Genetically engineered T cells with PTPN2 knockout have improved functionality and anti-tumor activity

Abstract

A population of genetically engineered T cells, comprising a disrupted protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene and optionally a disrupted TRAC gene, a disrupted 2M gene, and/or a disrupted CD70 gene. Also provided herein are methods for making such genetically engineered T cells and therapeutic uses thereof.

Claims

1. A population of genetically engineered T cells, comprising: a nucleic acid encoding a chimeric antigen receptor (CAR); a disrupted protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene; a disrupted T cell receptor alpha chain constant region (TRAC) gene; and a disrupted beta-2-microglobulin (2M) gene, wherein the cells exhibit enhanced survival in the presence of natural killer (NK) cells.

2. The population of genetically engineered T cells of claim 1, wherein the disrupted PTPN2 gene is genetically edited in exon 1, exon 2, and/or exon 3.

3. The population of genetically engineered T cells of claim 1, wherein the disrupted PTPN2 gene is genetically edited by CRISPR/Cas-mediated gene editing.

4. The population of genetically engineered T cells of claim 3, wherein the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the PTPN2 gene that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-13.

5. The population of genetically engineered T cells of claim 4, wherein the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28, 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 56, 57, 60, 61, 64-67, 70, and 71.

6. The population of genetically engineered T cells of claim 5, wherein the gRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26, 27, 30, 31, 34, 38, 39, 42, 43, 46, 47, 50, 51, 54, 55, 58, 59, 32, 63, 68, 69, 192, 193.

7. The population of genetically engineered T cells of claim 1, wherein the disrupted TRAC gene is genetically edited by a CRISPR/Cas-mediated gene editing system.

8. The population of genetically engineered T cells of claim 7, wherein the CRISPR/Cas-mediated gene editing system comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 76 or 77.

9. The population of genetically engineered T cells of claim 8, wherein the disrupted TRAC gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 93 to 100.

10. The population of genetically engineered T cells of claim 1, wherein the disrupted T cell TRAC gene has a deleted fragment comprising SEQ ID NO: 87.

11. The population of genetically engineered T cells of claim 1, wherein the nucleic acid is inserted in the genome of the T cells.

12. The population of genetically engineered T cells of claim 11, wherein the disrupted TRAC gene comprises the nucleic acid encoding the CAR.

13. The population of genetically engineered T cells of claim 12, wherein the nucleic acid encoding the CAR replaces the deleted fragment in the disrupted TRAC gene.

14. The population of genetically engineered T cells of claim 1, wherein the disrupted 2M gene is genetically edited by CRISPR/Cas-mediated gene editing.

15. The population of genetically engineered T cells of claim 14, wherein the CRISPR/Cas-mediated gene editing comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 80 or 81.

16. The population of genetically engineered T cells of claim 15, wherein the disrupted 2M gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 101 to 106.

17. The population of genetically engineered T cells of claim 1, wherein the T cells further comprise a disrupted CD70 gene.

18. The population of genetically engineered T cells of claim 17, wherein the disrupted CD70 gene is genetically edited by CRISPR/Cas-mediated gene editing.

19. The population of genetically engineered T cells of claim 18, wherein the CRISPR/Cas-mediated gene editing comprises a gRNA comprising the nucleotide sequence of SEQ ID NO: 72 or 73.

20. The population of genetically engineered T cells of claim 19, wherein the disrupted CD70 gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 107 to 112.

21. The population of genetically engineered T cells of claim 1, wherein the CAR comprises an extracellular antigen binding domain specific to a tumor antigen, a co-stimulatory signaling domain of 4-1BB or CD28, and a cytoplasmic signaling domain of CD35.

22. The population of genetically engineered T cells of claim 21, wherein the tumor antigen is CD19, BCMA, or CD70.

23. The population of genetically engineered T cells of claim 22, wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds CD19, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 137.

24. The population of genetically engineered T cells of claim 23, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 135.

25. The population of genetically engineered T cells of claim 22, wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds CD70, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 157 or 159.

26. The population of genetically engineered T cells of claim 25, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 155.

27. The population of genetically engineered T cells of claim 22, wherein the extracellular antigen binding domain is a single chain variable fragment (scFv) that binds BCMA, and wherein the scFv comprises the amino acid sequence of SEQ ID NO: 165.

28. The population of genetically engineered T cells of claim 27, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 163.

29. The population of genetically engineered T cells of claim 1, wherein the genetically engineered T cells are derived from primary T cells of one or more human donors.

30. The population of genetically engineered T cells of claim 1, wherein the population of genetically engineered T cells expressing the CAR has enhanced CAR potency and/or increased CAR copies as compared to non-engineered T cell counterparts.

31. A method for preparing the population of genetically engineered T cells of claim 1, the method comprising: (a) providing a plurality of cells, which are T cells or precursor cells thereof; genetically editing the PTPN2, TRAC and 2M genes; and (b) producing the population of genetically engineered T cells having disrupted PTPN2, TRAC and 2M genes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photo showing expression of PTPN2 protein in T cells edited by the indicated various guide RNAs targeting PTPN2 using Simple Wes protein analysis.

(2) FIG. 2 is a photo showing PTPN2 KO in anti-CD70 CAR T cells as determined by Simple Wes Western Blot protein analysis. (WT: unedited cells; CART: anti-CD70 CAR T with unedited PTPN2; CART/PTPN2: anti-CD70 CAR T with PTPN2 KO)

(3) FIGS. 3A-3B include diagrams showing the in vitro effect of PTPN2 editing on anti-CD70 CAR T cell killing ability against CAKI-1 (FIG. 3A) and A498 (FIG. 3B) tumor cells at various E:T ratios as indicated. PTPN2 KO improves cytotoxicity of CAR-T cells.

(4) FIGS. 4A-4H include diagrams showing the effect of PTPN2 editing on cytokine secretion of CAR T cells in the presence of A498 tumor cells. The anti-tumor cytokine secretion of anti-CD70 CAR T cells was compared to anti-CD70 CAR T cells with PTPN2 KO. FIG. 4A: IL-2. FIG. 4B: IFN-. FIG. 4C: TNF-. FIG. 4D: IL-10. FIG. 4E: Granzyme A. FIG. 4F: Granzyme B. FIG. 4G: Granulysin. FIG. 4H: Perforin.

(5) FIGS. 5A-5F include diagrams showing the effect of PTPN2 editing on cytokine secretion of CAR T cells in the presence of CAKI-1 tumor cells. The anti-tumor cytokine secretion of anti-CD70 CAR T cells was compared to anti-CD70 CAR T cells with PTPN2 KO.

(6) FIG. 5A: IL-2. FIG. 5B: IFN-. FIG. 5C: TNF-. FIG. 5D: IL-10. FIG. 5E: Granulysin. FIG. 5F: Perforin.

(7) FIGS. 6A-6B include diagrams showing analysis of exhaustion markers in CAR T cells with PTPN2 KO. Fluorescent cytometry analysis of exhaustion markers, PD1, LAG3, TIGIT and TIM3 in CD4+(FIG. 6A) and CD8+(FIG. 6B) CAR-T cells are shown. PTPN2 KO CAR T cells show higher expression of TIM3, and LAG3 in both CD4+ and CD8+ populations compared to anti-CD70 CAR-Ts without the knockout.

(8) FIG. 7 includes a photo showing knockout of PTPN2 in anti-CD70 CAR-T cells leads to higher phosphorylation levels of STAT5 and c-JUN. Diagram shows elevated phosphorylation levels of STAT5 in PTPN2 KO suggesting superior cytokine secretion. Phospho-JUN levels are elevated in PTPN2 KO CAR-T cells, which is more pronounced when the CAR-T cells are stimulated with IL-2. All samples were serum/cytokine starved for 24 hours.

(9) FIGS. 8A-8B include diagrams showing PTPN2 KO enhances in vivo anti-tumor killing function of anti-CD70 CAR T cells against CAKI-1 tumors (FIG. 8A) and prolongs survival of tumor bearing mice (FIG. 8B).

(10) FIGS. 9A-9B include diagrams showing that PTPN2 KO promotes survival of allogeneic anti-CD70 CAR-T cells against NK cell attack. FIG. 9A: Effector NK donor 1. FIG. 9B: Effector NK donor 2.

(11) FIGS. 10A-10B include diagrams showing that PTPN2 KO enhances in vivo anti-tumor killing function of anti-BCMA CAR-T cells (FIG. 10A) and in-vivo expansion of anti-BCMA CAR-T cells (FIG. 10B).

(12) FIGS. 11A-11B include diagrams showing that PTPN2 KO enhances in vivo anti-tumor killing function of anti-CD19 CAR-T cells (FIG. 11A) and prolongs survival of NALM6 tumor bearing mice (FIG. 11B).

DETAILED DESCRIPTION OF THE INVENTION

(13) The present disclosure aims at establishing genetically engineered T cells having improved growth activity, cytotoxicity, or both, particularly in the presence of TGF and/or cells capable of secreting such (e.g., fibroblasts). Such a T cell may use bona fide T cells as the starting material, for example, non-transformed T cells, terminally differentiated T cells, T cells having stable genome, and/or T cells that depend on cytokines and growth factors for proliferation and expansion. Alternatively, such a T cell may use T cells generated from precursor cells such as hematopoietic stem cells (e.g., iPSCs), e.g., in vitro culture. The T cells disclosed herein may confer one or more benefits in CAR-T cell clinical applications.

(14) The genetically engineered T cells having a disrupted PTPN2 gene, and optionally one or more additional genetic edits, for example, a disrupted TRAC gene, a disrupted 2M gene, a disrupted CD70 gene, an inserted nucleic acid coding for a chimeric antigen receptor (CAR), or a combination thereof. Unexpectedly, T cells having a disrupted PTPN2 gene showed advantageous features disclosed herein. Given such advantageous features, the genetically engineered T cells (e.g., CAR-T cells) disclosed herein, having a disrupted PTPN2 gene and optionally other genetic edits as disclosed herein, would be expected to exhibit superior therapeutic effects, for example, superior anti-tumor effects.

(15) Accordingly, provided herein are genetically engineered T cells (e.g., CAR-T cells) having a disrupted PTPN2 gene and optionally one or more additional genetic edits, e.g., a disrupted TRAC gene, a disrupted 2M gene, a disrupted CD70 gene, or a combination thereof; compositions comprising such; and therapeutic uses of such genetically engineered T cells, for example, in tumor treatment. Components and processes (e.g., the CRISPR approach for gene editing and components used therein) for making the T cells disclosed herein are also within the scope of the present disclosure.

(16) I. Genetically Engineered T Cells

(17) In some aspects, provided herein are genetically engineered T cells having a disrupted PTPN2 gene, and optionally one or more additional genetic edits as disclosed herein. As shown by the studies disclosed herein, such genetically engineered T cells show improved cytotoxicity; enhanced persistence; increased cytokine production; enhanced expression of LAGS and/or TIM3; enhanced survival in the presence of NK cells, or a combination thereof. When the engineered T cells are CAR-T cells expressing a chimeric antigen receptor (CAR), disruption of the PTPN2 gene led to enhanced CAR potency, increased in vivo CAR expansion, enhanced CAR-T cell activation, or a combination thereof.

(18) The genetically engineered T cells may be derived from parent T cells (e.g., non-edited wild-type T cells) obtained from a suitable source, for example, one or more mammal donors. In some examples, the parent T cells are primary T cells (e.g., non-transformed and terminally differentiated T cells) obtained from one or more human donors (e.g., healthy donors). Alternatively, the parent T cells may be differentiated from precursor T cells obtained from one or more suitable donor or stem cells such as hematopoietic stem cells or inducible pluripotent stem cells (iPSC), which may be cultured in vitro. In other examples, T cells from a T cell bank can be used as the starting material for preparing the genetically engineered T cells disclosed herein.

(19) In some embodiments, the genetically engineered T cells carry a disrupted PTPN2 gene, and optionally, one or more disrupted genes involved in cell exhaustion (e.g., CD70). Such genetically engineered T cells may further comprise one or more disrupted genes, for example, TRAC and/or 2M. Any of the genetically engineered T cells may be generated via gene editing (including genomic editing), a type of genetic engineering, in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell. Targeted gene editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. Targeted integration refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence is present.

(20) (a) Genetically Edited Genes

(21) In some aspects, the present disclosure provides genetically engineered T cells (e.g., CAR-T cells) that comprise a disrupted PTPN2 gene. In some instances, the genetically engineered T cells disclosed herein may further comprise a disrupted CD70 gene, a disrupted 2M gene, a disrupted TRAC gene, or a combination thereof.

(22) As used herein, a disrupted gene refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, disrupting a gene refers to a method of inserting, deleting or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art and described herein.

(23) In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g., in an immune assay using an antibody binding to the encoded protein or by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell.

(24) PTPN2 Gene Editing

(25) In some embodiments, the genetically engineered T cells may comprise a disrupted PTPN2 gene, which encodes protein tyrosine phosphatase non-receptor type 2 (PTPN2). PTPN2 is a member of the protein tyrosine phosphatase (PTP) family, members of which share a highly conserved catalytic domain. PTPN2 is a signaling molecule that regulates various cellular processes, including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPN2 can dephosphorylate several tyrosine kinases, including receptor protein tyrosine kinases such as INSR, EGFR, CSF1R, PDGFR and non-receptor protein tyrosine kinases such as JAK1, JAK2, JAK3, Src family kinases, STAT1, STAT3 and STAT6, either in the nucleus or the cytoplasm. Some of the tyrosine kinases play roles in cancer-related signaling pathways. As such, PTPN2 is deemed as a tumor suppressor. Veenstra et al., J. Cancer Res. Clin. Oncol. 145(7): 1845-1856 (2019).

(26) Structures of PTPN2 genes are known in the art. For example, human PTPN2 gene is located on chromosome 18p11.21. The gene contains 10 exons. Additional information can be found in GenBank under ENSG00000175354.

(27) In some examples, the genetically engineered T cells may comprise a disrupted PTPN2 gene such that the expression of PTPN2 in the T cells is substantially reduced or eliminated completely. The disrupted PTPN2 gene may comprise one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions) that disrupt expression of the PTPN2 gene. Such target sites may be identified based on the gene editing approach for use in making the genetically engineered T cells. Exemplary target sites for the genetic edits may include exon 1, exon 2, exon 3, or a combination thereof. In some examples, one or more genetic editing may occur in exon 1. In some examples, one or more genetic editing may occur in exon 2. Such genetic editing may be induced by a gene editing technology, (e.g., the CRISPR/Cas technology) using a suitable guide RNA, for example, those targeting the PTPN2 sites listed in Table 3. Exemplary PTPN2-targeting guide RNAs are provided in Table 4, which are also within the scope of the present disclosure.

(28) CD70 Gene Editing

(29) T cell exhaustion is a process of stepwise and progressive loss of T cell functions, which may be induced by prolonged antigen stimulation or other factors. Genes involved in T cell exhaustion refer to those that either positively regulate or negatively regulate this biological process. The genetically engineered T cells disclosed herein may comprise genetic editing of a gene that positively regulates T cell exhaustion to disrupt its expression. Alternatively or in addition, the genetically engineered T cells may comprise genetic editing of a gene that negatively regulates T cell exhaustion to enhance its expression and/or biologic activity of the gene product.

(30) In some embodiments, the genetically engineered T cells may comprise an edited gene involved in T cell exhaustion, e.g., disruption of a gene that positively regulates T cell exhaustion. Such a gene may be a Cluster of Differentiation 70 (CD70) gene. CD70 is a member of the tumor necrosis factor superfamily and its expression is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27. CD70 and its receptor CD27 have multiple roles in immune function in multiple cell types including T cells (activated and T.sub.reg cells), and B cells. In some embodiments, an edited CD70 gene may comprise a nucleotide sequence selected from the following sequences in Table 8. In other embodiments, an edited CD70 gene may have a nucleic acid encoding a CAR (e.g., those disclosed herein) inserted, leading to disruption of the CD70 gene expression.

(31) It was also found that disrupting the CD70 gene in immune cells engineered to express an antigen targeting moiety enhanced anti-tumor efficacy against large tumors and induced a durable anti-cancer memory response. Specifically, the anti-cancer memory response prevented tumor growth upon re-challenge. Further, it has been demonstrated disrupting the CD70 gene results in enhanced cytotoxicity of immune cells engineered to express an antigen targeting moiety at lower ratios of engineered immune cells to target cells, indicating the potential efficacy of low doses of engineered immune cells. See, e.g., WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

(32) Structures of CD70 genes are known in the art. For example, human CD70 gene is located on chromosome 19p13.3. The gene contains four protein encoding exons. Additional information can be found in GenBank under Gene ID: 970.

(33) In some examples, the genetically engineered T cells may comprise a disrupted CD70 gene such that the expression of CD70 in the T cells is substantially reduced or eliminated completely. The disrupted CD70 gene may comprise one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions) that disrupt expression of the CD70 gene. Such target sites may be identified based on the gene editing approach for use in making the genetically engineered T cells. Exemplary target sites for the genetic edits may include exon 1, exon 2, exon 3, exon 4, or a combination thereof. See also WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

(34) 2M Gene Edit

(35) In some embodiments, the genetically engineered T cells disclosed herein may further comprise a disrupted 2M gene. P2M is a common (invariant) component of MHC I complexes. Disrupting its expression by gene editing will prevent host versus therapeutic allogeneic T cells responses leading to increased allogeneic T cell persistence. In some embodiments, expression of the endogenous 2M gene is eliminated to prevent a host-versus-graft response.

(36) In some embodiments, an edited 2M gene may comprise a nucleotide sequence selected from the sequences in Table 7. It is known to those skilled in the art that different nucleotide sequences in an edited gene such as an edited 2M gene (e.g., those in Table 7) may be generated by a single gRNA. See also WO2019097305, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. In other embodiments, an edited 2M gene may have a nucleic acid encoding a CAR (e.g., those disclosed herein) inserted, leading to disruption of the 2M gene expression.

(37) TRAC Gene Edit

(38) In some embodiments, the genetically engineered T cells as disclosed herein may further comprise a disrupted TRAC gene. This disruption leads to loss of function of the TCR and renders the engineered T cell non-alloreactive and suitable for allogeneic transplantation, minimizing the risk of graft versus host disease. In some embodiments, expression of the endogenous TRAC gene is eliminated to prevent a graft-versus-host response. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. In some embodiments, an edited TRAC gene may comprise a nucleotide sequence selected from the following sequences in Table 6. In other embodiments, an edited TRAC gene may have a nucleic acid encoding a CAR (e.g., those disclosed herein) inserted, leading to disruption of the TRAC gene expression.

(39) It should be understood that more than one suitable target site/gRNA can be used for each target gene disclosed herein, for example, those known in the art or disclosed herein. Additional examples can be found in, e.g., WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein.

(40) The genetically engineered T cells disclosed herein may further comprise one or more additional gene edits (e.g., gene knock-in or knock-out) to improve T cell function. Examples include knock-in or knock-out genes to improve target cell lysis, knock-in or knock-out genes to enhance performance of therapeutic T cells such as CAR-T cells prepared from the genetically engineered T cells.

(41) (b) Exemplary Improved Features of Genetically Engineered T Cells Disclosed Herein

(42) Any of the genetically engineered T cell having a disrupted PTPN2 gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted 2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may have improved growth and expansion activities, both in vitro and in vivo, as compared with the non-engineered counterpart, which refers to T cells having the same genetic background except for an undisrupted PTPN2 gene. Further, such genetically engineered T cells (e.g., CAR-T cells) may exhibit enhanced cytotoxicity activity, for example, against undesired cells (e.g., tumor cells) expressing an antigen targeted by the CAR expressed in the CAR-T cells, as compared with the non-engineered counterpart.

(43) In some embodiments, the genetically engineered T cells may further comprise one or more disrupted genes (e.g., CD70) to improve T cell persistency. T cell persistence as used herein refers to the tendency of T cells to continue to grow, proliferate, self-renew, expand, and maintain healthy activity in culture. In some instances, T cell persistence can be represented by the longevity that T cells can grow and expand in vitro, which can be measured by conventional methods and/or assays described herein. In other instances, T cell persistence can be represented by the reduction of cell death (e.g., apoptosis) or reduction in cell states characterized by exhaustion or replicative senescence. In yet other instances, T cell persistence can be presented by the maintenance of T cell activation capacity in culture.

(44) In some examples, the genetically engineered T cells having a disrupted PTPN2 gene, and optionally one or more additional genetic edits as disclosed herein exhibit one or more of the following features: improved cytotoxicity, enhanced persistence; increased cytokine production; enhanced expression of LAGS and/or TIM3; enhanced survival in the presence of NK cells, or a combination thereof. When the engineered T cells are CAR-T cells expressing a chimeric antigen receptor (CAR), disruption of the PTPN2 gene led to enhanced CAR potency, increased CA copy numbers, or a combination thereof. See also Examples below.

(45) (c) Methods of Making Genetically Engineered T Cells

(46) The genetically engineered T cells disclosed herein can be prepared by genetic editing of parent T cells or precursor cells thereof via a conventional gene editing method or those described herein.

(47) (i) T Cells

(48) In some embodiments, T cells for generating the genetically engineered T cells disclosed herein can be derived from one or more suitable mammals, for example, one or more human donors. T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL separation. In some instances, the T cell population comprises primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, not transformed, depend on cytokines and/or growth factors for growth, and/or have stable genomes.

(49) In some examples, T cells can be isolated from a mixture of immune cells (e.g., those described herein) to produce an isolated T cell population. For example, after isolation of peripheral blood mononuclear cells (PBMC), both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.

(50) A specific subpopulation of T cells, expressing one or more of the following cell surface markers: TCR, CD3, CD4, CD8, CD27 CD28, CD38 CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I proteins and/or MCH-II proteins, can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of TCR, CD4 and/or CD8, is further isolated by positive or negative selection techniques. In some embodiments, subpopulations of T cells may be isolated by positive or negative selection prior to genetic engineering and/or post genetic engineering.

(51) An isolated population of T cells may express one or more of the T cell markers, including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof. In some embodiments, the T cells are isolated from a donor, or subject, and first activated and stimulated to proliferate in vitro prior to undergoing gene editing.

(52) In other embodiments, the T cells for use in generating the genetically engineered T cells disclosed herein may be derived from a T cell bank. A T cell bank may comprise T cells with genetic editing of certain genes (e.g., genes involved in cell self renewal, apoptosis, and/or T cell exhaustion or replicative senescence) to improve T cell persistence in cell culture. A T cell bank may be produced from bona fide T cells, for example, non-transformed T cells, terminally differentiated T cells, T cells having stable genome, and/or T cells that depend on cytokines and growth factors for proliferation and expansion. Alternatively, such a T cell bank may be produced from precursor cells such as hematopoietic stem cells (e.g., iPSCs), e.g., in vitro culture. In some examples, the T cells in the T cell bank may comprise genetic editing of one or more genes involved in cell self-renewal, one or more genes involved in apoptosis, and/or one or more genes involved in T cell exhaustion, so as to disrupt or reduce expression of such genes, leading to improved persistence in culture. Examples of the edited genes in a T cell bank include, but are not limited to, Tet2, Fas, CD70, Reg1, or a combination thereof. Compared with the non-edited T counterpart, T cells in a T cell bank may have enhanced expansion capacity in culture, enhanced proliferation capacity, greater T cell activation, and/or reduced apoptosis levels. Additional information of T cell bank may be found in International Application No. PCT/IB2020/058280, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

(53) In yet other embodiments, the T cells for generating the genetically engineered T cells disclosed herein may be derived from stem cells (e.g., HSCs or iPSCs) via in vitro differentiation.

(54) T cells from any suitable source (e.g., those disclosed herein) can be subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. In some embodiments, T cells can be activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of the genome editing compositions into the T cells.

(55) In some embodiments, T cells can be activated and expanded for about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours prior to introduction of the gene editing compositions into the T cells. In some embodiments, T cells are activated at the same time that genome editing compositions are introduced into the T cells. In some instances, the T cell population can be expanded and/or activated after the genetic editing as disclosed herein. T cell populations or isolated T cells generated by any of the gene editing methods described herein are also within the scope of the present disclosure.

(56) (ii) Gene Editing Methods

(57) Any of the genetically engineered T cells can be prepared using conventional gene editing methods or those described herein to edit one or more of the target genes disclosed herein (targeted editing). Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.

(58) Alternatively, the nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare-cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing also utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

(59) In some embodiments, gene disruption may occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95:e52118).

(60) Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases may also be used for targeted integration. Some exemplary approaches are disclosed in detail below.

(61) CRISPR-Cas9 Gene Editing System

(62) The CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon re-introduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

(63) crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5 20 nt in the crRNA allows targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM). tracrRNA hybridizes with the 3 end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

(64) Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB), where both strands of the DNA terminate in a base pair (a blunt end). After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

(65) NHEJ is a robust repair mechanism that appears highly active in the majority of cell types, including non-dividing cells. NHEJ is error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications are typically <20 nt. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operant.

(66) Endonuclease for Use in CRISPR

(67) In some embodiments, the Cas9 (CRISPR associated protein 9) endonuclease is used in a CRISPR method for making the genetically engineered T cells as disclosed herein. The Cas9 enzyme may be one from Streptococcus pyogenes, although other Cas9 homologs may also be used. It should be understood, that wild-type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 may be substituted with another RNA-guided endonuclease, such as Cpf1 (of a class II CRISPR/Cas system).

(68) In some embodiments, the CRISPR/Cas system comprises components derived from a Type-I, Type-II, or Type-III system. Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR/Cas systems have single protein effectors. Cas proteins of Types II, V, and VI are single-protein, RNA-guided endonucleases, herein called Class 2 Cas nucleases. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

(69) In some embodiments, the Cas nuclease is from a Type-II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR/Cas9 system). In some embodiments, the Cas nuclease is from a Class 2 CRISPR/Cas system (a single-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein.

(70) In some embodiments, a Cas nuclease may comprise more than one nuclease domain. For example, a Cas9 nuclease may comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that is capable of introducing a single-stranded break (a nick) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain is substituted to reduce or alter a nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease).

(71) TABLE-US-00001 AminoacidsequenceofCas9 nuclease(SEQIDNO:1): MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNP DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ KGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVV KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD

(72) In some embodiments, the Cas nuclease is from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease is derived from a Type-VI CRISPR/Cas system.

(73) Guide RNAs (gRNAs)

(74) The CRISPR technology involves the use of a genome-targeting nucleic acid that can direct the endonuclease to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be a RNA. A genome-targeting RNA is referred to as a guide RNA or gRNA herein. A guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence.

(75) In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.

(76) As is understood by the person of ordinary skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

(77) In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. A double-molecule guide RNA comprises two strands of RNA molecules. The first strand comprises in the 5 to 3 direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3 tracrRNA sequence and an optional tracrRNA extension sequence.

(78) In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA. A single-molecule guide RNA (referred to as a sgRNA) in a Type II system comprises, in the 5 to 3 direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3 tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins. A single-molecule guide RNA in a Type V system comprises, in the 5 to 3 direction, a minimum CRISPR repeat sequence and a spacer sequence.

(79) A spacer sequence in a gRNA is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest. In some embodiments, the spacer sequence range from 15 to 30 nucleotides. For example, the spacer sequence may contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence contains 20 nucleotides.

(80) The target sequence is in a target gene that is adjacent to a PAM sequence and is the sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The target sequence is on the so-called PAM-strand in a target nucleic acid, which is a double-stranded molecule containing the PAM-strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. For example, if the target sequence is 5-AGAGCAACAGTGCTGTGGCC**-3 (SEQ ID NO: 87), then the gRNA spacer sequence is 5-AGAGCAACAGUGCUGUGGCC**-3 (SEQ ID NO: 79). The spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.

(81) In a CRISPR/Cas system herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5 of a PAM recognizable by a Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5-NRG-3, where R comprises either A or G, where N is any nucleotide and N is immediately 3 of the target nucleic acid sequence targeted by the spacer sequence.

(82) In some embodiments, the target nucleic acid sequence has 20 nucleotides in length. In some embodiments, the target nucleic acid has less than 20 nucleotides in length. In some embodiments, the target nucleic acid has more than 20 nucleotides in length. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5 of the first nucleotide of the PAM. For example, in a sequence comprising 5-NNNNNNNNNNNNNNNNNNNNNRG-3, the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

(83) The guide RNA disclosed herein may target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

(84) For any of the gRNA sequences provided herein, those that do not explicitly indicate modifications are meant to encompass both unmodified sequences and sequences having any suitable modifications.

(85) The length of the spacer sequence in any of the gRNAs disclosed herein may depend on the CRISPR/Cas9 system and components used for editing any of the target genes also disclosed herein. For example, different Cas9 proteins from different bacterial species have varying optimal spacer sequence lengths. Accordingly, the spacer sequence may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence may have 18-24 nucleotides in length. In some embodiments, the targeting sequence may have 19-21 nucleotides in length. In some embodiments, the spacer sequence may comprise 20 nucleotides in length.

(86) In some embodiments, the gRNA can be an sgRNA, which may comprise a 20 nucleotide spacer sequence at the 5 end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a less than 20 nucleotide spacer sequence at the 5 end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a more than 20 nucleotide spacer sequence at the 5 end of the sgRNA sequence. In some embodiments, the sgRNA comprises a variable length spacer sequence with 17-30 nucleotides at the 5 end of the sgRNA sequence. Examples are provided in Table 5 below. In these exemplary sequences, the fragment of n refers to the spacer sequence at the 5 end.

(87) In some embodiments, the sgRNA comprises comprise no uracil at the 3 end of the sgRNA sequence. In other embodiments, the sgRNA may comprise one or more uracil at the 3 end of the sgRNA sequence. For example, the sgRNA can comprise 1-8 uracil residues, at the 3 end of the sgRNA sequence, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3 end of the sgRNA sequence.

(88) Any of the gRNAs disclosed herein, including any of the sgRNAs, may be unmodified. Alternatively, it may contain one or more modified nucleotides and/or modified backbones. For example, a modified gRNA such as an sgRNA can comprise one or more 2-O-methyl phosphorothioate nucleotides, which may be located at either the 5 end, the 3 end, or both.

(89) In certain embodiments, more than one guide RNAs can be used with a CRISPR/Cas nuclease system. Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different.

(90) In some embodiments, the gRNAs disclosed herein target a PTPN2 gene, for example, target a site within any one of exons 1-10, for example, exon 1, exon 2, or exon 3 of the PTPN2 gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 2 of a PTPN2 gene, or a fragment thereof. Exemplary target sequences of PTPN2 and exemplary gRNA sequences are provided in Table 3 and Table 4 below:

(91) In some embodiments, the gRNAs disclosed herein target a CD70 gene, for example, target a site within exon 1 or exon 3 of a CD70 gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 1 or exon 3 of a CD70 gene, or a fragment thereof. Exemplary target sequences in a CD70 gene and exemplary gRNAs specific to the CD70 gene are provided in Table 5 below. See also WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

(92) In some embodiments, the gRNAs disclosed herein target a 2M gene, for example, target a suitable site within a 2M gene. See also WO2019097305, the relevant disclosure s of which are incorporated by reference herein for the purpose and subject matter referenced herein. Other gRNA sequences may be designed using the 2M gene sequence located on Chromosome 15 (GRCh38 coordinates: Chromosome 15: 44,711,477-44,718,877; Ensembl: ENSG00000166710). In some embodiments, gRNAs targeting the 2M genomic region and RNA-guided nuclease create breaks in the 2M genomic region resulting in Indels in the 2M gene disrupting expression of the mRNA or protein. Exemplary spacer sequences and gRNAs targeting a 2M gene are provided in Table 5 below.

(93) In some embodiments, the gRNAs disclosed herein target a TRAC gene. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the subject matter and purpose referenced herein. Other gRNA sequences may be designed using the TRAC gene sequence located on chromosome 14 (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734). In some embodiments, gRNAs targeting the TRAC genomic region and RNA-guided nuclease create breaks in the TRAC genomic region resulting Indels in the TRAC gene disrupting expression of the mRNA or protein. Exemplary spacer sequences and gRNAs targeting a TRAC gene are provided in Table 5 below.

(94) By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system, or other smaller RNAs can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art.

(95) In some examples, the gRNAs of the present disclosure can be are produced in vitro transcription (IVT), synthetic and/or chemical synthesis methods, or a combination thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods are utilized. In one embodiment, the gRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in WO2013/151666. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors are used to in vitro transcribe a gRNA described herein.

(96) Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs disclosed herein during synthesis or post-synthesis. In certain embodiments, modifications are on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification is introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in WO2013/052523. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

(97) In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

(98) In some embodiments of the present disclosure, a CRISPR/Cas nuclease system for use in genetically editing any of the target genes disclosed here may include at least one guide RNA. In some examples, the CRISPR/Cas nuclease system may contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. Such multiple gRNAs may target different sites in a same target gene. Alternatively, the multiple gRNAs may target different genes. In some embodiments, the guide RNA(s) and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. The guide RNA(s) may guide the Cas protein to a target sequence(s) on one or more target genes as those disclosed herein, where the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR/Cas complex is a Cpf1/guide RNA complex. In some embodiments, the CRISPR complex is a Type-II CRISPR/Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex.

(99) In some embodiments, the indel frequency (editing frequency) of a particular CRISPR/Cas nuclease system, comprising one or more specific gRNAs, may be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA yields a gene editing frequency of higher than 80%. For example, a gRNA is considered to be highly efficient if it yields a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

(100) Delivery of Guide RNAs and Nucleases to T Cells

(101) The CRISPR/Cas nuclease system disclosed herein, comprising one or more gRNAs and at least one RNA-guided nuclease, optionally a donor template as disclosed below, can be delivered to a target cell (e.g., a T cell) for genetic editing of a target gene, via a conventional method. In some embodiments, components of a CRISPR/Cas nuclease system as disclosed herein may be delivered to a target cell separately, either simultaneously or sequentially. In other embodiments, the components of the CRISPR/Cas nuclease system may be delivered into a target together, for example, as a complex. In some instances, gRNA and a RNA-guided nuclease can be pre-complexed together to form a ribonucleoprotein (RNP), which can be delivered into a target cell.

(102) RNPs are useful for gene editing, at least because they minimize the risk of promiscuous interactions in a nucleic acid-rich cellular environment and protect the RNA from degradation. Methods for forming RNPs are known in the art. In some embodiments, an RNP containing an RNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) and one or more gRNAs targeting one or more genes of interest can be delivered a cell (e.g., a T cell). In some embodiments, an RNP can be delivered to a T cell by electroporation.

(103) In some embodiments, an RNA-guided nuclease can be delivered to a cell in a DNA vector that expresses the RNA-guided nuclease in the cell. In other examples, an RNA-guided nuclease can be delivered to a cell in an RNA that encodes the RNA-guided nuclease and expresses the nuclease in the cell. Alternatively or in addition, a gRNA targeting a gene can be delivered to a cell as a RNA, or a DNA vector that expresses the gRNA in the cell.

(104) Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be through direct injection or cell transfection using known methods, for example, electroporation or chemical transfection. Other cell transfection methods may be used.

(105) Other Gene Editing Methods

(106) Besides the CRISPR method disclosed herein, additional gene editing methods as known in the art can also be used in making the genetically engineered T cells disclosed herein. Some examples include gene editing approaching involve zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, and the like.

(107) ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496. A selected zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the FokI nuclease with a zinc finger DNA binding domain.

(108) A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A transcription activator-like effector DNA binding domain, TAL effector DNA binding domain, or TALE DNA binding domain is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). TALENs are described in greater detail in US Patent Application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.

(109) Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and WWSPBc/TP901-1, whether used individually or in combination.

(110) Any of the nucleases disclosed herein may be delivered using a vector system, including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.

(111) Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor templates in cells (e.g., T cells). Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

(112) Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Some specific examples are provided below.

(113) II. Genetically Engineered T Cells Expressing a Chimeric Antigen Receptor (CAR)

(114) The genetically engineered T cells having a disrupted PTPN2 gene and optionally one or more of additional disrupted genes, e.g., 2M, TRAC, CD70, or a combination thereof as disclosed herein, may further express a chimeric antigen receptor (CAR) targeting an antigen of interest or cells expressing such an antigen.

(115) (a) Chimeric Antigen Receptor (CAR)

(116) A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by undesired cells, for example, disease cells such as cancer cells. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed on T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains. There are various generations of CARs, each of which contains different components.

(117) First generation CARs join an antibody-derived scFv to the CD3zeta or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional co-stimulatory domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3 chain. Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2):151-155). Any of the various generations of CAR constructs is within the scope of the present disclosure.

(118) Generally, a CAR is a fusion polypeptide comprising an extracellular domain that recognizes a target antigen (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain comprising a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3) and, in most cases, a co-stimulatory domain. (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CAR construct may further comprise a hinge and transmembrane domain between the extracellular domain and the intracellular domain, as well as a signal peptide at the N-terminus for surface expression. Examples of signal peptides include SEQ ID NO: 113 and SEQ ID NO: 114 as provided in Table 9 below. Other signal peptides may be used.

(119) (i) Antigen Binding Extracellular Domain

(120) The antigen-binding extracellular domain is the region of a CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on cell surface. In some instances, a signal peptide may be located at the N-terminus to facilitate cell surface expression. In some embodiments, the antigen binding domain can be a single-chain variable fragment (scFv, which may include an antibody heavy chain variable region (V.sub.H) and an antibody light chain variable region (V.sub.L) (in either orientation). In some instances, the V.sub.H and V.sub.L fragment may be linked via a peptide linker. The linker, in some embodiments, includes hydrophilic residues with stretches of glycine and serine for flexibility as well as stretches of glutamate and lysine for added solubility. The scFv fragment retains the antigen-binding specificity of the parent antibody, from which the scFv fragment is derived. In some embodiments, the scFv may comprise humanized V.sub.H and/or V.sub.L domains. In other embodiments, the V.sub.H and/or V.sub.L domains of the scFv are fully human.

(121) The antigen-binding extracellular domain may be specific to a target antigen of interest, for example, a pathologic antigen such as a tumor antigen (e.g., a solid tumor antigen). In some embodiments, a tumor antigen is a tumor associated antigen, referring to an immunogenic molecule, such as a protein, that is generally expressed at a higher level in tumor cells than in non-tumor cells, in which it may not be expressed at all, or only at low levels. In some embodiments, tumor-associated structures, which are recognized by the immune system of the tumor-harboring host, are referred to as tumor-associated antigens. In some embodiments, a tumor-associated antigen is a universal tumor antigen, if it is broadly expressed by most types of tumors. In some embodiments, tumor-associated antigens are differentiation antigens, mutational antigens, overexpressed cellular antigens or viral antigens. In some embodiments, a tumor antigen is a tumor specific antigen or TSA, referring to an immunogenic molecule, such as a protein, that is unique to a tumor cell. Tumor specific antigens are exclusively expressed in tumor cells, for example, in a specific type of tumor cells.

(122) Exemplary tumor antigens include, but are not limited to, CD19, BCMA, and CD70. Any known antibodies specific to such tumor antigens, for example, those approved for marketing and those in clinical trials, can be used for making the CAR constructs disclosed herein. Non-limiting examples of CAR constructs are provided in WO2019097305 and WO2019215500, and WO2020/095107, the relevant disclosures of which are herein incorporated by reference for the purposes and subject matter referenced herein.

(123) (ii) Transmembrane Domain

(124) The CAR polypeptide disclosed herein may contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability of the CAR containing such.

(125) In some embodiments, the transmembrane domain of a CAR as provided herein can be a CD8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain. In yet other embodiments, the transmembrane domain is a chimera of a CD8 and CD28 transmembrane domain. Other transmembrane domains may be used as provided herein. In some embodiments, the transmembrane domain is a CD8a transmembrane domain containing the sequence of SEQ ID NO: 115 as provided below in Table 9. Other transmembrane domains may be used.

(126) (iii) Hinge Domain

(127) In some embodiments, a hinge domain may be located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR. A hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain in the polypeptide chain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof.

(128) In some embodiments, a hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domain(s) may be included in other regions of a CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

(129) (iv) Intracellular Signaling Domains

(130) Any of the CAR constructs contain one or more intracellular signaling domains (e.g., CD3, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

(131) CD3 is the cytoplasmic signaling domain of the T cell receptor complex. CD3 contains three (3) immunoreceptor tyrosine-based activation motif (ITAM)s, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In many cases, CD3 provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signaling.

(132) In some embodiments, the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains. For example, the co-stimulatory domains of CD28 and/or 4-1BB may be used to transmit a full proliferative/survival signal, together with the primary signaling mediated by CD3. In some examples, the CAR disclosed herein comprises a CD28 co-stimulatory molecule. In other examples, the CAR disclosed herein comprises a 4-1BB co-stimulatory molecule. In some embodiments, a CAR includes a CD3 signaling domain and a CD28 co-stimulatory domain. In other embodiments, a CAR includes a CD3 signaling domain and 4-1BB co-stimulatory domain. In still other embodiments, a CAR includes a CD3 signaling domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

(133) Table 9 provides examples of signaling domains derived from 4-1BB, CD28 and CD3-zeta that may be used herein.

(134) In specific examples, the anti-CD19 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 135, which may be encoded by the nucleotide sequence of SEQ ID NO: 134. In other examples, the anti-BCMA CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 163, which may be encoded by the nucleotide sequence of SEQ ID NO: 162. In other examples, the anti-CD70 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 155, which may be encoded by the nucleotide sequence of SEQ ID NO: 190. See sequence Table 9 provided below.

(135) (b) Delivery of CAR Construct to T Cells

(136) In some embodiments, a nucleic acid encoding a CAR can be introduced into any of the genetically engineered T cells disclosed herein by methods known to those of skill in the art. For example, a coding sequence of the CAR may be cloned into a vector, which may be introduced into the genetically engineered T cells for expression of the CAR. A variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into an immune effector cell. Non-limiting examples of methods for introducing nucleic acid into a cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.

(137) In specific examples, a nucleic acid encoding a CAR construct can be delivered to a cell using an adeno-associated virus (AAV). AAVs are small viruses which integrate site-specifically into the host genome and can therefore deliver a transgene, such as CAR. Inverted terminal repeats (ITRs) are present flanking the AAV genome and/or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids which confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, the AAV for use in delivering the CAR-coding nucleic acid is AAV serotype 6 (AAV6).

(138) Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy.

(139) A nucleic acid encoding a CAR can be designed to insert into a genomic site of interest in the host T cells. In some embodiments, the target genomic site can be in a safe harbor locus.

(140) In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a genomic site of interest. In some instances, the nucleic acid may comprise a left homologous arm and a right homologous arm flanking the nucleotide sequence encoding the CAR. The left and right homologous arms are homologous to the upstream and downstream sequences of the genomic site where the CAR-coding sequence is to be inserted. In some examples, the genomic site where the CAR-coding sequence is to be inserted is also the target site of a guide RNA such that the CAR-coding nucleic acid can be inserted at the guide RNA targeting site. In some examples, the left homologous arm and the right homologous arm may be homologous to the sequences immediately flank the guide RNA targeting site. In some instances, the guide RNA targeting site can be deleted and replaced by the CAR-encoding nucleic acid after gene editing.

(141) In some embodiments, a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a TRAC gene to disrupt the TRAC gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of TRAC leads to loss of function of the endogenous TCR. For example, a disruption in the TRAC gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more TRAC genomic regions. Any of the gRNAs specific to a TRAC gene and the target regions disclosed herein can be used for this purpose.

(142) In some examples, a genomic deletion in the TRAC gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the TRAC gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more TRAC genomic regions and inserting a CAR coding segment into the TRAC gene.

(143) A donor template as disclosed herein can contain a coding sequence for a CAR. In some examples, the CAR-coding sequence may be flanked by two regions of homology to allow for efficient HDR at a genomic location of interest, for example, at a TRAC gene using a gene editing method known in the art. In some examples, a CRISPR-based method can be used. In this case, both strands of the DNA at the target locus can be cut by a CRISPR Cas9 enzyme guided by gRNAs specific to the target locus. HDR then occurs to repair the double-strand break (DSB) and insert the donor DNA coding for the CAR. For this to occur correctly, the donor sequence is designed with flanking residues which are complementary to the sequence surrounding the DSB site in the target gene (hereinafter homology arms), such as the TRAC gene. These homology arms serve as the template for DSB repair and allow HDR to be an essentially error-free mechanism. The rate of homology directed repair (HDR) is a function of the distance between the mutation and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing.

(144) In other embodiments, the nucleic acid encoding the CAR may be inserted at a different genomic site, for example, at the disrupted CD70 locus or the disrupted 2M locus via the same CRISPR/Cas9-mediated gene editing and homologous recombination approach disclosed above.

(145) Alternatively, a donor template may have no regions of homology to the targeted location in the DNA and may be integrated by NHEJ-dependent end joining following cleavage at the target site.

(146) A donor template can be DNA or RNA, single-stranded and/or double-stranded, and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3 terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

(147) A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, a donor template can be introduced into a cell as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

(148) A donor template, in some embodiments, can be inserted at a site nearby an endogenous prompter (e.g., downstream or upstream) so that its expression can be driven by the endogenous promoter. In other embodiments, the donor template may comprise an exogenous promoter and/or enhancer, for example, a constitutive promoter, an inducible promoter, or tissue-specific promoter to control the expression of the CAR gene. In some embodiments, the exogenous promoter is an EF1 promoter, see, e.g., SEQ ID NO: 184 provided in Table 10 below. Other promoters may be used.

(149) Furthermore, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

(150) When needed, additional gene editing (e.g., gene knock-in or knock-out) can be introduced into therapeutic T cells as disclosed herein to improve T cell function and therapeutic efficacy. For example, if 2M knockout can be performed to reduce the risk of or prevent a host-versus-graft response. Other examples include knock-in or knock-out genes to improve target cell lysis, knock-in or knock-out genes to enhance performance of therapeutic T cells such as CAR-T cells.

(151) In some embodiments, a donor template for delivering an anti-CD19 CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-CD19 CAR, and optionally regulatory sequences for expression of the anti-CD19 CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 87). In some specific examples, the donor template for delivering the anti-CD19 CAR may comprise a nucleotide sequence of SEQ ID NO: 185, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 87.

(152) In some embodiments, a donor template for delivering an anti-BCMA CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-BCMA CAR, and optionally regulatory sequences for expression of the anti-BCMA CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 87. In some specific examples, the donor template for delivering the anti-BCMA CAR may comprise a nucleotide sequence of SEQ ID NO: 187, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 87.

(153) In some embodiments, a donor template for delivering an anti-CD70 CAR may be an AAV vector inserted with a nucleic acid fragment comprising the coding sequence of the anti-CD70 CAR, and optionally regulatory sequences for expression of the anti-CD70 CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table), which can be flanked by homologous arms for inserting the coding sequence and the regulatory sequences into a genomic locus of interest. In some examples, the nucleic acid fragment is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 87. In some specific examples, the donor template for delivering the anti-CD70 CAR may comprise a nucleotide sequence of SEQ ID NO: 186, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 87.

(154) The genetically engineered T cells having a disrupted PTPN2 gene, one or more additional disrupted genes, e.g., 2M, TRAC, and/or CD70, and further expressing a chimeric antigen receptor (CAR) can be produced by sequential targeting of the genes of interest. For example, in some embodiments, the PTPN2 gene may be disrupted first, followed by disruption of TRAC, and/or 2M genes and CAR insertion. In other embodiments, TRAC and 2M genes may be disrupted first, followed by CAR insertion and disruption of the PTPN2 gene. In other embodiments, CD70 may be disrupted first, followed by TRAC and 2M genes disruption and CAR insertion, as well as disruptions of the PTPN2 gene. Accordingly, in some embodiments, the genetically engineered T cells disclosed herein may be produced by multiple, sequential electroporation events with multiple RNPs targeting the genes of interest, e.g., PTPN2, 2M, TRAC, and CD70 etc.

(155) In other embodiments, the genetically engineered CAR T cells disclosed herein may be produced by a single electroporation event with an RNP complex comprising an RNA-guided nuclease and multiple gRNAs targeting the genes of interest, e.g., PTPN2, 2M, TRAC, and CD70.

(156) (c) Exemplary Genetically Engineered T Cells Expressing a Chimeric Antigen Receptor

(157) It should be understood that gene disruption encompasses gene modification through gene editing (e.g., using CRISPR/Cas gene editing to insert or delete one or more nucleotides). A disrupted gene may contain one or more mutations (e.g., insertion, deletion, or nucleotide substitution, etc.) relative to the wild-type counterpart so as to substantially reduce or completely eliminate the activity of the encoded gene product. The one or more mutations may be located in a non-coding region, for example, a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, the one or more mutations may be located in a coding region (e.g., in an exon). In some instances, the disrupted gene does not express or expresses a substantially reduced level of the encoded protein. In other instances, the disrupted gene expresses the encoded protein in a mutated form, which is either not functional or has substantially reduced activity. In some embodiments, a disrupted gene is a gene that does not encode functional protein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g. by antibody, e.g., by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell. For example, a cell having a 2M gene edit may be considered a 2M knockout cell if 2M protein cannot be detected at the cell surface using an antibody that specifically binds 2M protein.

(158) In some embodiments, the genetically engineered immune cells (e.g., T cells such as human T cells) may comprise a disrupted PTPN2 gene, a disrupted TRAC gene, a disrupted 2M gene, and express an anti-CD19 CAR, e.g., those disclosed herein (anti-CD19 CAR-T cells). In some examples, the population of anti-CD19 CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-CD19 CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-CD19 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 87) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD19 CAR (e.g., SEQ ID NO: 185). Alternatively or in addition, the population of anti-CD19 CAR T cells may comprise a disrupted 2M gene via CRISPR/Cas9 technology using the gRNA of 2M-1. Table 5. Such anti-CD19 CAR T cells may comprise Indels in the 2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 101-106. See Table 7. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

(159) In some embodiments, the genetically engineered immune cells (e.g., T cells such as human T cells) may comprise a disrupted PTPN2 gene, a disrupted TRAC gene, a disrupted 2M gene, and express an anti-BCMA CAR, e.g., those disclosed herein (anti-BCMA CAR-T cells). In some examples, the population of anti-BCMA CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-BCMA CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-BCMA CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 87) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-BCMA CAR (e.g., SEQ ID NO: 187). Alternatively or in addition, the population of anti-BCMA CAR T cells may comprise a disrupted 2M gene via CRISPR/Cas9 technology using the gRNA of 2M-1. Table 5. Such anti-BCMA CAR T cells may comprise Indels in the 2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 101-106. See Table 7. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

(160) In some embodiments, the genetically engineered immune cells (e.g., T cells such as human T cells) may comprise a disrupted PTPN2 gene, a disrupted TRAC gene, a disrupted 2M gene, a disrupted CD70 gene, and express an anti-CD70 CAR, e.g., those disclosed herein (anti-CD70 CAR-T cells). In some examples, the population of anti-CD70 CAR T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the anti-BCMA CAR T cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the anti-CD70 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 87) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD70 CAR (e.g., SEQ ID NO: 186). Alternatively or in addition, the population of anti-CD70 CAR T cells may comprise a disrupted 2M gene via CRISPR/Cas9 technology using the gRNA of 2M-1. Such anti-CD70 CAR T cells may comprise Indels in the 2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 101-106. See Table 7. See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein. Further, the population of anti-CD70 CAR T cells may comprise a disrupted CD70 gene via CRISPR/Cas9 technology using a gRNA targeting the CD70 locus, for example, CD70-7. See Table 5. Such anti-CD70 CAR T cells may comprise Indels in the CD70 gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 107-112. See Table 8. See also WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

(161) III. Therapeutic Applications

(162) The therapeutic T cells disclosed herein can be administered to a subject for therapeutic purposes, for example, treatment of a tumor such as a solid tumor targeted by the CAR construct expressed by the therapeutic CAR-T cells. As reported herein, disruption of the PTPN2 gene led to improved T cell persistence, increased cytokine secretion, enhanced CAR potency and/or CAR copy numbers, etc., leading to improved anti-tumor efficacy as observed in animal models.

(163) The step of administering may include the placement (e.g., transplantation) of the therapeutic T cells into a subject by a method or route that results in at least partial localization of the therapeutic T cells at a desired site, such as a tumor site, such that a desired effect(s) can be produced. Therapeutic T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the subject, i.e., long-term engraftment. For example, in some aspects described herein, an effective amount of the therapeutic T cells can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

(164) In some embodiments, the therapeutic T cells are administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous.

(165) A subject may be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some instances, the human patient has a cancer involving CD19.sup.+ cancer cells (e.g., B cell malignancy such as B-cell leukemia, non-Hodgkin lymphoma, e.g., diffuse large B cell lymphoma (DLBCL), B cell lymphoma, or transformed follicular lymphoma, or T cell malignancy). CAR-T cells expressing an anti-CD19 CAR (e.g., disclosed herein) may be used to treat such a patient. In some instances, the human patient has a cancer involving BCMA.sup.+ cancer cells (e.g., multiple myeloma). CAR-T cells expressing an anti-BCMA CAR (e.g., disclosed herein) may be used to treat such a patient. In some instances, the human patient has a CD70.sup.+ hematological tumor (e.g., cutaneous T cell lymphoma, peripheral T-cell lymphoma, or T cell leukemia) or a solid tumor (e.g., renal cell carcinoma). CAR-T cells expressing an anti-CD70 CAR (e.g., disclosed herein) may be used to treat such a patient.

(166) In some instances, the therapeutic T cells may be autologous (self) to the subject, i.e., the cells are from the same subject. Alternatively, the therapeutic T cells can be non-autologous (non-self, e.g., allogeneic, syngeneic or xenogeneic) to the subject. Allogeneic means that the therapeutic T cells are not derived from the subject who receives the treatment but from different individuals (donors) of the same species as the subject. A donor is an individual who is not the subject being treated. A donor is an individual who is not the patient. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors, e.g., two or more donors, are used.

(167) In some embodiments, an engineered T cell population being administered according to the methods described herein comprises allogeneic T cells obtained from one or more donors. Allogeneic refers to a cell, cell population, or biological samples comprising cells, obtained from one or more different donors of the same species, where the genes at one or more loci are not identical to the recipient (e.g., subject). For example, an engineered T cell population, being administered to a subject can be derived from one or more unrelated donors, or from one or more non-identical siblings. In some embodiments, syngeneic cell populations may be used, such as those obtained from genetically identical donors, (e.g., identical twins). In some embodiments, the cells are autologous cells; that is, the engineered T cells are obtained or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same.

(168) An effective amount refers to the amount of a population of engineered T cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject having a medical condition. An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

(169) The efficacy of a treatment using the therapeutic T cells disclosed herein can be determined by the skilled clinician. A treatment is considered effective, if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease (e.g., cancer) are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

(170) Combination therapies are also encompassed by the present disclosure. For example, the therapeutic T cells disclosed herein may be co-used with other therapeutic agents, for treating the same indication, or for enhancing efficacy of the therapeutic T cells and/or reducing side effects of the therapeutic T cells.

(171) IV. Kits

(172) The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses,

(173) In some embodiments, a kit provided herein may comprise components for performing genetic edit of a PTPN2 gene, and optionally components for editing one or more additional genes, including TRAC gene, 2M gene, and/or CD70 gene. The kit may also comprise a population of immune cells to which the genetic editing will be performed (e.g., a leukopak or a T cell bank). A leukopak sample may be an enriched leukapheresis product collected from peripheral blood. It typically contains a variety of blood cells including monocytes, lymphocytes, platelets, plasma, and red cells. The components for genetically editing one or more of the target genes may comprise a suitable endonuclease such as an RNA-guided endonuclease and one or more nucleic acid guides, which direct cleavage of one or more suitable genomic sites by the endonuclease. For example, the kit may comprise a Cas enzyme such as Cas 9 and one or more gRNAs targeting PTPN2, and optionally gRNAs targeting TRAC, 2M, and/or a CD70. Any of the gRNAs specific to these target genes can be included in the kit.

(174) In some embodiments, a kit provided herein may comprise a population of genetically engineered T cells as disclosed herein, and one or more components for producing the therapeutic T cells as also disclosed herein. Such components may comprise an endonuclease suitable for gene editing and a nucleic acid coding for a CAR construct of interest. The CAR-coding nucleic acid may be part of a donor template as disclosed herein, which may contain homologous arms flanking the CAR-coding sequence. In some instances, the donor template may be carried by a viral vector such as an AAV vector. The kit may further comprise gRNAs specific to a TRAC gene for inserting the CAR-coding sequence into the TRAC gene.

(175) In yet other embodiments, the kit disclosed herein may comprise a population of therapeutic T cells as disclosed for the intended therapeutic purposes.

(176) Any of the kit disclosed herein may further comprise instructions for making the therapeutic T cells, or therapeutic applications of the therapeutic T cells. In some examples, the included instructions may comprise a description of using the gene editing components to genetically engineer one or more of the target genes (e.g., TRAC, 2M, CD70, or a combination thereof). In other examples, the included instructions may comprise a description of how to introduce a nucleic acid encoding a CAR construction into the T cells for making therapeutic T cells.

(177) In some embodiments, a kit as disclosed herein may comprise a population of genetically engineered T cells (e.g., CAR-T cells) for use to eliminate undesired cells targeted by the CAR construct (e.g., for treatment of cancer such as a solid tumor). Such a kit may comprise one or more containers in which the genetically engineered T cells can be placed. The kit may further comprise instructions for administration of the therapeutic T cells as disclosed herein to achieve the intended activity, e.g., eliminating disease cells targeted by the CAR expressed on the therapeutic T cells. Alternatively or in addition, the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions relating to the use of the therapeutic T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the therapeutic T cells are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.

(178) The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device for administration of the therapeutic T cells. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

(179) Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

(180) General Techniques

(181) The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984; Animal Cell Culture (R. I. Freshney, ed. (1986; Immobilized Cells and Enzymes (IRL Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).

(182) Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

Example 1. Screening of PTPN2 Targeting Site for CRISPR/Cas-Mediated Gene Editing

(183) This example discloses screening of exemplary guide RNAs for knocking out PTPN2 by CRISPR/Cas-mediated gene editing.

(184) (A) Efficient Knockout of PTPN2 by Cas9:sgRNA RNPs in T Cells

(185) The PTPN2 gene was efficiently edited in primary human T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segments of the PTPN2 gene containing the six (6) protein coding exons were used as input in gRNA design software. Desired gRNAs were those that would lead to insertions or deletions in the coding sequence, disrupting the amino acid sequence of PTPN2, leading to out-of-frame/loss-of-function allele(s) (referred to as PTPN2 knockout (KO) alleles). All twelve (12) in silico-identified gRNA spacer sequences targeting the PTPN2 gene (targeting sites provided in Table 3) were synthesized, and the gRNAs were specifically modified, as indicated in Table 4. While the gRNAs in Table 4 were modified with 2-O-methyl phosphorothioate modifications, unmodified gRNAs, or gRNAs with other modifications, may be used (see also Table 4).

(186) Primary human T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the PTPN2 gene (sequences in Table 4) or controls (no Cas9, no gRNA). Five (5) days post transfection, cells were subjected to a TIDE analysis to assess indel frequency. Knockout of PTPN2 was also confirmed by Western blot protein analysis. All twelve guides significantly inhibited PTPN2 protein expression compared to the control, wild type T cells without the knockout (FIG. 1).

(187) (B) On-Target and Off-Target Editing of PTPN2 Guide RNAs

(188) On-target and off-target editing efficiencies of various PTPN2-targeting gRNAs noted above were examined following the method disclosed herein. Briefly, activated T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the PTPN2 gene (target sequences in Table 4) or controls (no Cas9, no gRNA).

(189) For genomic on- and off-target assessment, these electroporation methods were used to generate two cell populations of edited cells from two different donor T cells. Cells were gene edited with each of the twelve guides noted above, and then harvested ten (10) days post transfection. These samples were analyzed with hybrid capture, a homology-dependent method to enrich on- and off-target sites, combined with next-generation sequencing. Briefly, on- and off-target sites with homology to each gRNA target site were identified computationally, single-stranded RNA probes were used to enrich these sites from bulk genomic DNA, these enriched sites were sequenced with next-generation sequencing, and the data were analyzed for insertions and deletions (indels) indicating repair following CRISPR editing. Three (3) gRNAs showed no off-target effect with an on-target editing rate greater than 96%, which includes PTPN2-10, PTPN2-11 and PTPN2-13 as shown in Table 1 below.

(190) TABLE-US-00002 TABLE 1 Off-target analysis of gRNAs that target PTPN-2 Number of On-target predicted mean off target editing Name sites tested hyb cap .sup.a Detected off-targets PTPN2-1 102 98.9% Two 1-10% off-targets PTPN2-2 129 97.2% One >40% off-target PTPN2-3 339 96.4% Two <1% off-targets PTPN2-4 234 97.0% Two >50% off-targets PTPN2-5 346 97.6% One 1-10% off-target; five <1% off-targets PTPN2-10 7 98.4% None PTPN2-11 8 98.3% None PTPN2-12 17 94.6% Two <1% off-target PTPN2-13 17 96.9% None PTPN2-14 33 98.8% Two >10% off-targets PTPN2-15 70 98.9% One >10% off-target; one <1% off-target PTPN2-16 31 96.9% Two 1-10% off-targets; one <1% off-target .sup.a Average across donors 1 and 2.

Example 2: Cell Killing Function of Anti-CD70 CAR T Cells with PTPN2 Knock-Out

(191) Allogeneic human T cells that lack expression of the TRAC gene, 2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were generated. The edited CAR T cells further comprised knock out of PTPN2 gene. As in the examples above, activated human T cells were electroporated with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000), and Cas9:sgRNA RNPs (1 M Cas9, 5 M gRNA).

(192) Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 190 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 155). The following sgRNAs were used: TRAC (SEQ ID NO: 76), 2M (SEQ ID NO: 80), CD70 (SEQ ID NO:72).

(193) One week post-electroporation, T cells were analyzed for CAR expression by flow cytometry and PTPN2 protein expression by western blot protein analysis (Figure. 2). Both anti-CD70 CAR T cells with unedited PTPN2 and anti-CD70 CAR T cells that lack PTPN2 (using PTPN2-1 gRNA targeting: GGAAACTTGGCCACTCTATG; SEQ ID NO: 2) expressed the anti-CD70 CAR on their surface at day 7 post HDR at 69.6% and 62.9%, respectively, as determined by FACS.

(194) In Vitro Cell Killing Function of Anti-CD70 CAR T Cells with PTPN2 Knock-Out

(195) A cell killing assay was used to assess the ability of the TRAC/2M/CD70/PTPN2/anti-CD70 CAR+ cells to kill CD70+ adherent renal cell carcinoma (RCC)-derived cell lines (e.g.: Caki-1, or A498 cell lines). Adherent cells were seeded in 96-well plates at 50,000 cells per well and incubated overnight at 37 C. in cytokine-free medium. The next day edited anti-CD70 CAR T cells (cultured until day 12 post HDR or day 27 post HDR) were added to the wells containing target cells at 0.25:1, 1:1 or 4:1 CAR T: Target cell ratios. After 24 hours co-culture, CAR T cells were removed from the culture by aspiration and 100 L Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable target cells. The amount of light emitted per well was then quantified using a plate reader.

(196) Anti-CD70 CAR-T cells with PTPN2 knock out exhibited a more potent cell killing of RCC-derived cell lines, CAKI-1 and A498, following 24-hour co-incubation (FIGS. 3A-3B). This suggests that knocking-out the PTPN2 gene gives a maintained/persistent higher cell kill potency compared to anti-CD70 CAR+ T cells with an intact PTPN2 gene over time post HDR.

Example 3. PTPN2 Knockout and CAR-T Cytokine Secretion

(197) To determine the effect of knocking out PTPN2 on cytokine secretion by anti-CD70 CAR-T cells when engaged with tumor cells, cell culture supernatants from the co-culture experiment described in Example 2 were harvested and analyzed for secreted cytokines using the BIOLEGEND Legendplex system. CAR-T cells with PTPN2 KO secreted higher levels of cytokines such as IFN-g, TNF-, IL-10, Granzyme A and Perforin. This was observed against both A498 cells (FIGS. 4A-4H) and CAKI-1 cells (FIGS. 5A-5F).

Example 4. Effect of PTPN2 Knockout on Exhaustion Marker Expression

(198) The levels of the T cell exhaustion markers, PD1, LAG3, TIGIT and TIM3 were also assessed on TRAC/2M/CD70/anti-CD70 CAR+ and TRACVM/CD70/PTPN2/anti-CD70 CAR+ cells. Both CD4+ and CD8+ T cells were assessed by flow cytometry at Day 36 post HDR (FIGS. 6A and 6B).

(199) PTPN2 KO did not significantly alter expression of PD1 and TIGIT in CD4 T cells. However, PTPN2 KO elevated expression of LAG3 and TIM3 in both CD4+ and CD8+ CAR-T cells. This suggests that enhanced potency of the CAR T cells is not via reducing expression of exhaustion markers, PD1, LAG3 and TIM3. In fact, enhanced TIM3 expression via knocking out PTPN2 may contribute to potency enhancement of the anti-CD70 CAR-T cells.

Example 5. Effect of PTPN2 KO on Phosphorylation of Downstream Targets

(200) As a phosphatase, PTPN2 dephosphorylates target proteins such as LCK, FYN, STAT5, JAK and c-JUN, to modulate T cell activation and function. For example, phosphorylation of STAT5 and JAK induces cytokine secretion in activated T cells. To assess whether knockout of PTPN2 enhances phosphorylation of some of its target proteins, anti-CD70 CAR-T cells with and without PTPN2 KO were cultured in serum- and cytokine-free medium for 24 hours. Cells were then processed immediately or stimulated for 20 minutes at 37 degrees Celsius with 50 ng/ml concentration of IL-2, followed by protein isolation with RIPA buffer containing protease and phosphatase inhibitors. Protein analysis was conducted by western blot assay using antibodies specific for the phosphorylated forms of STAT5 and JUN proteins. As a control, GAPDH protein was also assessed to ensure equal amount of protein in samples with and without PTPN2 KO to avoid erroneous comparison of protein levels. Results showed that PTPN-2 KO CAR-T cells have elevated phospho-STAT5 and phospho-JUN expression compared to unstimulated samples; however, this difference was not detected in IL-2-stimulated samples. FIG. 7. These results suggest that knocking out PTPN2 interferes with its dephosphorylation function leading to increased phosphorylation of target proteins such as STAT5 and JUN which may promote optimal T cell activation, cytokine secretion and anti-tumor activity.

Example 6: In Vivo Effect of PTPN2 KO on Allogeneic Anti-CD70 CAR-T Cells in the CAKI-1 Human Renal Cell Carcinoma Tumor Xenograft Model in NSG Mice

(201) A xenograft mouse model was utilized to further assess the in vivo efficacy of allogeneic CART cells lacking (32M and TRAC, as well as PTPN2. The CAKI-1 human renal cell carcinoma tumor xenograft in NSG mice was used to demonstrate the efficacy of TRAC/2M/anti-CD70 CAR+ T cells (anti-CD70 CAR T cells) with or without editing of the PTPN2 locus. The PTPN2 gene was edited via CRISPR/Cas-mediated gene editing using PTPN2-1 (comprising the spacer of GGAAACUUGGCCACUCUAUG; SEQ ID NO: 28) guide RNA.

(202) Efficacy of the anti-CD70 CAR T cells was evaluated in the xenograft model using methods employed by Translations Drug Development, LLC (Scottsdale, AZ) and described herein. In brief, 5-8 week old female NSG mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 3 treatment groups of 5 mice each, as shown in Table 2. The mice were inoculated with 510.sup.6 CAKI-1 cells and monitored until tumor sizes of 150 mm.sup.3 were achieved. Thereafter, mice in Groups 2 and 3 received an intravenous injection of CAR T cells (1010.sup.6 CAR+ cells/mouse), as indicated in Table 2.

(203) TABLE-US-00003 TABLE 2 Treatment groups for intravenous tumor xenograft study CAKI-1 tumor cells CAR T cells (i.v.) Group (0.5 10.sup.6 cells/mouse) (10 10.sup.6 cells/mouse) N 1 X NA 5 2 X anti-CD70 CAR/TRAC/2M 5 (8e6 CAR+) 3 X anti-CD70 CAR/TRAC/2M/ 5 PTPN2(8e6 CAR+)

(204) During the course of the study, the mice were monitored daily and body weight was measured two times weekly. Tumor volumes were measured twice weekly beginning on Day 5 of the study. A significant endpoint between the time to peri-morbidity and the effect of T-cell engraftment was also assessed. The percentage of animal mortality and time to death were recorded for every group in the study. Mice were euthanized prior to reaching a moribund state. Mice may be defined as moribund and sacrificed if one or more of the following criteria were met: Loss of body weight of 20% or greater sustained for a period of greater than 1 week; Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate; Prolonged, excessive diarrhea leading to excessive weight loss (>20%); or Persistent wheezing and respiratory distress.

(205) Animals were also considered moribund if there was prolonged or excessive pain or distress, as defined by clinical observations such as: prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures and/or hemorrhages.

(206) Tumor Growth

(207) Tumor-bearing mice receiving a dose of TRAC/2M/PTPN2/anti-CD70 CAR+ T cells completely abrogated tumor growth 20 days after injection of CAR-T cells, which was sustained beyond 68 days post-injection (FIG. 8A).

(208) In Vivo Survival Rate

(209) Mice in groups receiving TRAC/2M/anti-CD70 CAR+ T cells with or without an additional PTPN2 knockout exhibited an increase in survival relative to mice in the untreated group (Group 1). Mice receiving a dose of TRAC/2M/PTPN2/anti-CD70 CAR+ T cells exhibited increased survival in comparison to TRAC/2M/anti-CD70 CAR+ T cells (FIG. 8B).

(210) These data demonstrate that the PTPN2 knockout in CAR T cells increases efficacy of these CAR T cells in vivo, decreasing tumor burden and increasing survival.

Example 7: In Vitro Effect of PTPN2 KO on Allogeneic CD70 CAR-T Cells Against NK Lysis

(211) Allogeneic CAR T cells lacking 2M and TRAC may be susceptible to cell mediated killing by donor NK cells. To test whether PTPN2 KO affects this susceptibility in CAR-T cells, anti-CD70 CAR/CD70/TRAC/132M CAR T cells and anti-CD70 CAR/CD70/TRAC/2M/PTPN2 cells (both generated from two different PBMC donors) were co-cultured with NK cells from a different donor, in the presence of IL-2 stimulation. After 24 hours, cell death of CAR-T cells was quantified by FACs analysis. Interestingly, NK-mediated killing of allogeneic CAR-T cells was reduced by 30% to 50% in CAR-T cells lacking PTPN2 (FIGS. 9A and 9B). This suggests that knocking out PTPN2 enhances survival of CAR-T cells in the presence of NK cells and may protect donor allogeneic CAR-T cells from encountering recipient NK cell attack.

Example 8: In Vivo Effect of PTPN2 KO on Allogeneic Anti-BCMA CAR-T Cells in Xenograft Mouse Tumor Model

(212) Allogenic CAR-T cells directed against BCMA were produced by knocking out TRAC & B2M loci, and HDR mediated insertion of a BCMA CAR sequence to the TRAC locus. To evaluate the effect of PTPN2 knockout on these cells, anti-BCMA CAR-T cells were produced with or without targeting the PTPN2 locus, using the PTPN2-1 sgRNA (e.g.: TRAC/B2M/PTPN2/anti-BCMA CAR+ T cells).

(213) The ability of the CAR-T cells to inhibit the growth of BCMA+ RPMI-8226 (multiple myeloma) tumor model was examined in NOG mice. Compared to allogenic CAR-T cells with a functional PTPN2 gene (TRAC/B2M/anti-BCMA CAR+ T cells), allogenic CAR-T cells with PTPN2 KO (TRAC/B2M/PTPN2/anti-BCMA CAR+ T cells) showed an improved ability to inhibit tumor progression (FIG. 10A). The enhanced CAR-T cell potency was accompanied by increased ability of the anti-BCMA CAR T cells to expand in-vivo in peripheral blood (FIG. 10B).

Example 9: In Vivo Effect of PTPN2 KO on Allogeneic Anti-CD19 CAR-T Cells in Xenograft Mouse Tumor Model

(214) This Example describes evaluation of anti-CD19 CAR T cells with and without a PTPN2 knockout in the intravenously disseminated Nalm-Fluc-GFP acute lymphoblastic leukemia tumor xenograft model.

(215) Allogenic CAR-T cells directed against CD19 were produced by knocking out TRAC & B2M loci, and HDR mediated insertion of a CD19 CAR sequence to the TRAC locus. To evaluate the effect of PTPN2 knockout on these cells, anti-CD19 CAR-T cells were produced with or without targeting the PTPN2 locus (e.g.: TRAC/B2M/PTPN2/anti-CD19 CAR+ T cells), using the PTPN2-13 sgRNA (spacer sequence SEQ ID NO: 60).

(216) The ability of the CAR-T cells (410.sup.6 cells injected) to inhibit the growth of the Nalm6 tumor model was examined in NOG mice by measuring the bioluminescence emitted by the tumor cells. Compared to allogenic CAR-T cells with a functional PTPN2 gene (TRAC/B2M/anti-CD19 CAR+ T cells), allogenic CAR-T cells with PTPN2 KO (TRAC/B2M/PTPN2/anti-CD19 CAR+ T cells) showed complete remission of the tumor until at least day 67 (FIG. 11A).

(217) Mice receiving a dose of TRAC/2M/PTPN2/anti-CD19 CAR+ T cells exhibited increased survival in comparison to TRAC/2M/anti-CD19 CAR+ T cells (FIG. 11B).

(218) These data demonstrate that the PTPN2 knockout in CAR T cells increases efficacy of these CAR T cells in vivo, decreasing tumor burden and increasing survival.

(219) Sequence Tables

(220) The following tables provide details for the various nucleotide and amino acid sequences disclosed herein.

(221) TABLE-US-00004 TABLE3 PTPN-2gRNATargetSequences SEQ SEQ ID ID NO NO (no (with Name sgRNAtargetsequence(PAM) PAM) PAM) PTPN2-1 GGAAACTTGGCCACTCTATG(AGG) 2 14 PTPN2-2 CCACTCTATGAGGATAGTCA(TGG) 3 15 PTPN2-3 CTCTTCTATGTCAACTAAAC(TGG) 4 16 PTPN2-4 CAGTTTAGTTGACATAGAAG(AGG) 5 17 PTPN2-5 TTGACATAGAAGAGGCACAA(AGG) 6 18 PTPN2-10 TTCGAACTCCCGCTCGATGG(TGG) 7 19 PTPN2-11 CTCTTCGAACTCCCGCTCGA(TGG) 8 20 PTPN2-12 AGTTGGATACTCAGCGTCGC(TGG) 9 21 PTPN2-13 CGAGCGGGAGTTCGAAGAGT(TGG) 10 22 PTPN2-14 AACTCCCGCTCGATGGTGGT(GGG) 11 23 PTPN2-15 CCGCTCGATGGTGGTGGGCA(TGG) 12 24 PTPN2-16 GAACTCCCGCTCGATGGTGG(TGG) 13 25

(222) TABLE-US-00005 TABLE4 sgRNASequencesforPTPN2 Name UnmodifiedSequence ModifiedSequence PTPN2-1 GGAAACUUGGCCACUCUAUGguuuuagagc G*G*A*AACUUGGCCACUCUAUGguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:26) cggugcU*U*U*U(SEQIDNO:27) PTPN2-1 GGAAACUUGGCCACUCUAUG(SEQID G*G*A*AACUUGGCCACUCUAUG(SEQID (spacer) NO:28) NO:29) PTPN2-2 CCACUCUAUGAGGAUAGUCAguuuuagagc C*C*A*CUCUAUGAGGAUAGUCAguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:30) cggugcU*U*U*U(SEQIDNO:31) PTPN2-2 CCACUCUAUGAGGAUAGUCA(SEQID C*C*A*CUCUAUGAGGAUAGUCA(SEQID (spacer) NO:32) NO:33) PTPN2-3 CUCUUCUAUGUCAACUAAACguuuuagagc C*U*C*UUCUAUGUCAACUAAACguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:34) cggugcU*U*U*U(SEQIDNO:35) PTPN2-3 CUCUUCUAUGUCAACUAAAC(SEQID C*U*C*UUCUAUGUCAACUAAAC(SEQID (spacer) NO:36) NO:37) PTPN2-4 CAGUUUAGUUGACAUAGAAGguuuuagagc C*A*G*UUUAGUUGACAUAGAAGguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:38) cggugcU*U*U*U(SEQIDNO:39) PTPN2-4 CAGUUUAGUUGACAUAGAAG(SEQID C*A*G*UUUAGUUGACAUAGAAG(SEQID (spacer) NO:40) NO:41) PTPN2-5 UUGACAUAGAAGAGGCACAAguuuuagagc U*U*G*ACAUAGAAGAGGCACAAguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:42) cggugcU*U*U*U(SEQIDNO:43) PTPN2-5 UUGACAUAGAAGAGGCACAA(SEQID U*U*G*ACAUAGAAGAGGCACAA(SEQID (spacer) NO:44) NO:45) PTPN2-10 UUCGAACUCCCGCUCGAUGGguuuuagagc U*U*C*GAACUCCCGCUCGAUGGguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:46) cggugcU*U*U*U(SEQIDNO:47) PTPN2-10 UUCGAACUCCCGCUCGAUGG(SEQID U*U*C*GAACUCCCGCUCGAUGG(SEQID (spacer) NO:48) NO:49) PTPN2-11 CUCUUCGAACUCCCGCUCGAguuuuagagc C*U*C*UUCGAACUCCCGCUCGAguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:50) cggugcU*U*U*U(SEQIDNO:51) PTPN2-11 CUCUUCGAACUCCCGCUCGA(SEQID C*U*C*UUCGAACUCCCGCUCGA(SEQID (spacer) NO:52) NO:53) PTPN2-12 AGUUGGAUACUCAGCGUCGCguuuuagagc A*G*U*UGGAUACUCAGCGUCGCguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:54) cggugcU*U*U*U(SEQIDNO:55) PTPN2-12 AGUUGGAUACUCAGCGUCGC(SEQID A*G*U*UGGAUACUCAGCGUCGC(SEQID (spacer) NO:56) NO:57) PTPN2-13 CGAGCGGGAGUUCGAAGAGUguuuuagagc C*G*A*GCGGGAGUUCGAAGAGUguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:58) cggugcU*U*U*U(SEQIDNO:59) PTPN2-13 CGAGCGGGAGUUCGAAGAGU(SEQID C*G*A*GCGGGAGUUCGAAGAGU(SEQID (spacer) NO:60) NO:61) PTPN2-14 AACUCCCGCUCGAUGGUGGUguuuuagagc A*A*C*UCCCGCUCGAUGGUGGUguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:62) cggugcU*U*U*U(SEQIDNO:63) PTPN2-14 AACUCCCGCUCGAUGGUGGU(SEQID A*A*C*UCCCGCUCGAUGGUGGU(SEQID (spacer) NO:64) NO:65) PTPN2-15 CCGCUCGAUGGUGGUGGGCAguuuuagagc C*C*G*CUCGAUGGUGGUGGGCAguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:192) cggugcU*U*U*U(SEQIDNO:193) PTPN2-15 CCGCUCGAUGGUGGUGGGCA(SEQID C*C*G*CUCGAUGGUGGUGGGCA(SEQID (spacer) NO:66) NO:67) PTPN2-16 GAACUCCCGCUCGAUGGUGGguuuuagagc G*A*A*CUCCCGCUCGAUGGUGGguuuuaga uagaaauagcaaguuaaaauaaggcuaguc gcuagaaauagcaaguuaaaauaaggcuagu cguuaucaacuugaaaaaguggcaccgagu ccguuaucaacuugaaaaaguggcaccgagu cggugcUUUU(SEQIDNO:68) cggugcU*U*U*U(SEQIDNO:69) PTPN2-16 GAACUCCCGCUCGAUGGUGG(SEQID G*A*A*CUCCCGCUCGAUGGUGG(SEQID (spacer) NO:70) NO:71)

(223) TABLE-US-00006 TABLE5 sgRNASequencesandTargetGeneSequencesforTRAC,2M,andCD70 SEQ ID NO: sgRNASequences CD70sgRNA Modified G*C*U*UUGGUCCCAUUGGUCGCguuuuagagcuagaaau 72 (CD70-7) agcaaguuaaaauaaggcuaguccguuaucaacuugaaaa aguggcaccgagucggugcU*U*CJ*U Unmodified GCUUUGGUCCCAUUGGUCGCguuuuagagcuagaaauagc 73 aaguuaaaauaaggcuaguccguuaucaacuugaaaaagu ggcaccgagucggugcUUUU CD70sgRNA Modified G*C*U*UUGGUCCCAUUGGUCGC 74 spacer Unmodified GCUUUGGUCCCAUUGGUCGC 75 TRACsgRNA Modified A*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaau 76 (TA-1) agcaaguuaaaauaaggcuaguccguuaucaacuugaaaa aguggcaccgagucggugcU*U*U*U Unmodified AGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagc 77 aaguuaaaauaaggcuaguccguuaucaacuugaaaaagu ggcaccgagucggugcUUUU TRACsgRNA Modified A*G*A*GCAACAGUGCUGUGGCC 78 spacer Unmodified AGAGCAACAGUGCUGUGGCC 79 2MsgRNA Modified G*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaau 80 (2M-1) agcaaguuaaaauaaggcuaguccguuaucaacuugaaaa aguggcaccgagucggugcU*U*U*U Unmodified GCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagc 81 aaguuaaaauaaggcuaguccguuaucaacuugaaaaagu ggcaccgagucggugcUUUU 2MsgRNA Modified G*C*U*ACUCUCUCUUUCUGGCC 82 spacer Unmodified GCUACUCUCUCUUUCUGGCC 83 TargetSequences(PAM) CD70target GCTTTGGTCCCATTGGTCGC(GGG) 84 sequencewith (PAM) CD70target GCTTTGGTCCCATTGGTCGC 85 sequence TRACtarget AGAGCAACAGTGCTGTGGCC(TGG) 86 sequencewith (PAM) TRACtarget AGAGCAACAGTGCTGTGGCC 87 sequence 2Mtarget GCTACTCTCTCTTTCTGGCC(TGG) 88 sequencewith (PAM) 2Mtarget GCTACTCTCTCTTTCTGGCC 89 sequence ExemplarysgRNAFormulas sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaag 90 sequence gcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaag 91 sequence gcuaguccguuaucaacuugaaaaaguggcaccgagucggugc sgRNA n(17-30)guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguua 92 sequence ucaacuugaaaaaguggcaccgagucggugcu(1-8) *indicates a nucleotide with a 2-O-methyl phosphorothioate modification, nrefers to the spacer sequence at the 5end.

(224) TABLE-US-00007 TABLE6 EditedTRACGeneSequence. Sequence(Deletions indicatedby SEQ dashes(-); Descrip- insertionsindicated ID tion bybold) NO: TRACgene AA------------------- 93 edit --GAGCAACAAATCTGACT TRACgene AAGAGCAACAGTGCTGT-GCC 94 edit TGGAGCAACAAATCTGACT TRACgene AAGAGCAACAGTG-------C 95 edit TGGAGCAACAAATCTGACT TRACgene AAGAGCAACAGT------GCC 96 edit TGGAGCAACAAATCTGACT TRACgene AAGAGCAACAGTG-------- 97 edit --------------CTGACT TRACgene AAGAGCAACAGTGCTGTGGG 98 edit CCTGGAGCAACAAATCTGACT TRACgene AAGAGCAACAGTGC--TGGC 99 edit CTGGAGCAACAAATCTGACT TRACgene AAGAGCAACAGTGCTGTGTG 100 edit CCTGGAGCAACAAATCTGACT

(225) These sequences are not present in the disrupted TRAC gene if a CAR-coding sequence is inserted at the TRAC targeting site.

(226) TABLE-US-00008 TABLE7 Edited2MGeneSequence. Sequence(Deletions indicatedbydashes(-); SEQ Descrip- insertionsindicated ID tion bybold) NO: 2M CGTGGCCTTAGCTGTGCTCGCGCTACTCT 101 gene- CTCTTTCT-GCCTGGAGGCTATCCAGCGT edit GAGTCTCTCCTACCCTCCCGCT 2M CGTGGCCTTAGCTGTGCTCGCGCTACTCT 102 gene- CTCTTTC--GCCTGGAGGCTATCCAGCGT edit GAGTCTCTCCTACCCTCCCGCT 2M CGTGGCCTTAGCTGTGCTCGCGCTACTCT 103 gene- CTCTTT-----CTGGAGGCTATCCAGCGT edit GAGTCTCTCCTACCCTCCCGCT 2M CGTGGCCTTAGCTGTGCTCGCGCTACTCT 104 gene- CTCTTTCTGGATAGCCTGGAGGCTATCCA edit GCGTGAGTCTCTCCTACCCTCCCGCT 2M CGTGGCCTTAGCTGTGCTCGC-------- 105 gene- -----------------GCTATCCAGCG edit TGAGTCTCTCCTACCCTCCCGCT 2M CGTGGCCTTAGCTGTGCTCGCGCTACTC 106 gene- TCTCTTTCTGTTGGCCTGGAGGCTACCA edit GCGTGAGTCTCTCCTACCCTCCCGCT

(227) TABLE-US-00009 TABLE8 EditedCD70GeneSequence. Sequence(Deletionsindicated SEQ bydashes(-);insertions ID Description indicatedbybold) NO: CD70 CACACCACGAGGCAGATCACCAAGCCCGCG-- 107 gene-edit CAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGCGA 108 gene-edit ACCAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATC------------ 109 gene-edit ACCAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGCG- 110 gene-edit CCAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGC- 111 gene-edit ACCAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCA--------- 112 gene-edit -----------------AGCCCGCAGGACG

(228) TABLE-US-00010 TABLE9 ChimericAntigenReceptorSequences SEQ ID NO Description Sequence 113 signalpeptide MLLLVTSLLLCELPHPAFLLIP 114 signalpeptide MALPVTALLLPLALLLHAARP 115 CD8a IYIWAPLAGTCGVLLLSLVITLY transmembrane domain 116 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTAT nucleotide GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGAT sequence TTCCAGAAGAAGAAGAAGGAGGATGTGAACTG 117 4-1BBamino KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL acid sequence 118 CD28nucleotide TCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCC sequence TCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCC CACGAGACTTCGCTGCGTACAGGTCC 119 CD28aminoacid SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS sequence 120 CD3-zeta CGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGG Nucleotide ACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGT sequence ATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGT AAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCA GAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCG AACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGT ACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCC TCCCAGA 121 CD3-zetaamino RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG acidsequence KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 122 anti-CD19VL RASQDISKYLN CDR1(Kabat) 123 anti-CD19VL HTSRLHS CDR2(Kabat) 124 anti-CD19VL QQGNTLPYT CDR3(Kabat) 125 anti-CD19VH DYGVS CDR1(Kabat) 126 anti-CD19VH VIWGSETTYYNSALKS CDR2(Kabat) 127 anti-CD19VH HYYYGGSYAMDY CDR3(Kabat) 128 anti-CD19VL RASQDISKYLN CDR1(Chothia) 129 anti-CD19VL HTSRLHS CDR2(Chothia) 130 anti-CD19VL QQGNTLPYT CDR3(Chothia) 131 anti-CD19VH GVSLPDY CDR1(Chothia) 132 anti-CD19VH WGSET CDR2(Chothia) 133 anti-CD19VH HYYYGGSYAMDY CDR3(Chothia) 134 Anti-CD19CAR ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCC FMC63-28Z AGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTA (FMC63-CDS[tm]- GCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCA CD28[co- AGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGA stimulatory CGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCG domain]-CD3z) GAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCC TTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTG TCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAAC TCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGA GAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGG TCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTG GTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCG CGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAAC GTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATA ACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGAC GATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAG TTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTA GTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACG ACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCA ACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTG CTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGG GCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTAT TACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGT TGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACA AGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTA CAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATC AGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGC GAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAAT GGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATG AACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATG AAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGG GTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGG CCCTGCCTCCCAGA 135 Anti-CD19CAR MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVHSCRA (withno FMC63-28Z SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS signal (FMC63-CD8[tm]- LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSG peptide) CD28[co- EGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP 188 stimulatory RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD (withsignal domain]-CD3z) DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTT peptide) AminoAcid TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 136 Anti-CD19scFv GATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGG codingsequence AGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAAT ACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTC ATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTC AGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCG AGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTC CCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCAC CAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCG AGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAA AGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTA TGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGC TTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTC AAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTT CCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATT GTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGG GGGCAGGGGACTTCTGTCACAGTCAGTAGT 137 CD19scFvamino DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL acidsequence IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL Linkerunderlined PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQ SLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS 138 CD8aextracellular GCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGAC +CD8a TCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAAC transmembrane+5 CTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCT Linker(underlined) GTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGC TCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTA CTTTGTATTGTAATCACAGGAATCGC 139 CD8aextracellular TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCC +CD8a GCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTC transmembrane TTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACG (withoutlinker) AGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGC GGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATT GTAATCACAGGAATCGC 140 CD8aextracellular FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT +CD8a RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR transmcmbrane 141 CD19VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEW LGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSS 142 CD19VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEIT 143 CD19linker GSTSGSGKPGSGEGSTKG 144 CD70VLCDR1 RASKSVSTSGYSFMH (Kabat) 145 CD70VLCDR1 SKSVSTSGYSF (Chothia) 146 CD70VLCDR2 LASNLES (Kabat) N/A CD70VLCDR2 LAS (Chothia) 147 CD70VLCDR3 QHSREVPWT (Kabat) 148 CD70VLCDR3 SREVPW (Chothia) 149 CD70VHCDR1 NYGMN (Kabat) 150 CD70VHCDR1 GYTFTNYGMN (Chothia) 151 CD70VHCDR2 WINTYTGEPTYADAFKG (Kabat) 152 CD70VHCDR2 NTYTGE (Chothia) 153 CD70VHCDR3 DYGDYGMDY (Kabat) 154 CD70VHCDR3 CARDYGDYGMDYWG (Chothia) 155 CD70CARamino MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASG (withno acidsequence YTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRD signal (CD70BscFvwith TSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGG peptide) 41BB) GGSGGGGSGGGGSGDIVMTQSPDSLAVSLGERATINCRASKSVSTSG 189 YSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTIS (withsignal SLQAEDVAVYYCQHSREVPWTFGQGTKVEIKSAAAFVPVFLPAKPTT peptide) TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 190 CD70CAR ATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCT nucleotidesequence CCACGCAGCAAGGCCGCAGGTCCAGTTGGTGCAAAGCGGGGCGGAGG (CD70BscFvwith TGAAAAAACCCGGCGCTTCCGTGAAGGTGTCCTGTAAGGCGTCCGGT 41BB) TATACGTTCACGAACTACGGGATGAATTGGGTTCGCCAAGCGCCGGG GCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCGAAC CTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGAT ACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGA CGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCA TGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGA GGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACAT AGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGA GGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGA TATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAA GCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACC GATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGT TCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAG AGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAA GTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACG ACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCA ACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTG CTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGG GCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTAT TACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGG AGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGG CATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGA CGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCC GGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCT ACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATA GGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTA CCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATA TGCAGGCCCTGCCTCCCAGATAA 156 CD70A GATATAGTTATGACCCAATCACCCGATAGTCTTGCGGTAAGCCTGGG scFvnucleotide GGAGCGAGCAACAATAAACTGTCGGGCATCAAAATCCGTCAGTACAA sequence GCGGGTATTCATTCATGCACTGGTATCAACAGAAACCCGGTCAGCCA CCCAAGCTCCTGATTTATCTTGCGTCTAATCTTGAGTCCGGCGTCCC AGACCGGTTTTCCGGCTCCGGGAGCGGCACGGATTTTACTCTTACTA TTTCTAGCCTTCAGGCCGAAGATGTGGCGGTATACTACTGCCAGCAT TCAAGGGAAGTTCCTTGGACGTTCGGTCAGGGCACGAAAGTGGAAAT TAAAGGCGGGGGGGGATCCGGCGGGGGAGGGTCTGGAGGAGGTGGCA GTGGTCAGGTCCAACTGGTGCAGTCCGGGGCAGAGGTAAAAAAACCC GGCGCGTCTGTTAAGGTTTCATGCAAGGCCAGTGGATATACTTTCAC CAATTACGGAATGAACTGGGTGAGGCAGGCCCCTGGTCAAGGCCTGA AATGGATGGGATGGATAAACACGTACACCGGTGAACCTACCTATGCC GATGCCTTTAAGGGTCGGGTTACGATGACGAGAGACACCTCCATATC AACAGCCTACATGGAGCTCAGCAGATTGAGGAGTGACGATACGGCAG TCTATTACTGTGCAAGAGACTACGGCGATTATGGCATGGATTACTGG GGCCAGGGCACTACAGTAACCGTTTCCAGC 157 CD70A DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQP scFvaminoacid PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQH sequence SREVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGQVQLVQSGAEVKKP (linkerunderlined) GASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYA DAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYW GQGTTVTVSS 158 CD70B CAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGC scFvnucleotide TTCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACT sequence ACGGGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGG ATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGC TTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCG CATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTAC TATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCA GGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGG GGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCC CCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTG TCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATT GGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTG GCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGG AAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGG ATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACC TTCGGTCAAGGCACGAAAGTAGAAATTAAA 159 CD70B QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKW scFvaminoacid MGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVY sequence YCARDYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMTQS (linkerunderlined) PDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYL ASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWT FGQGTKVEIK 160 CD70VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKW MGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVY YCARDYGDYGMDYWGQGTTVTVSS 161 CD70VL DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQP PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQH SREVPWTFGQGTKVEIK 162 BCMACAR ATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCT nucleotidesequence CCACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGC TCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGC AACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGG CCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGA CCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGAT AAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGA GGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCT TTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGC GGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGT GATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGG CCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGC AACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAG GCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCA GGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGC AGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAG CCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAA GTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACG ACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCA ACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTG CTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGG GCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTAT TACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGG AGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGG CATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGA CGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCC GGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCT ACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATA GGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTA CCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATA TGCAGGCCCTGCCTCCCAGA 163 BCMACARamino MALPVTALLLPLALLLHAARPQVQLVQSGAELKKPGASVKVSCKASG (withno acidsequence NTLTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRD signal KSASTAYMELSSLRSEDTAVYYCTRWDWDGFFDPWGQGTTVTVSSGG peptide) GGSGGGGSGGGGSEIVMTQSPATLSVSPGERASISCRASQSLVHSNG 191 NTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTIS (withsignal SVESEDFAVYYCSQTSHIPYTFGGGTKLEIKSAAAFVPVFLPAKPTT peptide) TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 164 BCMA CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGC scFvnucleotide CTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACT sequence ACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGG ATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAA GTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCG CCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTAC TACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCA GGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCG GCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCC GCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAG GGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACT GGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGC GTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGG AAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGG ACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACC TTCGGCGGCGGCACCAAGCTGGAGATCAAA 165 BCMA QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEW scFvaminoacid MGYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVY sequence YCTRWDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSP (linkerunderlined) ATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYS VSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYT FGGGTKLEIK 166 BCMAVH QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEW MGYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVY YCTRWDWDGFFDPWGQGTTVTVSS 167 BCMAVL EIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQ APRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCS QTSHIPYTFGGGTKLEIK 168 BCMAVLCDR1 RASQSLVHSNGNTHLH (Kabat&Chothia) 169 BCMAVLCDR2 SVSNRFS (Kabat&Chothia) 170 BCMAVLCDR3 SQTSHIPYT (Kabat) 171 BCMAVLCDR3 SQTSHIPYT (Chothia) 172 BCMAVHCDR1 NYVIH (Kabat) 173 BCMAVHCDR1 GNTLTNY (Chothia) 174 BCMAVHCDR2 YILPYNDLTKYSQKFQG (Kabat) 175 BCMAVHCDR2 LPYNDL (Chothia) 176 BCMAVHCDR3 WDWDGFFDP (Kabat) 177 BCMAVHCDR3 WDWDGFFDP (Chothia)

(229) TABLE-US-00011 TABLE10 AAVDonorTemplateSequences 178 LeftITR TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAC (5ITR) CAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA GCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 179 LeftITR CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTC (5ITR) GGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA (alternate) GGGAGTGGCCAACTCCATCACTAGGGGTTCCT 180 RightITR AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC (3ITR) GCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 181 RightITR AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC (3ITR) GCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCC (alternate) CGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 182 TRAC-LHA GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA (800bp) CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAA GTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGG CCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGT TTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAG CCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAAC GTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTC CCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCC TTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGG AAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATG AGGTCTATGGACTTCA 183 TRAC-RHA TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCA (800bp) TTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGG TGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCA GAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCC TTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTT GTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAG GTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTC CTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAG GCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTG TCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCAC TCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGT GTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAA GCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAA TAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTAC CTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAA GATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGA CAGGAGCTCAATGAGAAAGG 184 EF1a GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCG AGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTG GCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTT CCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTG TGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCT TGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCG GGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCT TCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCG TGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGG CAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGG TTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATG TTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGG TAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGT GTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGC GTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAA TGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAA GGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGA GTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGT ACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCC ACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTA ATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGT GA 185 CD19 GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA LHAtoRHA CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAA GTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGG CCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGT TTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAG CCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAAC GTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTC CCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCC TTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGG AAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATG AGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCG GTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCA GTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTA TGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTC TTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGG CGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCG CTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGC GACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTG CACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTG CGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACC GAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCT GGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCC GGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGC TGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTT CATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTT CTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAG CTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGG ATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTT CCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTT GCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAG ATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAA CAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTA CCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCA AGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAA CTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGAC ATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGA ACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCA GTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCC CGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGT GGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGC GAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTA TTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCC AAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCG CTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGAT GGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCC TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGC GCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCG CCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGC TTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGT GCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAG GAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATG ACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCC CCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAG CGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAA CTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGG GGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGA AGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCA GAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCC TCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCA TATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAG ATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGC ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTC CCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTG CTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAA AACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCT CTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGAC ACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCC CAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGA CTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCC AAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGC TCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGAT TGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAG TCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCC CATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTT TAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAG GGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAG GGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG 186 CD70 GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA LHAtoRHA CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC (CD70B CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC scFVwith TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAA 41BB) GTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGG CCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGT TTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAG CCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAAC GTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTC CCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCC TTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGG AAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATG AGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCG GTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCA GTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTA TGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTC TTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGG CGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCG CTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGC GACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTG CACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTG CGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACC GAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCT GGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCC GGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGC TGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTT CATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTT CTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAG CTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGG ATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTT CCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCT CCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTCCAGTTG GTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTTCCGTGAAGGTGT CCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGGGATGAATTGGGT TCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGGTGGATAAATACC TACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTA TGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACT CCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGAT TATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTG GTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGA CATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAG AGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGAT ATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCT GCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTT TCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGC AGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCC CTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAAGTGCTGCTGCC TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGC GCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCG CCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGC TTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGT GCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAG GAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTC CCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTAT AACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAAC GCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCC CCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCC TACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACG ATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGC ACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCA TCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGA CTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACC TTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTT TCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGAT GTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAA AACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAG AATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCAC GTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTG CTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCC TTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTT CCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATC ACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATT AAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGG GGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAAT GTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCA GGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAA GGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAA AGG 187 BCMA GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA RHAtoLHA CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAA GTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGG CCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGT TTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAG CCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAAC GTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTC CCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCC TTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGG AAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATG AGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCG GTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCA GTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTA TGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTC TTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGG CGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCG CTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGC GACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTG CACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTG CGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACC GAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCT GGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCC GGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGC TGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGT GAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTT CATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTT CTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAG CTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGG ATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTT CCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCT CCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTGCAGCTG GTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGA GCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTGATCCACTGGGT GAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCC TACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCA TCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCT GAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGAC GGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCG GCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAAT CGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGG GCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCA ACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCT GCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTT AGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGG AGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCC TTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCC TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGC GCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCG CCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGC TTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGT GCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAG GAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTC CCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTAT AACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAAC GCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCC CCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCC TACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACG ATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGC ACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCA TCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGA CTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACC TTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTT TCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGAT GTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAA AACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAG AATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCAC GTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTG CTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCC TTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTT CCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATC ACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATT AAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGG GGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAAT GTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCA GGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAA GGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAA AGG

Other Embodiments

(230) All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

(231) From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

(232) While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

(233) All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

(234) All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

(235) The indefinite articles a and an, as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean at least one.

(236) The phrase and/or, as used herein in the specification and in the claims, should be understood to mean either or both of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with and/or should be construed in the same fashion, i.e., one or more of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the and/or clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with open-ended language such as comprising can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

(237) As used herein in the specification and in the claims, or should be understood to have the same meaning as and/or as defined above. For example, when separating items in a list, or or and/or shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as only one of or exactly one of, or, when used in the claims, consisting of, will refer to the inclusion of exactly one element of a number or list of elements. In general, the term or as used herein shall only be interpreted as indicating exclusive alternatives (i.e. one or the other but not both) when preceded by terms of exclusivity, such as either, one of, only one of, or exactly one of. Consisting essentially of, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

(238) The term about as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, about can mean within an acceptable standard deviation, per the practice in the art. Alternatively, about can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term about is implicit and in this context means within an acceptable error range for the particular value.

(239) As used herein in the specification and in the claims, the phrase at least one, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase at least one refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B, or, equivalently at least one of A and/or B) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

(240) It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.