or absence of 10 ng / ml TGFβ. In the presence of excess TGFβ, primary T cells and anti-BCMA CAR T cells exhibited the expected inhibitory effect of reduced production of GzmB (Figure 13A) and IFNγ (Figure 13B). However, in DN cells or CRISPR-edited anti-BCMA CAR T cells, the inhibitory effect of TGFβ is rescued, restoring GzmB and IFNγ production to levels seen in the absence of TGFβ.

[0420] To further demonstrate the utility of rescuing the inhibitory effects of TGFβ signaling, we analyzed the killing activity of anti-BCMA CAR T cells in a DN background or a CRISPR-edited background. Anti-BCMA CAR-expressing T cells with an unedited TGFBR2 gene were used as a control. Anti-BCMA CAR T cells were co-cultured with RPMI 8226 cells at a ratio of one anti-BCMA CAR T cell to four RPMI cells in the presence or absence of 10 ng / ml TGFβ. In the presence of TGFβ, the lytic activity of anti-BCMA CAR T cells was maintained in both the DN and CRISPR-edited backgrounds. Surprisingly, the CRISPR-edited anti-BCMA CAR T cells exhibited superior lytic activity compared to the DN cells (Figure 14). The lytic activity of the CRISPR-edited anti-BCMA CAR T cells was higher than that of the control cells, even without the addition of excess TGFβ.

[0421] Repeated antigen stimulation of T cells, including CAR T cells, can reduce T cell persistence and cause activation-induced cell death (AICD) (Gargett et al. Mol. Ther. 24:1 135-1149. 2016). Strategies to maintain T cell activity against specific antigens are crucial for improving the efficacy of T cell-based therapies. Anti-BCMA CAR T cells in a DN background or a CRISPR-edited background were analyzed for their proliferative capacity in the presence of repeated stimulation with TGFβ. Anti-BCMA CAR-expressing T cells with an unedited TGFBR2 gene were used as a control. While unmodified anti-BCMA CAR T cells did not proliferate over several days of TGFβ stimulation, TGFBR2 DN cells or CRISPR-edited cells continued to proliferate over time in the presence of TGFβ (Figures 15A-C). Furthermore, the percentage of anti-BCMA CAR T cells did not decrease during repeated stimulation with TGFβ (Figures 15D-F).

[0422] Using a single-gRNA approach rather than a dual-gRNA approach, we again analyzed IFNγ production in the DN and gene-edited backgrounds. As described in Figure 13, we used an additional multiple myeloma cell line, OPM2, as a comparison to the previously used RPMI 8226 cell line. Consistent with previous results, inhibition of TGFβ signaling effectively maintained IFNγ production in the presence of excess TGFβ. This was true for both the RPMI (Figure 16A) and OPM2 (Figure 16B) cell lines.

[0423] We also analyzed CD25 expression in DN and gene-edited backgrounds using a single gRNA approach. In RPMI (Figure 17A) and OPM2 (Figure 17B) cell lines, CD25 expression was maintained in the presence of excess TGFβ. Surprisingly, CD25 expression in DN and gene-edited cells was increased compared to unmodified T cells.

[0424] PD-1 expression was similarly detected in DN and gene-edited backgrounds. Inhibition of TGFβ signaling by either approach effectively prevented the increase in PD-1 expression in the presence of excess TGFβ (Figures 18A and 18B).

[0425] Previous experiments used TGFβ at a set dose of 10 ng / ml for stimulation. To better understand how TGFBR2 gene editing affects TGFβ signaling under physiological conditions, we used a range of TGFβ from 0 to 100 ng / ml. This range is based on previous studies demonstrating that TGFβ serum levels range from approximately 3 to 88 ng / ml (Aref et al. Hematological Oncology. 35: 51-57. 2017) and approximately 3 to 10 ng / ml (Bruns et al. Blood. 120: 2620-2630. 2012) in multiple myeloma bone marrow. A cell proliferation assay was used to determine the relative fold expansion of cells in the DN background and the gene-edited background. Anti-BCMA CAR T cells were co-cultured with RPMI 8226 cells at a 1:1 ratio. Unexpectedly, a positive effect on cell proliferation can be observed at TGFβ concentrations as low as 0.1 ng / ml, i.e., concentrations well below physiological levels in normal or cancer environments (Figure 19A). This responsiveness is further demonstrated by increased production of IFNγ (Figure 19B).

[0426] Using the same range of TGFβ concentrations, we analyzed PD-1 expression on CD4+ and CD8+ CAR T cells. Again, in the TGFBR2 DN and gene-edited backgrounds, low levels of PD-1 expression could be maintained with TGFβ as low as 0.1 ng / ml (Figures 20A and 20B).

[0427] Using the DN TGFBR2 strategy, we tested cell proliferation, granzyme B expression, and Smad 2 / 3 phosphorylation in T cells transduced to express three different CARs. DN TGFBR2 was able to rescue the antiproliferative effects of excess TGFβ in the CAR T cell background tested. DN was able to maintain granzyme B expression in the presence of excess TGFβ in the CAR T cell background tested. Finally, DN was able to effectively suppress TGFβ signaling in the presence of excess TGFβ, as measured by a decrease in Smad 2 / 3 phosphorylation in the CAR T cell background tested (Figure 22). Although data for only one of the three CAR T cell backgrounds are shown, similar results were observed in the other two CAR T cell backgrounds.

[0428] Example 5 - Transcriptional profiling of BCMA CAR T cells in vivo For the TGFBR2 gene editing strategy disclosed herein to be an effective treatment, the TGFβ immunosuppressive pathway must be present in an in vivo setting. If the TGFβ immunosuppressive pathway is present, the TGFBR2 gene editing strategy must effectively release T cells from the TGFβ immunosuppressive effects. To address both of these concerns, transcriptional profiling of anti-BCMA CAR T cells was performed on anti-BCMA CAR T cells isolated from the tumor microenvironment in mice. Tumor xenograft models were generated by implanting multiple myeloma RPMI 8226 cells into mice and allowing tumors to grow / form for 21 days. After a 21-day incubation period, wild-type, AAVS gene-edited control, TGFBR2 DN background, or TGFBR2 gene-edited background anti-BCMA CAR T cells were injected into tumor-bearing mice. After tumor regression for 14 days, tumor-infiltrating leukocytes (TILs) and CAR T cells were isolated from the tumor and spleen (non-cancerous negative control tissue). After isolation, cells were subjected to RNAseq analysis to determine the gene expression profile of TGFβ signaling pathway members.

[0429] Transcriptional profiling results reveal that TGFBR2 gene editing of CAR T cells successfully restricts the expression of several TGFβ signaling pathway members within tumors where TGFβ inhibitory signaling occurs (Figure 21). As shown in Figure 21, TGFβ signaling pathway members are upregulated in anti-BCMA CAR-expressing T cells isolated from tumors but not from the spleen. The data indicate that anti-BCMA CAR-expressing T cells are exposed to a TGFβ-rich tumor microenvironment (TME). Furthermore, anti-BCMA CAR-expressing T cells with a TGFBR2 DN background or a TGFBR2 gene-edited background reverse the upregulation of TGFβ signaling pathway members. The data indicate that the TGFβ signaling pathway is effectively abrogated.

[0430] Example 6 - Selective advantage of TGFBR2 gene editing strategy To determine whether TGFBR2 gene-edited CAR T cells have a selective growth advantage, anti-BCMA CAR T cells were gene-edited to generate various indel frequency percentages. These distinct indel percentage populations of CAR T cells were co-cultured with RPMI 8226 cells at a 1:1 ratio for stimulation in the presence or absence of 10 ng / ml TGFβ. Cells were assessed approximately every 7 days using high-throughput sequencing to determine which portions of the cells were gene-edited and which portions remained wild-type (Figure 23). Cells were restimulated with fresh RPMI cells and readjusted to a 1:1 ratio weekly. The results demonstrate that in an immune-stimulating environment, TGFBR2 gene editing confers a selective growth advantage over wild-type cells (see Figure 24).

[0431] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0432] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the claims appended hereto.

Claims

1. a gRNA comprising a targeting domain complementary to a target sequence of the transforming growth factor beta receptor II (TGFBR2) gene; RNA-guided nucleases and A genome editing system comprising: the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103; Genome editing system.

2. The genome editing system of claim 1, the targeting domain is configured to create a double-stranded or single-stranded break within at least 10 bp of the TGFBR2 target locus, thereby modifying the TGFBR2 gene; or the RNA-guided nuclease is a Streptococcus pyogenes (S. pyogenes) Cas9 nuclease; or the RNA-guided nuclease is Staphylococcus aureus (S. aureus) Cas9 nuclease; or the RNA-guided nuclease is a mutant Cas9 nuclease; or the gRNA is a modular or chimeric gRNA, or the genome editing system comprises two, three, or four gRNAs; or TGFBR2 expression is reduced by 30% or more compared to baseline measurements, or A frameshift mutation is introduced into the TGFBR2 gene. Genome editing system.

3. 3. The genome editing system of claim 1 or 2, for use in modifying the TGFBR2 gene in a cell.

4. A composition comprising a gRNA comprising a targeting domain complementary to a target sequence of a TGFBR2 gene, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103.

5. 5. The composition of claim 4, wherein the composition comprises one, two, three, or four gRNAs.

6. 6. The composition of claim 4 or 5, further comprising a Cas9 nuclease, or further comprising one or both of a wild-type Cas9 nuclease and a mutant Cas9 nuclease.

7. 7. The composition of any one of claims 4 to 6, comprising at least one Streptococcus pyogenes Cas9 nuclease.

8. 8. The composition of any one of claims 4 to 7 for use in reducing or eliminating TGFBR2 gene expression in a cell.

9. A vector comprising a polynucleotide encoding a gRNA comprising a targeting domain complementary to a target sequence of the TGFBR2 gene, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103.

10. 10. The vector of claim 9, wherein the vector further comprises a polynucleotide encoding a Cas9 nuclease.

11. The vector of claim 9 or 10, wherein the vector is a viral vector.

12. 12. The vector of any one of claims 9 to 11 for use in reducing or eliminating TGFBR2 gene expression in a cell.

13. (i) a genome editing system comprising a guide RNA (gRNA) comprising a targeting domain complementary to a target sequence of a transforming growth factor beta receptor II (TGFBR2) gene, and an RNA-guided nuclease, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103; or (ii) A vector comprising a polynucleotide encoding a gRNA comprising a targeting domain complementary to a target sequence of the TGFBR2 gene and a polynucleotide encoding an RNA-guided nuclease, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103. A method for modifying expression of the TGFBR2 gene in a cell in vitro, comprising administering to the cell one of the following:

14. 14. The method of claim 13, The modification comprises knocking out or knocking down TGFBR2 gene expression, or the cells are derived from a subject suffering from cancer, or The gRNA and RNA-guided nuclease comprise a ribonucleoprotein (RNP) complex; method.

15. A cell comprising a genome editing system according to any one of claims 1 to 3, a composition according to any one of claims 4 to 8, or a vector according to any one of claims 9 to 12.

16. A cell modified according to the method of claim 13 or 14.

17. An RNA-guided nuclease-mediated method for modifying TGFBR2 gene expression in a cell in vitro, the method comprising: a) a sufficient amount of a gRNA that targets TGFBR2, wherein the gRNA comprises a targeting domain that is complementary to a target sequence of the TGFBR2 gene; and RNA-guided nucleases contacting the cell with b) creating a first DNA double-strand break near a TGFBR2 target location in a TGFBR2 gene of the cell, wherein the first DNA double-strand break is repaired by NHEJ, and the repair alters expression of the TGFBR2 gene. Including, The method, wherein the gRNA comprises a targeting domain comprising a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 5103.

18. 18. The method of claim 17, further comprising creating a second DNA double-strand break near the TGFBR2 target location.

19. 19. The method of claim 17 or 18, the RNA-guided nuclease is Streptococcus pyogenes Cas9 nuclease, or the RNA-guided nuclease is Staphylococcus aureus Cas9 nuclease; or the RNA-guided nuclease is a mutant Cas9 nuclease; or the NHEJ repair results in an insertion or deletion at a frequency of 20% or more; method.

20. 1. Use of engineered immune cells in the manufacture of a medicament for use in a method of treating cancer in a subject, comprising: The engineered immune cells have reduced expression of the TGFBR2 gene by the method of claim 13, wherein the engineered immune cells optionally express an engineered T cell receptor (eTCR) or a chimeric antigen receptor (CAR), and the engineered immune cells have an insertion or deletion near or at a target location in the TGFBR2 gene. use.

21. 21. The use according to claim 20, the engineered immune cells comprise T cells or NK cells, and optionally the T cells are CD4+ T cells and / or CD8+ T cells; or The eTCR or CAR has antigen specificity for cancer cells, or the cancer is selected from the group consisting of leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, low-grade B-cell lymphoma, B-cell malignancies, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, and brain cancer, ovarian cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, mesothelioma, and / or any cancer type that expresses TGF-β, or the engineered immune cells maintain or have enhanced lytic activity against target cancer cells compared to non-engineered immune cells, or the engineered immune cells maintain or increase expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to non-engineered immune cells; or the engineered immune cells maintain or have improved persistence to repeated antigen stimulation compared to non-engineered immune cells; or the engineered immune cells maintain or have increased expression of CD25 compared to non-engineered immune cells, or the engineered immune cells maintain or have reduced expression of PD-1 compared to non-engineered immune cells; or the engineered immune cells maintain or increase proliferation compared to non-engineered immune cells; use.

22. 20. A composition comprising a plurality of engineered T cells modified according to the method of any one of claims 17-19, wherein the engineered T cells exhibit reduced TGFBR2 gene expression compared to non-engineered T cells.

23. 23. The composition of claim 22, the engineered T cells exhibit a TGFBR2 gene expression level that is at least or at most 50%, 40%, 30%, 20%, 10%, or 5% of the TGFBR2 expression level in unengineered T cells; or a) enhanced lytic activity against target cancer cells compared to non-engineered T cells; b) sustained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to unmanipulated T cells; c) sustained or increased persistence to repeated antigen stimulation compared to unmanipulated T cells; d) maintained or increased expression of CD25 compared to unmanipulated T cells; e) maintained or decreased expression of PD-1 compared to unmanipulated T cells, and / or f) Sustained or increased proliferation compared to unmanipulated T cells The engineered T cells are further characterized by having composition.

24. 1. A composition comprising a plurality of engineered T cells, the engineered T cells exhibit reduced transforming growth factor beta receptor II (TGFBR2) gene expression compared to unengineered T cells; the engineered T cells A guide RNA (gRNA) comprising a targeting domain complementary to a target sequence of the TGFBR2 gene, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103; and RNA-guided nucleases by contacting unmanipulated T cells with a genome editing system comprising composition.

25. 25. The composition of claim 24, the engineered T cells are further transduced with a vector expressing an eTCR or a CAR, or The RNA-guided nuclease is Streptococcus pyogenes Cas9 nuclease. composition.

26. 14. A composition comprising a plurality of engineered T cells, wherein the engineered T cells have a TGFBR2 gene modified by the method of claim 13, wherein the engineered T cells are defective in TGFBR2 signaling, and wherein the defective TGFBR2 signaling is mediated by expressing a dominant-negative (DN) form of TGFBR2 in the engineered T cells.

27. The engineered T cells are further transduced with a vector expressing an eTCR or a CAR, or the T cells are CD4+ T cells and / or CD8+ T cells, or a) enhanced lytic activity against target cancer cells compared to non-engineered T cells; b) sustained or increased expression of granzyme B and / or interferon gamma in the presence of TGFβ compared to unmanipulated T cells; c) sustained or increased persistence to repeated antigen stimulation compared to unmanipulated T cells; d) maintained or increased expression of CD25 compared to unmanipulated T cells; e) maintained or decreased expression of PD-1 compared to unmanipulated T cells, and / or f) Sustained or increased proliferation compared to unmanipulated T cells The engineered T cells are further characterized by having:

27. The composition of claim 26.

28. A ribonucleoprotein (RNP) complex comprising a gRNA comprising a targeting domain complementary to a target sequence of a TGFBR2 gene and an RNA-guided nuclease, wherein the targeting domain comprises a nucleotide sequence identical to SEQ ID NO: 5103.

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