Genetically Engineered T Cells With Regnase-1 And/or Tgfbrii Disruption Have Improved Functionality And Persistence

Abstract

A population of genetically engineered T cells, comprising a disrupted Reg1 gene and/or a disrupted TGFBRII gene. Such genetically engineered T cells may comprise further genetic modifications, for example, a disrupted CD70 gene. The population of genetically engineered T cells exhibit one or more of (a) improved cell growth activity; (b) enhanced persistence; and (c) reduced T cell exhaustion, (d) enhanced cytotoxicity activity, (e) resistant to inhibitory effects induced by TGF-b, and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby, as compared to non-engineered T cell counterparts.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing dates of U.S. Provisional Application No. 63/082,357, filed Sep. 23, 2020, U.S. Provisional Application No. 63/124,429, filed Dec. 11, 2020, and U.S. Provisional Application No. 63/225,673, filed Jul. 26, 2021. The entire contents of each of the prior applications are incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

[0002] The application contains a Sequence Listing that has been filed electronically in the form of a text file, created Sep. 23, 2021, and named "095136-0369-026US1_SEQ.TXT" (236,320 bytes), the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0003] Chimeric antigen receptor (CAR) T-cell therapy uses genetically modified T cells to more specifically and efficiently target and kill cancer cells. After T cells have been collected from the blood, the cells are engineered to include CARs on their surface. The CARs may be introduced into the T cells using CRISPR/Cas9 gene editing technology. When these allogeneic CAR T cells are injected into a patient, the receptors enable the T cells to kill cancer cells.

[0004] T cells having improved persistence in culture are desired in CAR T therapy. Such T cells live longer in both in vitro and in vivo, thereby conferring benefits in CAR T cell manufacturing and clinical applications. However, it remains challenging to improve persistence of T cells in culture.

SUMMARY OF THE INVENTION

[0005] The present disclosure is based, at least in part, on the development of genetically edited T cells carrying a disrupted Regnase 1 (Reg1) gene (e.g., "Reg1 Knockout T cells"), a disrupted TGFBRII gene (e.g., "TGFBRII Knockout T cells", or genetically edited T cells carrying both a disrupted Reg1 gene and a disrupted TGFBRII gene, and effective methods of producing such genetically edited T cells via CRISPR/Cas-mediated gene editing using guide RNAs, for example, those targeting specific sites within the Reg1 gene with high on-target editing efficiency and low off-target editing efficiency, and/or those targeting specific sites within the TGFBRII gene with high on-target editing efficiency and low off-target editing efficiency.

[0006] Such genetically engineered T cells exhibits at least one of the following advantageous features: (a) improved cell growth activity; (b) enhanced persistence; (c) reduced T cell exhaustion; (d) resistant to inhibitory effects induced by TGF-.beta.; (e) enhanced cell killing capacity; and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby.

[0007] The Reg1 disrupted T cells, the TGFBRII disrupted T cells, or the Reg1/TGFBRII double disrupted T cells disclosed herein can further be genetically engineered to express a chimeric antigen receptor (CAR) targeting an antigen of interest, e.g., an antigen associated with an undesired cell such as a cancer cell, and to comprise one or more additional disrupted genes, for example, TRAC, .beta.2M, CD70, or a combination thereof. The resultant CAR-expressing, Reg1 disrupted T cells exhibit enhanced cytotoxic activity against target cells and anti-tumor activity as compared with CAR-T cells having a wild-type Reg1 gene.

[0008] In some aspects, the current disclosure is related to the development of genetically engineered CAR T cells that comprise a disrupted Reg1 gene. The genetically engineered CAR T cells, in certain aspects, are further genetically engineered to comprise a disrupted cluster of differentiation 70 (CD70) gene. In some aspects, the CAR T cells described herein may express anti-CD70 CAR, anti-cluster of differentiation 19 (CD19) CAR or anti-B-cell maturation antigen (anti-BCMA) CAR.

[0009] The genetically edited T cells disclosed herein showed enhanced cell expansion, longevity and proliferation capacity in culture, enhanced potency (e.g., enhanced cytotoxicity), and enhanced CAR-T efficacy in animal models (via, e.g., longer persistence). Further, the genetically edited T cells showed cytokine-dependent growth, indicating safety. In addition, disrupting both the Reg1 and TGFBRII genes exhibited synergistic effects in anti-tumor activity and CAR-T cell expansion and persistence as observed in animal models.

[0010] Accordingly, the present disclosure provides, in some aspects, a population of genetically engineered T cells, comprising: (i) a disrupted Regnase-1 (Reg1) gene; and/or (ii) a disrupted Transforming Growth Factor Beta Receptor II (TGFBRII) gene. In some embodiments, the population of genetically engineered T cells comprises (i). In some embodiments, population of genetically engineered T cells comprises (ii). In other embodiments, the population of genetically engineered T cells comprises both (i) and (ii). Any of the genetically engineered T cells may be further engineered to express a chimeric antigen receptor (CAR).

[0011] The population of genetically engineered T cells disclosed herein, as compared to non-engineered T cell counterparts, has one or more of the following features: (a) improved cell growth activity; (b) enhanced persistence; (c) reduced T cell exhaustion; (d) resistant to inhibitory effects induced by TGF-.beta.; (e) enhanced cell killing capacity; and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby.

[0012] In some embodiments, the disrupted Reg1 gene is genetically edited in exon 1, exone 2, exon 3, or exon 4. In some examples, the disrupted Reg1 gene is genetically edited in exon 2 and/or exon 4. Alternatively or in addition, the disrupted TGFBRII gene is genetically edited in exon 1, exon 2, exon 3, exon 4, or exon 5. In some examples, the disrupted TGFBRII gene is genetically edited in exon 4. In other examples, the disrupted TGFBRII gene is genetically edited in exon 5. The disrupted Reg1 gene, the disrupted TGFBRII gene, or both can be genetically edited by a CRISPR/Cas-mediated gene editing system.

[0013] In some instances, the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the Reg1 gene that comprises a nucleotide sequence listed in Table 22 (with or without PAM) (e.g., SEQ ID NO: 320, 322, 323, or 327, or the corresponding ones with PAM). For example, the gRNA targeting the Reg1 gene comprises a spacer that comprises the nucleotide sequence of listed in Table 22 (e.g., SEQ ID NO: 24, 32, 36, or 52). In some examples, the disrupted Reg1 gene comprises a nucleotide sequence selected from those listed in Tables 29-38 (e.g., Table 31, 33, 34, or 38).

[0014] In some instances, the CRISPR/Cas-mediated gene editing system comprises a guide RNA (gRNA) targeting a site in the TGFBRII gene that comprises a nucleotide sequence listed in Table 39 (with or without PAM). For example, the gRNA targeting the TGFBRII gene comprises a spacer listed in Table 39, for example, having a nucleotide sequence of any one of SEQ ID NOs: 270, 302, 308, and 312. In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 (e.g., Table 43).

[0015] Any of the gRNAs disclosed herein may further comprise a scaffold sequence. For example, the gRNA targeting the Reg1 gene comprises any of the nucleotide sequences listed in Table 22. Examples include 22, 23, 30, 31, 34, 35, 50, and 51. Alternatively or in addition, the gRNA targeting the TGFBRII gene may comprise any of the nucleotide sequences provided in Table 39. Examples include SEQ ID NOs: 270, 271, 300, 301, 306, 307, 312, and 313.

[0016] Any of the populations of genetically engineered T cells disclosed herein may further comprise: (iii) a disrupted T cell receptor alpha chain constant region (TRAC) gene, (iv) a disrupted beta-2-microglobulin (.beta.2M) gene, (v) a disrupted CD70 gene, or (vi) a combination of any of (iii)-(v). In some embodiments, the T cells comprise a disrupted T cell receptor alpha chain constant region (TRAC) gene. Alternatively or in addition, the T cells comprise a disrupted beta-2-microglobulin (.beta.2M) gene. Any of the T cells disclosed herein may also comprise a disrupted CD70 gene. In some examples, the disrupted TRAC gene, the disrupted .beta.2M gene, and/or the disrupted CD70 gene is genetically edited by one or more CRISPR/Cas-mediated gene editing system

[0017] In some embodiments, the genetically engineered T cells may comprise a nucleic acid encoding the CAR, and wherein the nucleic acid is inserted in the genome of the T cells. In some instances, the nucleic acid encoding the CAR is inserted in the disrupted Reg1 gene, the disrupted TGFBRII gene, the disrupted TRAC gene, the disrupted .beta.2M, or the disrupted CD70 gene. In some examples, the nucleic acid encoding the CAR is inserted in the disrupted TRAC gene. In specific examples, the nucleic acid encoding the CAR may replace the deleted fragment comprising SEQ ID NO: 69 in the TRAC gene. In some examples, the disrupted Reg1 gene may comprise a nucleotide sequence listed in Tables 29-38 (e.g., Table 31, 33, 34, or 38).

[0018] In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 (e.g., Table 43). In some examples, the disrupted TRAC gene may comprise a nucleotide sequence of any one of SEQ ID NOs: 75-82 (see Table 24). In some examples, the disrupted .beta.2M may comprise a nucleotide sequence of any one of SEQ ID NOs: 83-88 (see Table 25). In some examples, the disrupted CD70 gene may comprise a nucleotide sequence of any one of SEQ ID NOs: 89-94 (see Table 26).

[0019] Any of the CAR constructs disclosed herein may comprise 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 CD3.zeta.. In some examples, the tumor antigen is CD19. In some examples, the tumor antigen is BCMA. In some examples, the tumor antigen is CD70. In some examples, the tumor antigen is CD33. In some examples, the tumor antigen is PTK7.

[0020] In some embodiments, the CAR binds CD19 (anti-CD19 CAR). The extracellular antigen binding domain in the anti-CD19 CAR can be a single chain variable fragment (scFv) that binds CD19 (anti-CD19 scFv). In some instances, the anti-CD19 scFv may comprise (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 125. In some examples, the V.sub.H comprises the amino acid sequence of SEQ ID NO: 124 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 125. In one example, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 120. In one specific example, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 118 (with an N-terminal signal peptide) or SEQ ID NO:353 (without N-terminal signal peptide).

[0021] In some embodiments, the CAR binds CD70 (anti-CD70 CAR). The extracellular antigen binding domain in the anti-CD70 CAR can be a single chain variable fragment (scFv) that binds CD70 (anti-CD70 scFv). In some instances, the anti-CD70 scFv comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 144. In some examples, the V.sub.H comprises the amino acid sequence of SEQ ID NO: 143 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 144. In one example, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 140 or 142. In one specific example, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 138 (with an N-terminal signal peptide) or SEQ ID NO:354 (without N-terminal signal peptide).

[0022] In some embodiments, the CAR binds BCMA (anti-BCMA CAR). The extracellular antigen binding domain in the anti-BCMA CAR can be a single chain variable fragment (scFv) that binds BCMA (anti-BCMA CAR). In some instances, the anti-BCMA scFv comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 149; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 150. In some examples, the V.sub.H comprises the amino acid sequence of SEQ ID NO: 149 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 150. In one example, the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 148. In one specific example, the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 146 (with an N-terminal signal peptide) or SEQ ID NO:355 (without N-terminal signal peptide).

[0023] The genetically engineered T cells disclosed herein may be derived from primary T cells of one or more human donors. In some instances, the genetically engineered T cells show cytokine-dependent growth.

[0024] In other aspects, the present disclosure provides a method for preparing any of the populations of genetically engineered T cells disclosed herein. In some instances, the method may comprise: (a) providing a plurality of cells, which are T cells or precursor cells thereof; (b) genetically editing the Reg1 gene and/or the TGFBRII gene; and (c) producing the population of genetically engineered T cells having disrupted Reg1 gene and/or the TGFBRII gene. In some examples, the T cells of step (a) are derived from primary T cells of one or more human donors. In some examples, step (b) comprises genetically editing the Reg1 gene. In some examples, step (b) comprises genetically editing the TGFBRII gene. In some examples, step (b) comprises genetically editing both the Reg1 gene and the TGFBRII gene.

[0025] In some embodiments, step (b) is performed by one or more CRISPR/Cas-mediated gene editing systems. For example, step (b) can be performed by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the Reg1 gene. In some instances, the gRNA targeting the Reg1 gene may be specific to an exon of the Reg1 gene, e.g., exon 2 or exon 4. In some examples, the gRNA targeting the Reg1 gene comprises a spacer that comprises a nucleotide sequence listed in Table 22 (e.g., SEQ ID NO: 24, 32, 36, or 52).

[0026] Alternatively or in addition, step (b) can be performed, inter alia, by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the TGFBRII gene. For example, the gRNA targeting the TGFBRII gene may be specific to an exon of the TGFBRII gene, e.g., exon 1, exon 2, exon 3, exon 4, and exon 5. In one example, the gRNA targeting the TGFBRII gene is specific to exon 4. In another example, the gRNA targeting the TGFBRII gene is specific to exon 5. In some instances, the gRNA targeting the TGFBRII gene comprises a spacer listed in Table 39. Examples include SEQ ID NOs: 272, 302, 308, and 314.

[0027] Any of the gRNAs disclosed herein may further comprise a scaffold sequence. For example, the gRNA targeting the Reg1 gene may comprise any of the nucleotide sequences listed in Table 22. Examples include SEQ ID NO: 22, 23, 30, 31, 34, 35, 50, and 51. Alternatively or in addition, the gRNA targeting the TGFBRII gene may comprise any of the nucleotide sequences provided in Table 39. Examples include SEQ ID NOs: 270, 271, 300, 301, 306, 307, 312, and 313.

[0028] In any of the methods disclosed herein, the plurality of T cells in step (a) comprises one or more of the following genetic modifications: (i) engineered to express a chimeric antigen receptor (CAR); (ii) has a disrupted T cell receptor alpha chain constant region (TRAC) gene; (iii) has a disrupted .beta.2M gene; and (iv) has a disrupted CD70 gene.

[0029] Alternatively, any of the methods disclosed herein may further comprise: [0030] (i) delivering to the T cells a nucleic acid encoding a chimeric antigen receptor (CAR); [0031] (ii) genetically editing a TRAC gene to disrupt its expression; [0032] (iii) genetically editing a .beta.2M gene to disrupt its expression; [0033] (iv) genetically editing a CD70 gene to disrupt its expression; or [0034] (v) a combination thereof.

[0035] In some embodiments, one or more of (i)-(iv) are performed by one or more CRISPR/Cas-mediated gene editing system comprising one or more RNA-guided nucleases and one or more gRNAs targeting the TRAC gene, the .beta.2M gene, and/or the CD70 gene. In some examples, the gRNA targeting the TRAC gene comprises a spacer that comprises the nucleotide sequence of SEQ ID NO: 61. In some examples, the gRNA targeting the .beta.2M gene comprises a spacer that comprises the nucleotide sequence of SEQ ID NO: 65. In some examples, the gRNA targeting the CD70 gene comprises a spacer that comprises the nucleotide sequence of SEQ ID NO: 57. See Table 23.

[0036] In some embodiments, the method disclosed herein may comprise delivering to the T cells one or more ribonucleoprotein particles (RNP), which may comprise the RNA-guided nuclease, one or more of the gRNAs, and the nucleic acid encoding the CAR. In some examples, the RNA-guided nuclease is a Cas9 nuclease, for example, a S. pyogenes Cas9 nuclease.

[0037] In some embodiments, the nucleic acid encoding the CAR is in an AAV vector. In some instances, the nucleic acid encoding the CAR comprises a left homology arm and a right homology arm flanking the nucleotide sequence encoding the CAR. The left homology arm and the right homology arm are homologous to a genomic locus in the T cells, allowing for insertion of the nucleic acid into the genomic locus. In some examples, the genomic locus is in the Reg1 gene. In some examples, the genomic locus is in the TGFBRII gene. In some examples, the genomic locus is in the TRAC gene. In some examples, the genomic locus is in the .beta.2M gene. In some examples, the genomic locus is in the CD70 gene.

[0038] In some examples, the method comprising disrupting the TRAC gene by a CRISPR/Cas-mediated gene editing system comprising a gRNA comprising the nucleotide sequence of SEQ ID NO: 59 and the nucleic acid encoding the CAR is inserted at the site targeted by the gRNA. Alternatively or in addition, the method may comprise delivering to the T cells a nucleic acid encoding a CAR, which is specific to CD70, and genetically editing the CD70 gene to disrupt its expression.

[0039] Any population of the genetically engineered T cells prepared by a method disclosed herein is also within the scope of the present disclosure.

[0040] Further, the present disclosure provides a method for eliminating undesired cells in a subject, the method comprising administering to a subject in need thereof any of the populations of genetically engineered T cells disclosed herein. In some embodiments, the undesired cells are cancer cells, for example, hematopoietic cancer cells or solid tumor cells. In some embodiments, the undesired cells are CD19.sup.+. In some embodiments, the undesired cells are BCMA.sup.+. In some embodiments, the undesired cells are CD70.sup.+. In some embodiments, the undesired cells are CD33.sup.+. In some embodiments, the undesired cells are PTK7.sup.+.

[0041] In yet other aspects, provided herein is a guide RNA (gRNA) targeting a Reg1 gene, comprising a nucleotide sequence specific to a fragment in exon 2 or exon 4 of the Reg1 gene. In some embodiments, the gRNA comprises a spacer listed in Table 22 (e.g., SEQ ID NO: 24, 32, 36 or 52). Such a gRNA may further comprise a scaffold sequence. Alternatively or in addition, the gRNA comprises one or more modified nucleotides. For example, the gRNA comprises one or more 2'-O-methyl phosphorothioate residues at the 5' and/or 3' terminus of the gRNA. Examples of gRNAs targeting Reg1 include any of those listed in Table 22 (e.g., SEQ ID NO: 22, 23, 30, 31, 34, 35, 50, or 51; see also disclosures herein).

[0042] In still other aspects, provided herein is a guide RNA (gRNA) targeting a TGFBRII gene, comprising a nucleotide sequence specific to a fragment in exon 1, exon 2, exon 3, exon 4, or exon5 of the TGFBRII gene. In some examples, the gRNA comprises a nucleotide sequence specific to exon 4 of the TGFBRII gene. In other examples, the gRNA comprises a nucleotide sequence specific to exon 5 of the TGFBRII gene. In some instances, the gRNA comprises a spacer having the nucleotide sequence listed in Table 39 (e.g., SEQ ID NOs: 272, 302, 308, and 314). Such a gRNA may further comprise a scaffold sequence. Alternatively or in addition, the gRNA comprises one or more modified nucleotides. For example, the gRNA comprises one or more 2'-O-methyl phosphorothioate residues at the 5' and/or 3' terminus of the gRNA. Examples of gRNAs targeting the TGFBRII gene include any of those listed in Table 39 (e.g., SEQ ID NOs: 270, 271, 300, 301, 306, 307, 312, and 313).

[0043] Also within the scope of the present disclosure are any of the genetically engineered T cells, gRNAs targeting Reg1, or gRNAs targeting TGFBRII for use in treating a target disease as disclosed herein (e.g., cancer such as those disclosed herein), or uses of such for manufacturing a medicament for the intended therapeutic purposes

[0044] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIGS. 1A and 1B include diagrams showing that exemplary CAR T cells (anti-CD70 CAR T cells) with Reg1 KO exhibit superior in vitro expansion FIG. 1A: Proliferation of anti-CD70 CAR T cells (CAR T) with Reg1 KO using one of the 10 guides (Z01-Z10) targeting Reg1 as indicated. CAR T indicates anti-CD70 CAR T cells with an unedited (wild-type) Reg1 gene. FIG. 1B: Proliferation of anti-CD70 CAR T cells with Reg1 KO using guide REG1-Z10 (Z10) up to 52 days post HDR. Anti-CD70 CAR T cells with an unedited Reg1 gene are also shown (CAR T). (A) and (B) refer to duplicative assays.

[0046] FIGS. 2A-2E include diagrams showing that exemplary CAR T cells (anti-CD70 CAR T cells) with Reg1 KO (+reg1 KO) exhibit superior in vitro potency against tumor cell lines relative to CAR T cells with an unedited Reg1 gene (CAR T). FIG. 2A: Cell lysis of ACHN cells by anti-CD70 CAR T cells with Reg1 KO, using Regnase guides Z03 or Z10, relative to CAR T cells with an unedited (wild-type) Reg1 gene (CAR T). Cell lysis was measured after 24 h co-culture at day 12 post HDR. FIG. 2B: Cell lysis of ACHN cells by anti-CD70 CAR T cells with Regnase 1 KO using Regnase guides Z05 or Z06 relative to CAR T cells with an unedited Reg1 gene. Cell lysis was measured after 24 h co-culture at day 12 post HDR. FIG. 2C: Cell lysis of ACHN cells by anti-CD70 CAR T cells with Regnase 1 KO, using Regnase guides Z03, Z05, Z06 or Z10 relative to CAR T cells with an unedited Reg1 gene. Cell lysis was measured after 24 h co-culture at day 27 post HDR. FIG. 2D: Cell lysis of caki-1 cells by anti-CD70 CAR T cells with Regnase 1 KO using Regnase guides Z03, Z05, Z06 or Z10 relative to CAR T cells with an unedited Reg1 gene. Cell lysis was measured after 24 h co-culture at day 27 post HDR. FIG. 2E: Cell lysis of 769P cells by anti-CD70 CAR T cells with Regnase 1 KO using Regnase guides Z03, Z05, Z06 or Z10 relative to CAR T cells with an unedited Reg1 gene. Cell lysis was measured after 24 h co-culture at day 27 post HDR.

[0047] FIGS. 3A-3D include diagrams showing that exemplary CAR T cells (anti-CD70 CAR T cells) with Reg1 KO (CAR T+Reg KO, using Z10 guide as an example) express lower levels of T cell exhaustion markers in vitro relative to Reg1 wild-type counterparts (CAR T). FIG. 3A: Day 13 post HDR PD1 expression in CD4+ and CD8+ anti-CD70 CAR T cells with Reg1 KO (+Reg KO) relative to wild-type counterparts. FIG. 3B: Day 26 post HDR PD1 expression in CD4+ and CD8+ anti-CD70 CAR T cells with Reg1 KO (+Reg KO) relative to wild-type counterparts. FIG. 3C: Day 13 post HDR Tim3 expression in CD4+ and CD8+ anti-CD70 CAR T cells with Reg1 KO (+Reg KO) relative to wild-type counterparts. FIG. 3D: Day 26 post HDR Tim3 expression in CD4+ and CD8+ anti-CD70 CAR T cells with Reg1 KO (+Reg KO) relative to wild-type counterparts.

[0048] FIG. 4 is a diagram showing that exemplary CAR T cells (anti-CD19 CAR T cells) with Reg1 KO showed enhanced expansion in the presence of cytokines in vitro and continue to depend on cytokines for in vitro expansion. Anti-CD19 CAR T cells with a Reg1 KO (Anti-CD19 CAR T/Reg KO) and anti-CD19 CAR T cells with a wild-type Reg1 gene (Anti-CD19 CAR T) were cultured in the presence and absence (No cytokines) of cytokines for 40 days.

[0049] FIGS. 5A-5D include diagrams showing that exemplary CAR T cells (anti-CD19 CAR T cells) with Reg1 KO (Anti-CD19 CAR T/Reg KO) provide superior in vivo survival and decreased tumor burden relative to Reg1 wild-type counterparts (Anti-CD19 CAR T) in the intravenous disseminated Nalm-6 human acute lymphoblastic leukemia tumor xenograft mouse model. FIG. 5A: Probability of survival of untreated mice, mice dosed with 4e6 anti-CD19 CAR T cells, and 4e6 anti-CD19 CAR T/Reg KO cells. FIG. 5B: Probability of survival of untreated mice, mice dosed with 8e6 anti-CD19 CAR T cells, and 8e6 anti-CD19 CAR T/Reg KO cells. FIG. 5C: Bioluminescence signal from bioluminescent model leukemia cells in mice treated with 4e6 anti-CD19 CAR T cells or 4e6 anti-CD19 CAR T/Reg KO cells. FIG. 5D: Bioluminescence signal from bioluminescent model leukemia cells in mice treated with 8e6 anti-CD19 CAR T cells or 8e6 anti-CD19 CAR T/Reg KO cells.

[0050] FIGS. 6A-6B include diagrams showing that exemplary CAR T cells (anti-CD70 CAR T cells) with Reg1 KO (CAR T+Reg KO) exhibit superior in vitro potency against tumor cell lines relative to Reg1 wild-type counterparts (CAR T). FIG. 6A: Cell lysis of ACHN cells by anti-CD70 CAR T cells with Reg1 KO using guide REG1-Z10 (CAR T+Reg KO) relative to Reg1 wild-type counterparts (CAR T). Cell lysis was measured after 24 h co-culture at day 19 and 26 post HDR. FIG. 6B: Cell lysis of caki-1 cells by anti-CD70 CAR T cells with Reg1 KO using guide REG1-Z10 (CAR T+Reg KO) relative to Reg1 wild-type counterparts (CAR T). Cell lysis was measured after 24 h co-culture at day 13, 19 and 26 post HDR.

[0051] FIGS. 7A and 7B include diagrams showing knock out of TGFBRII using various guide RNAs as indicated. FIG. 7A: Indel rates of edited TGFBRII gene by eight gRNAs that target different TGFBRII gene exons as indicated. From left to right, EX1_T1, EX1_T3, EX2_T1, EX3_T1, EX3_T2, EX4_T1, EX4_T2, and EX5_T1, the nucleotide sequence of each of which is provided in Table 32. FIG. 7B: immunoblot of TGFBRII expression in gene-edited T cells. GAPDH was used as a loading control. The mock sample is unedited T cells with wild-type TGFBRII.

[0052] FIGS. 8A-8K include diagrams showing the effect of TGF-.beta. on CAR T cell expansion. Anti-CD70 CAR T cells were exposed to different concentrations of recombinant human TGF-.beta. (10, 20, 50, 100 ng/ml) and cell number was recorded at different time points (FIG. 8A). T cells with or without TGFBRII knock-out, generated using different TGFBRII gRNAs as indicated, were incubated with 0 or 50 ng/ml of TGFB-.beta. and cell expansion was recorded over time (FIGS. 8B-8K).

[0053] FIG. 9 is a diagram showing the effect of TGFBRII KO on CAR T cell killing ability against A498 cells at various E:T ratios as indicated. TGFBRII KO improves cytotoxicity of CAR-T cells.

[0054] FIGS. 10A-10E include diagrams showing the effect of TGFBRII KO on CAR T cell kill ability against multiple tumor cell lines. The cell kill capacity of anti-CD70 CAR T cells was compared to anti-CD70 CAR T cells with TGFBRII KO. Cell killing activity of the CAR T cells was assessed against CAM-1 (FIG. 10A) H1975 (FIG. 10B), Hs-766T (FIG. 10C), 786-O (FIG. 10D) and SK-OV3 (FIG. 10E). TGFBRII KO improves cytotoxicity of CAR-T cells.

[0055] FIG. 11 is a graph showing the effect of TGFBRII KO on CAR T cell phenotype. Anti-CD70 CAR T cells with or without TGFBRII KO were exposed to 50 ng/ml recombinant human TGF-.beta. and the expression of CD25 was assessed by flow cytometry. TGFBRII KO protects CAR T from TGF-.beta. inhibitory effect on cell phenotype.

[0056] FIG. 12 is a graph showing that TGFBRII KO protects CAR T cells against TGF-.beta. inhibitory effect on cytotoxicity. Anti-CD70 CAR T cells was co-cultured with target tumor cells (A498) in the presences or absence of TGF-.beta. (0, 1, 10, 50 ng/ml) The ability of anti-CD70 CAR T cells with unedited TGFBRII to kill target cells, were compared to anti-CD70 CAR T with TGFBRII KO using an exemplary guide RNA as indicated.

[0057] FIGS. 13A-13C include diagrams showing that TGFBRII KO anti-CD70 CAR T cells are resistant to TGF-.beta. inhibitory effects on effector function. Anti-CD70 CAR T cells were co-cultured with target cells (A498) with TGF-.beta. (50 ng/ml) or without TGF-.beta. and compared to anti-CD70 CAR T with TGFBRII KO (e.g.: anti-CD70 CAR+TGFBRII_EX4_T1) in their ability to kill target cells. T cell proliferation (FIG. 13A) and effector cytokine secretion was assessed by Luminx (FIGS. 13B and 13C).

[0058] FIG. 14 is a graph showing fibroblasts reduce CAR-T cell cytolytic activity. Anti-CD70 CAR T was co-cultured with target cells (A498) with or without fibroblast (CCL-190) placed in a transwell plate at 0.25:1, fibroblast: anti-CD70 CAR T.

[0059] FIGS. 15A-15C include graphs showing that TGFBRII KO protects CAR-T cells against the inhibitory effect of fibroblasts. Anti-CD70 CAR T was co-cultured with target cells (A498) at 0.1:1 (E:T) in presence of different volumes of conditioned media from CCL-190 (2.5, 5, 10 .mu.L) and the cell kill capacity was evaluated and compared to cells with TGFBRII KO. The ability of anti-CD70 CAR T cells (with or without TGFBRII KO) to kill target cells is shown in Hs-766T pancreatic tumor cells (FIG. 15A), A498 kidney tumor cells (FIG. 15B), and H1975 lung tumor cells (FIG. 15C).

[0060] FIG. 16A-16B include diagrams showing synergistic effects of TGFBRII and Regnase double disruptions with in vitro rechallenge of CAR T Cells with ACHN. FIG. 16A: improved potency. FIG. 16B: improved CAR expansion.

[0061] FIG. 17A-17B include diagrams showing synergistic effects of disrupting both TGFBRII and Regnase genes in cancer xenograph models. FIG. 17A: CAKI-1 renal cell carcinoma xenograph model with anti-CD70 CAR T cells. FIG. 17B: H1975 lung cancer xenograph model with anti-CD70 CAR T cells.

[0062] FIG. 18A-18B include diagrams showing synergistic effects of disrupting both TGFBRII and Regnase genes in an RCC rechallenge xenograph model. FIG. 18A: reduction in RCC (A498) tumor size. FIG. 18B: inhibition of RCC tumor cell growth following rechallenge with ACHN cells.

[0063] FIG. 19A-19B include diagrams showing impact of Reg1 and/or TGFBRII disruption on CAR-T cell differentiation and expansion in vivo. FIG. 19A: CAR-T cell differentiation. FIG. 19B: CAR-T cell expansion.

[0064] FIG. 20A-20B include diagrams showing synergistic effects of TGFBRII and Regnase double knock-out in a Nalm6-leukemia (B-ALL) mouse model. FIG. 20A: reduction in tumor size. FIG. 20B: Survival rates.

[0065] FIG. 21 is a diagram showing survival advantage arising from TGFBRII and Regnase double disruptions in a NOG Mantle cell lymphoma (MCL) tumor xenograft mouse model.

[0066] FIGS. 22A-22B include diagrams showing increased in vivo expansion of CAR-T cells having TGFBRII and/or Regnase knock-out. FIG. 22A shows CAR T cell expansion in the Jeko-1 xenograph model. FIG. 22B shows CAR T cell expansion in the nalm-6 xenograph model.

[0067] FIGS. 23A-23D include diagrams showing consistent rates of CRISPR/Cas editing in anti-BCMA CAR-T cells with Reg-1 and/or TGFBRII disruption as determined by flowcytometry. FIG. 23A: levels of TCR.sup.- cells. FIG. 23B: levels of .beta.2M.sup.- cells. FIG. 23C: levels of CAR.sup.+ cells. FIG. 23D: ratio of CD4.sup.+/CD8.sup.+ cells.

[0068] FIGS. 24A-24B include diagrams showing consistent edit editing rates in anti-BCMA CAR-T cells with Reg-1 and/or TGFBRII disruptions. FIG. 24A: TGFBRII disruption efficiency. FIG. 24B: Reg-1 disruption efficiency.

[0069] FIGS. 25A-25D include diagrams showing superior cell cytotoxicity of TRAC-/.beta.2M-/Reg-1- TGFBRII- anti-BCMA CAR+ T-cells. FIGS. 25A-25B: cytotoxicity against MM1s (multiple myeloma cell line) cells (25A) relative to K562 cells (25B). FIGS. 25C-25D: cytotoxicity against JeKo-1 cells (mantle cell lymphoma cell line) (25C) relative to K562 cells (25D).

[0070] FIGS. 26A-26C include diagrams showing that the combined disruption of Regnase-1 and TGFBRII improved anti-BCMA CAR-T activity against murine multiple myeloma in an animal model. FIG. 26A: tumor volume reduction. FIG. 26B: survival rate. FIG. 26C: CAR-T cell expansion in peripheral blood.

[0071] FIGS. 27A-27F include diagrams showing that the combined disruption of Regnase and TGFBRII improves anti-BCMA CAR-T activity against murine mantle cell lymphoma in an animal model. FIG. 27A: tumor volume reduction. FIG. 27B: survival rate. FIG. 27C: CAR-T cell expansion in peripheral blood. FIG. 27D: PD-1 and LAG-3 levels in CAR T cells. FIG. 27E: levels of circulating T cells at three weeks post CAR-T injection. FIG. 27F: levels of exhaustion markers (LAG-3 and PD-1) on circulating T cells at three weeks post CAR-T injection.

[0072] FIG. 28 is a diagram showing disruption of TGFBRII and Reg-1 genes increases proliferation of anti-PTK7 CAR T cells.

[0073] FIGS. 29A-29D include diagrams showing impact of TGFBRII disruption, optionally in combination with Reg-1 disruption, in long-term in vitro rechallenge assays. FIG. 29A: TGFBRII disruption alone improves anti-PTK7 CAR T-cell potency in a long-term in vitro rechallenge assay. FIG. 29B: TGFBRII disruption improves anti-PTK7 CAR T-cell persistence and expansion in a long-term in vitro rechallenge assay as measured by humCD45+ cells. FIG. 29C: TGFBRII disruption enhances cytotoxic CD8+ T cells expressing the anti-PTK7 CAR. FIG. 29D: CD4+ cells expressing the anti-PTK7 CAR remains consistent regardless of TGFBRII disruption.

[0074] FIGS. 30A-30B include diagrams showing anti-tumor activity of anti-PTK7 CAR T-cells with or without TGFBRII disruption. FIG. 30A: effect of treatment on tumor volume. FIG. 30B: effect of treatment on body weight. .circle-solid. Group 1: no treatment. .smallcircle.: Group 2: anti-PTK7 CAR-T cells, 5.times.10.sup.6 cells/mouse (iv) Day 1. .box-solid.: Group 3: anti-PTK7 CAR/TGFBRII KO T cells, 5.times.10.sup.6 cells/mouse (iv) Day 1. UTA chow was administered 9 days prior to CAR-T cell treatment to applicable groups.

[0075] FIGS. 31A-31B include diagrams showing T cell fractions in a pancreatic cell carcinoma (Hs766T) tumor xenograft animal model treated with anti-PTK7 CAR T cells with or without TGFBRII disruption. FIG. 31A: number of humCD45+ cells/ul in murine blood at Day 47 post dose. FIG. 31B: CAR-T cell differentiation at Day 47 post dose.

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present disclosure aims at establishing genetically engineered T cells having improved growth activity, persistence, reduced T cell exhaustion, and enhanced potency, a long-felt need in CAR-T therapy. 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 both CAR-T cell manufacturing and clinical applications.

[0077] Conventional allogenic CAR T cells are produced wherein a single donor leukopak is edited in most cases so that the cells can avoid components of the patient immune system and thus do not cause GvHD. The process of expanding these CAR T cells can yield 10s to 100s of vialed drug product. Patients may receive a single dose or multiple doses. During the manufacturing process, these CAR T cells lose potential due to various mechanisms, for example, apoptosis, exhaustion, replicative senescence, and other processes where the cells become less fit.

[0078] The genetically engineered T cells having a disrupted TGFBRII gene, a disrupted Reg1 gene, or a combination thereof, and optionally one or more additional genetic edits, for example, a disrupted TRAC gene, a disrupted .beta.2M gene, a disrupted CD70 gene, and/or an inserted nucleic acid coding for a chimeric antigen receptor (CAR), or a combination thereof.

[0079] Unexpectedly, the present disclosure reports that knocking out Reg1 in T cells led to various advantageous features in T cell-mediated cell therapy such as CAR-T therapy. Examples include, but are not limited to: improved cell culture growth and in vitro expansion including faster expansion, longer viability, faster proliferation and/or increased resistance to apoptosis, which are beneficial for manufacturing and production of therapeutic T-cell based products such as CAR-T cells; T cell potency advantages related to maintaining therapeutic T cells (e.g., CAR-T cells) in vitro and in vivo potency and activity (target cell killing) for a more effective and persistent T-cell based therapeutic products; production and/or retention of more central memory cells; lower expression of T cell exhaustion markers (such as, PD-1, Tim-3); improved efficacy of T cell therapeutics in vivo, related to decreasing tumor burden and increasing survival of CAR T treated subjects.

[0080] Further, unexpectedly, T cells having a disrupted TGFBRII gene showed advantageous features, including improved cell growth and expansion, enhanced cytotoxicity activity, resistant to the inhibitory effect mediated by TGF.beta., and/or mediated by fibroblasts. Given such advantageous features, the genetically engineered T cells (e.g., CAR-T cells) disclosed herein, having a disrupted TGFBRII gene and optionally other genetic edits as disclosed herein, would be expected to exhibit superior therapeutic effects, for example, superior anti-tumor effects, e.g., in TME of a solid tumor.

[0081] Moreover, CAR-T cells having both a disrupted Reg1 gene and a disrupted TGFBRII gene showed much higher anti-tumor activities, as well as CAR-T cell expansion in animal models as relative to CAR-T cells having a disrupted Reg1 gene or a disrupted TGFBRII gene.

[0082] Other unlimited advantageous features of the T cells provided herein include:

[0083] (a) Improved quality and consistency of CAR-T cell-based therapeutics.

[0084] (b) Greater potency and longer-lived potency of CAR-T cells produced from the T cells in human patients.

[0085] (c) Reduced dosage requirement. Because the T cells disclosed herein have enhanced proliferation and expansion capacities, they can live longer in vivo. As such, a lower dose relative to standard CAR-T therapy may be used to achieve substantially similar therapeutic effects relative to a high dose of conventional CAR-T cell therapy.

[0086] (d) Increased efficacy resulting from enhanced proliferation and expansion of the CAR-T cells disclosed herein, enhanced cytotoxicity, and prolonged persistence in vivo. Further, the T cells would provide the benefit of titratable dosing in patients to optimize safety and efficacy as noted above.

[0087] (e) Extended therapeutic effects due to reduced exhaustion and/or replicative senescence and prolonged persistence of the T cells both in vitro and in vivo.

[0088] (f) Enhanced anti-tumor activity, e.g., reduction of tumor size and/or elongated survival rates.

[0089] Accordingly, provided herein are T cells having improved persistence in culture, methods of producing such T cells, and methods of using such T cells for producing therapeutic T cells such as CAR-T cells. 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.

I. Genetically Engineered T Cells Having Enhanced Features

[0090] The T cells disclosed herein comprises genetically engineered T cells having enhanced persistence in culture. Such genetically engineered T cells may have genetic editing of the Reg1 gene or genetic editing of the TGFBRII gene. In some instances, such genetically engineered T cells may have genetic editing of both the Reg1 gene and the TGFBRII gene.

[0091] In some embodiments, the genetically engineered T cells may have genetic editing in one or more additional genes involved in T cell exhaustion, such as CD70. As shown by the studies disclosed herein, such genetically engineered T cells show one or more of the following superior features as relative to the T cell counterparts having a wild-type Regnase 1 gene: enhanced expansion capacity in culture (e.g., expandable in culture for at least 4 weeks, e.g., at least 6 weeks; and/or at least 10-fold expandable, for example, at least 15-fold expandable, relative to the non-edited counterpart), enhanced longevity, enhanced proliferation capacity, greater T cell activation, enhanced potency, enhanced expression of central memory T cell markers, and reduced expression of T cell exhaustion markers.

[0092] 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. 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.

[0093] In some embodiments, the genetically engineered T cells carry a disrupted Reg1 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 or .beta.2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7).

[0094] In some embodiments, the genetically engineered T cells carry a disrupted TGFBRII 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 or .beta.2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7). In some examples, the genetically engineered T cells may express an anti-PTK7 CAR such as those disclosed herein. In some instances, such genetically engineered T cells may have a wild-type endogenous Reg-1 gene.

[0095] In some embodiments, the genetically engineered T cells carry a disrupted Reg1 gene and a disrupted TGFBRII 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 or .beta.2M. Such genetically engineered T cells may further express a chimeric antigen receptor (CAR), which may be capable of binding to an antigen of interest, for example, a tumor associated antigen (e.g., CD19, BCMA, CD70, CD33, or PTK7). 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.

[0096] (a) Genetically Edited Genes

[0097] In some aspects, the present disclosure provides genetically engineered T cells that may comprise a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof. In some embodiments, the genetically engineered T cells provided herein comprise both a disrupted Reg1 gene and a disrupted TGFBRII gene. In some instances, the genetically engineered T cells disclosed herein may further comprise a disrupted CD70 gene, a disrupted .beta.2M gene, a disrupted TRAC gene, or a combination thereof.

[0098] 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.

[0099] 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.

[0100] Reg1 Gene Editing

[0101] In some embodiments, the genetically engineered T cells may comprise a disrupted gene involved in mRNA decay. Such a gene may be Reg1. Reg1 contains a zinc finger motif, binds RNA and exhibits ribonuclease activity. Reg1 plays roles in both immune and non-immune cells and its expression can be rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation. Human Reg1 gene is located on chromosome 1p34.3. Additional information can be found in GenBank under Gene ID: 80149.

[0102] In some examples, the genetically engineered T cells may comprise a disrupted Reg1 gene such that the expression of Reg1 in the T cells is substantially reduced or eliminated completely. The disrupted Reg1 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 Reg1 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, exon 5, exon 6, or a combination thereof. In some examples, one or more genetic editing may occur in exon 2 or exon 4. Such genetic editing may be induced by the CRISPR/Cas technology using a suitable guide RNA, for example, those listed in Table 22. The resultant edited Reg1 gene using a gRNA listed in Table 22 may comprise one or more edited sequences provided in Tables 29-38 below.

[0103] TGFBRII Gene Editing

[0104] In some embodiments, the genetically engineered T cells may comprise a disrupted TGFBRII gene, which encodes Transforming Growth Factor Receptor Type II (TGFBRII). TGFBRII receptors are a family of serine/threonine kinase receptors involved in the TGF.beta. signaling pathway. These receptors bind growth factor and cytokine signaling proteins in the TGF.beta. family, for example, TGF.beta.s (TGF.beta.1, TGF.beta.2, and TGF.beta.3), bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activin and inhibin, myostatin, anti-Mullerian hormone (AMH), and NODAL.

[0105] In some examples, the genetically engineered T cells may comprise a disrupted TGFBRII gene such that the expression of TGFBRII in the T cells is substantially reduced or eliminated completely. The disrupted TGFBRII 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 TGFBRII 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, exon 5, or a combination thereof. In some examples, one or more genetic editing may occur in exon 4 and/or exon 5. 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 listed in Table 39. The resultant edited TGFBRII gene using a gRNA listed in Table 39 may comprise one or more edited sequences provided in Tables 40-48 below.

[0106] CD70 Gene Editing

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] In some embodiments, the gRNA targeting CD70 listed in Table 23 (CD70-7) may be used for disrupting the CD70 gene via CRISPR/Cas9 gene editing. In some examples, an edited CD70 gene may comprise a nucleotide sequence selected from the following sequences in Table 26.

[0113] .beta.2M Gene Edit

[0114] In some embodiments, the genetically engineered T cells disclosed herein may further comprise a disrupted .beta.2M gene. .beta.2M 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 .beta.2M gene is eliminated to prevent a host-versus-graft response.

[0115] In some embodiments, an edited .beta.2M gene may comprise a nucleotide sequence selected from the following sequences in Table 25. It is known to those skilled in the art that different nucleotide sequences in an edited gene such as an edited .beta.2M gene (e.g., those in Table 25) may be generated by a single gRNA such as the one listed in Table 23 (.beta.2M-1). See also WO2019097305, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0116] 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.

[0117] TRAC Gene Edit

[0118] 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.

[0119] In some embodiments, an edited TRAC gene may comprise a nucleotide sequence selected from the following sequences in Table 24. It is known to those skilled in the art that different nucleotide sequences in an edited gene such as an edited TRAC gene (e.g., those in Table 24) may be generated by a single gRNA such as the one listed in Table 23 (TA-1). 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.

[0120] (b) Exemplary Improved Features of Genetically Engineered T Cells Disclosed Herein

[0121] Any of the genetically engineered T cell having a disrupted Reg1 gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted .beta.2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may be expandable in culture for greater than 4 weeks, for example, greater than 5 weeks, greater than 6 weeks, greater than 8 weeks, and greater than 10 weeks. In some examples, the genetically engineered T cells comprise a disrupted Reg1 (optionally, disruptions in CD70) and are expandable after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may maintain the ability to be activated after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Further, such genetically engineered T cells have an increased expansion capacity, which can be at least 10-fold (e.g., at least 15-fold) higher than the non-engineered counterparts, i.e., T cells having the same genetic background as the engineered T cells disclosed herein except that the counterpart T cells have a wild-type Reg1 gene.

[0122] Further, the genetically engineered T cells disclosed herein may exhibit enhanced T cell persistence. "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.

[0123] Alternatively or in addition, the genetically engineered T cells disclosed may grow faster and longer than the non-engineered T cells, for example, as observed in vitro cell culture. In some instances, the genetically engineered T cells may grow at least 50% (e.g., at least 1-fold, at least 2-fold, at least 5-fold, or more) than the non-engineered T cells in a conventional in vitro T cell culture (e.g., as described in Examples below). In other instances, the genetically engineered T cells may maintain a high growth rate (e.g., having substantially the same growth rate or with only a slight reduction) in vitro for at least 20 days (e.g., at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, or longer).

[0124] In addition, the genetically engineered T cells may exhibit a reduced level of cell exhaustion as relative to the non-engineered T cell counterpart. In some instances, a reduced level of cell exhaustion is reflected by a higher level of central memory T cells in the whole T cell population. The population of genetically engineered T cells disclosed may comprise a higher number of central memory T cells as compared to non-engineered T cell counterparts. For example, in some instances the population of genetically engineered T cells include a higher number of central memory T cells that are characterized by enhanced expression of CD27 and/or CD45RO as compared to non-engineered T cell counterparts. In some instances, the population of genetically engineered T cells disclosed exhibit reduced T cell exhaustion, which is characterized, for example, by reduced expression of PD-1 and/or TIM3 as compared to non-engineered T cell counterparts.

[0125] Any of the genetically engineered T cell having a disrupted TGFBRII gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted .beta.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 TGFBRII 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. Such genetically engineered T cells (e.g., CAR-T cells) may also be resistant to inhibitory effects mediated by the TGF.beta. signaling and/or by fibroblast (e.g., in TME). For example, the genetically engineered T cells with a disrupted TGFBRII gene may be resistant to inhibitory factors secreted by fibroblasts.

[0126] In some embodiments, the genetically engineered T cells may further comprise one or more disrupted genes (e.g., CD70, Reg1, or a combination thereof) 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.

[0127] For example, such genetically engineered T cells may be expandable in culture for greater than 4 weeks, for example, greater than 5 weeks, greater than 6 weeks, greater than 8 weeks, and greater than 10 weeks. In some examples, the genetically engineered T cells comprise a disrupted TGFBRII gene, and a disrupted CD70 gene, Reg1 gene, or both may be expandable after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may maintain the ability to be activated after 6 weeks (e.g., after 7 weeks, after 8 weeks, after 9 weeks, or after 10 weeks) in culture. Such genetically engineered T cells may exhibit more improved growth and expansion capacity relative to the T cells having the same genetic background except for an undisrupted TGFBRII gene, and an undisrupted CD70 gene and/or Reg1 gene.

[0128] In addition, any of the genetically engineered T cell having a disrupted TGFBRII gene and a disrupted Reg1 gene, and optionally one or more additional genetic edits, for example, a disrupted CD70 gene, a disrupted TRAC gene, a disrupted .beta.2M gene, a CAR-coding nucleic acid insertion, or a combination thereof, may have expansion advantage (e.g., in vivo) over counterpart T cells, i.e., having the disrupted TGFBRII gene or the disrupted Reg1 gene (but not both), as well as the other additional genetic edits. CAR-T cells having disruptions of both the TGFBRII gene and the Reg1 gene were found to be more potent in cancer treatment than the counterpart T cells as observed in xenograft mouse models. Accordingly, CAR-T cells having disruptions of both the TGFBRII gene and the Reg1 gene would be expected to show superior cancer treatment efficacy.

[0129] (c) Methods of Making Genetically Engineered T Cells

[0130] 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.

[0131] (a) T Cells

[0132] In some embodiments, T cells 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.TM. separation.

[0133] 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.

[0134] A specific subpopulation of T cells, expressing one or more of the following cell surface markers: TCRab, 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 TCRab, 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.

[0135] 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.

[0136] 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.

[0137] Alternatively, the T cells may be derived from stem cells (e.g., HSCs or iPSCs) via in vitro differentiation.

[0138] T cells from a suitable source 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.

[0139] In some embodiments, T cells are 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.

[0140] (b) Gene Editing Methods

[0141] 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.

[0142] 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.

[0143] 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).

[0144] 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.

[0145] CRISPR-Cas9 Gene Editing System

[0146] 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). 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).

[0147] 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.

[0148] 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).

[0149] 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).

[0150] 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.

[0151] Endonuclease for Use in CRISPR

[0152] 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).

[0153] 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.

[0154] 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.

[0155] 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).

TABLE-US-00001 Amino acid sequence of Cas9 nuclease (SEQ ID NO: 1): MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

[0156] 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.

[0157] Guide RNAs (gRNAs)

[0158] 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.

[0159] 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.

[0160] 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).

[0161] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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: 69), then the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC**-3' (SEQ ID NO: 61). 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 23 below. In these exemplary sequences, the fragment of "n" refers to the spacer sequence at the 5' end.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] In some embodiments, the gRNAs disclosed herein target a Reg1 gene, for example, target a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the Reg1 gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 2 or exon 4 of a Reg1 gene, or a fragment thereof. Exemplary target sequences of Reg1 and exemplary gRNA sequences are provided in Table 22 below.

[0176] In some embodiments, the gRNAs disclosed herein target a TGFBRII gene, for example, target a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the TGFBRII gene. Such a gRNA may comprise a spacer sequence complementary (complete or partially) to the target sequences in exon 4 or exon 5 of a TGFBRII gene, or a fragment thereof. Exemplary target sequences of TGFBRII and exemplary gRNA sequences are provided in Table 39 below.

[0177] 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 23 below.

[0178] In some embodiments, the gRNAs disclosed herein target a .beta.2M gene, for example, target a suitable site within a .beta.2M gene. See also WO2019097305, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. Other gRNA sequences may be designed using the .beta.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 .beta.2M genomic region and RNA-guided nuclease create breaks in the .beta.2M genomic region resulting in Indels in the .beta.2M gene disrupting expression of the mRNA or protein.

[0179] 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.

[0180] Exemplary spacer sequences and gRNAs targeting a .beta.2M gene or TRAC gene are provided in Table 23 below.

[0181] 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.

[0182] 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.

[0183] 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).

[0184] 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).

[0185] 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.

[0186] 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%.

[0187] Delivery of Guide RNAs and Nucleases to T Cells

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Other Gene Editing Methods

[0193] 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.

[0194] 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 Fold nuclease with a zinc finger DNA binding domain.

[0195] 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.

[0196] Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and W.beta./SPBc/TP901-1, whether used individually or in combination.

[0197] 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.

[0198] 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.

[0199] 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.

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

[0200] The genetically engineered T cells having a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination of disrupted Reg1 gene and disrupted TGFBRII gene. Optionally, such genetically engineered T cells may comprise one or more of additional disrupted genes, e.g., .beta.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.

[0201] (a) Chimeric Antigen Receptor (CAR)

[0202] 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.

[0203] There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta (.zeta. 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.zeta. 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.

[0204] 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.zeta.) 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: 95 and SEQ ID NO: 96 as provided in Table 27 below. Other signal peptides may be used.

[0205] (i) Antigen Binding Extracellular Domain

[0206] 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.

[0207] The antigen-binding extracellular domain may be specific to a target antigen of interest, for example, a pathologic antigen such as a 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.

[0208] In some embodiments, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds a tumor antigen as disclosed herein. The scFv may comprise an antibody heavy chain variable region (V.sub.H) and an antibody light chain variable region (V.sub.L), which optionally may be connected via a flexible peptide linker. In some instances, the scFv may have the V.sub.H to V.sub.L orientation (from N-terminus to C-terminus). Alternatively the scFv may have the V.sub.L to V.sub.H orientation (from N-terminus to C-terminus).

[0209] Exemplary tumor antigens include, but are not limited to, CD19, BCMA, CD70, CD33, and PTK7. 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, WO2020/095107, and International Patent Application No. PCT/IB2021/053849, the relevant disclosures of which are herein incorporated by reference for the purposes and subject matter referenced herein.

[0210] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD19. In some instances, the anti-CD19 scFv may comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 125. In some specific examples, the anti-CD19 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 108-110, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD19 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:105-107 as determined by the Kabat method. Alternatively, the anti-CD19 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 114-116, respectively as determined by the Chothia method. Alternatively or in addition, the anti-CD19 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:111-113 as determined by the Chothia method. In one specific example, the anti-CD19 scFv may comprise a V.sub.H comprising the amino acid sequence of SEQ ID NO: 124 and a V.sub.L comprises the amino acid sequence of SEQ ID NO: 125. See Sequence Table 27 below.

[0211] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD70. In some instances, the anti-CD70 scFv may comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 144. In some specific examples, the anti-CD70 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 132, 134, and 136, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD70 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:127, 129, and 130, respectively as determined by the Kabat method. Alternatively, the anti-CD70 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 133, 135, and 137, respectively as determined by the Chothia method. Alternatively or in addition, the anti-CD70 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NO:128, LAS, and SEQ ID NO:131, respectively as determined by the Chothia method. In one specific example, the anti-CD70 scFv may comprise a V.sub.H comprising the amino acid sequence of SEQ ID NO: 143 and a V.sub.L comprises the amino acid sequence of SEQ ID NO: 144. See Sequence Table 27 below.

[0212] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human BCMA. In some instances, the anti-BCMA scFv may comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 149; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 150. In some specific examples, the anti-BCMA antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 155, 157, and 159, respectively as determined by the Kabat method. Alternatively or in addition, the anti-BCMA antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:151, 152, and 153, respectively as determined by the Kabat method. Alternatively, the anti-BCMA antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 156, 158, and 160, respectively as determined by the Chothia method. Alternatively or in addition, the anti-BCMA antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:151, 152, and 154, respectively as determined by the Chothia method. In one specific example, the anti-BCMA scFv may comprise a V.sub.H comprising the amino acid sequence of SEQ ID NO: 149 and a V.sub.L comprises the amino acid sequence of SEQ ID NO: 150. See Sequence Table 27 below.

[0213] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD33. Exemplary anti-CD33 scFv and anti-CD33 CAR constructs can be found, for example, in Sequence Table 27 below and in WO2020/095107, the relevant disclosures of which are incorporated by reference for the subject matter and purpose noted herein.

[0214] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human CD33. In some instances, the anti-CD33 scFv may comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 334; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 335. In some specific examples, the anti-CD33 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 328-330, respectively as determined by the Kabat method. Alternatively or in addition, the anti-CD33 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:331-333, respectively as determined by the Kabat method. In one specific example, the anti-BCMA scFv may comprise a V.sub.H comprising the amino acid sequence of SEQ ID NO: 149 and a V.sub.L comprises the amino acid sequence of SEQ ID NO: 150. See Sequence Table 27 below.

[0215] In some examples, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds human PTK7. In some instances, the anti-PTK7 scFv may comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 346; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 347. In some specific examples, the anti-PTK7 antibody discloses herein may comprise the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 set forth as SEQ ID NOs: 340-342, respectively as determined by the Kabat method. Alternatively or in addition, the anti-PTK7 antibody discloses herein may comprise the light chain CDR1, light chain CDR2, and light chain CDR3 set forth as SEQ ID NOs:343-345, respectively as determined by the Kabat method. In one specific example, the anti-BCMA scFv may comprise a V.sub.H comprising the amino acid sequence of SEQ ID NO: 346 and a V.sub.L comprises the amino acid sequence of SEQ ID NO: 347. See Sequence Table 27 below.

[0216] Two antibodies having the same V.sub.H and/or V.sub.L CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf.org.uk/abs/ or abysis.org/abysis/sequence_input).

[0217] (ii) Transmembrane Domain

[0218] 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.

[0219] 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: 97 as provided below in Table 27. Other transmembrane domains may be used.

[0220] (iii) Hinge Domain

[0221] 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.

[0222] 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.

[0223] (iv) Intracellular Signaling Domains

[0224] Any of the CAR constructs contain one or more intracellular signaling domains (e.g., CD3.zeta., 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.

[0225] CD3.zeta. is the cytoplasmic signaling domain of the T cell receptor complex. CD3.zeta. 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.zeta. provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signaling.

[0226] 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.zeta.. 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.zeta. signaling domain and a CD28 co-stimulatory domain. In other embodiments, a CAR includes a CD3.zeta. signaling domain and 4-1BB co-stimulatory domain. In still other embodiments, a CAR includes a CD3.zeta. signaling domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

[0227] Table 27 provides examples of signaling domains derived from 4-1BB, CD28 and CD3-zeta that may be used herein.

[0228] In specific examples, the anti-CD19 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 118, which may be encoded by the nucleotide sequence of SEQ ID NO: 117. Alternatively, the anti-CD19 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:353.

[0229] In other examples, the anti-BCMA CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 146, which may be encoded by the nucleotide sequence of SEQ ID NO: 145. Alternatively, the anti-CDBCMA CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:355.

[0230] In other examples, the anti-CD70 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 138, which may be encoded by the nucleotide sequence of SEQ ID NO: 141. Alternatively, the anti-CD70 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:354.

[0231] In some examples, the anti-CD33 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 338 or 339. Alternatively, the anti-CD33 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:356 or 357.

[0232] In some examples, the anti-PTK7 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 349 or 350. Alternatively, the anti-PTK7 CAR may be a mature form without the N-terminal signal peptide, e.g., comprising the amino acid sequence of SEQ ID NO:358 or 359.

[0233] See sequence Table 27 provided below.

[0234] (b) Delivery of CAR Construct to T Cells

[0235] 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.

[0236] 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).

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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 .beta.2M gene to disrupt the .beta.2M gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of .beta.2M leads to loss of function of the endogenous MHC Class I complexes. For example, a disruption in the .beta.2M gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more .beta.2M genomic regions. Any of the gRNAs specific to a .beta.2M gene and the target regions disclosed herein can be used for this purpose.

[0242] In some examples, a genomic deletion in the .beta.2M 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 .beta.2M gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more .beta.2M genomic regions, and inserting a CAR coding segment into the .beta.2M gene.

[0243] 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 CD70 gene to disrupt the CD70 gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of CD70 leads to loss of function of the endogenous CD70 protein. For example, a disruption in the CD70 gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more CD70 genomic regions. Any of the gRNAs specific to a CD70 gene and the target regions disclosed herein can be used for this purpose.

[0244] In some examples, a genomic deletion in the CD70 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 CD70 gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more CD70 genomic regions, and inserting a CAR coding segment into the CD70 gene.

[0245] 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 Reg1 gene to disrupt the Reg1 gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of Reg1 leads to loss of function of the endogenous Reg1 protein. For example, a disruption in the Reg1 gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more Reg1 genomic regions. Any of the gRNAs specific to a Reg1 gene and the target regions disclosed herein can be used for this purpose.

[0246] In some examples, a genomic deletion in the Reg1 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 Reg1 gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more Reg1 genomic regions, and inserting a CAR coding segment into the Reg1 gene.

[0247] 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 TGFBRII gene to disrupt the TGFBRII gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of Reg1 leads to loss of function of the endogenous TGFBRII receptor. For example, a disruption in the TGFBRII gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more TGFBRII genomic regions. Any of the gRNAs specific to a TGFBRII gene and the target regions disclosed herein can be used for this purpose.

[0248] In some examples, a genomic deletion in the TGFBRII 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 TGFBRII gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more TGFBRII genomic regions, and inserting a CAR coding segment into the TGFBRII gene.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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 (IDLY)).

[0253] 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.alpha. promoter, see, e.g., SEQ ID NO: 167 provided in Table 28 below. Other promoters may be used.

[0254] 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.

[0255] 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 .beta.2M disruption 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.

[0256] 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 SEQ ID NO: 69. In some specific examples, the donor template for delivering the anti-CD19 CAR may comprise a nucleotide sequence of SEQ ID NO: 117, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69.

[0257] 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: 69. In some specific examples, the donor template for delivering the anti-BCMA CAR may comprise a nucleotide sequence of SEQ ID NO: 145, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69.

[0258] 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: 69. In some specific examples, the donor template for delivering the anti-CD70 CAR may comprise a nucleotide sequence of SEQ ID NO: 139, which can be inserted into a disrupted TRAC gene, for example, replacing the fragment of SEQ ID NO: 69.

[0259] The genetically engineered T cells having a disrupted Reg1 gene, additional disrupted genes, e.g., .beta.2M, TRAC, 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 Reg1 gene may be disrupted first, followed by disruption of TRAC and .beta.2M genes and CAR insertion. In other embodiments, TRAC and .beta.2M genes may be disrupted first, followed by CAR insertion and disruption of the Reg1 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., Reg1, .beta.2M, TRAC, CD70, etc.

[0260] 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., Reg1, .beta.2M, TRAC, CD70, etc.

[0261] (c) Exemplary Genetically Engineered T Cells Expression a Chimeric Antigen Receptor

[0262] 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 .beta.2M gene edit may be considered a .beta.2M knockout cell if .beta.2M protein cannot be detected at the cell surface using an antibody that specifically binds .beta.2M protein.

[0263] In some embodiments, a population of genetically engineered T cells disclosed herein express a CAR (e.g., anti-CD19, anti-BCMA, or anti-CD70 CAR), a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted TRAC gene, and optionally a disrupted .beta.2M gene, and optionally a disrupted CD70 gene. The nucleotide sequence encoding the CAR may be inserted in the disrupted TRAC gene (e.g., replacing the site targeted by a sgRNA such as TA-1). In some examples, such a population of genetically engineered T cells may comprise about 70-99% Reg1.sup.- cells, for example about 90-97% Reg1.sup.- cells, about 70-99% TGFBRII.sup.- cells, e.g., for example about 80-89% TGFBRII.sup.- cells, about 70-99% TCR.sup.- cells, for example about 90-99% TCR.sup.- cells, and/or optionally about 60-99% .beta.2M.sup.- cells, for example about 60-82% .beta.2M.sup.- cells, and/or optionally about 70-99% CD70.sup.- cells, for example about 90-99% CD70.sup.- cells. The cell population may also contain at least about 30%-50% (e.g., at least 60%) cells expressing the CAR.

[0264] i. Anti-CD19 CAR T Cells Having Reg1 and/or TGFBRII Gene Disruption

[0265] Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof, and expressing an anti-CD19 CAR, e.g., those disclosed herein. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-CD19 CAR, e.g., those disclosed herein. In some examples, the anti-CD19 CAR-T cells disclosed herein, which express any of the anti-CD19 CAR disclosed herein (e.g., the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 106), may also comprise a disrupted TRAC gene and/or a disrupted .beta.2M gene as also disclosed herein.

[0266] In some examples, the population of genetically engineered T cells are anti-CD19 CAR cells that further comprise a disrupted Regnanse-1 gene. In some examples, anti-CD19 CAR cells are CD19-directed T cells having disrupted TRAC gene and .beta.2M gene. The nucleic acid encoding the anti-CD19 CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-CD19 CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-CD19 CAR cells may comprise the nucleotide sequence of SEQ ID NO: 119.

[0267] Anti-CD19 CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, optionally TRAC and/or .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting Reg1 (e.g., REG1-Z03 (SEQ ID NO: 22), REG1-Z05 (SEQ ID NO: 30), REG1-Z06 (SEQ ID NO: 34) or REG1-Z10 (SEQ ID NO: 50)), and optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63), which targets the .beta.2M locus. 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.

[0268] Anti-CD19 CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, optionally TRAC and/or .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting TGFBRII (e.g., those listed in Table 39, for example, TGFBRII_EX1_T2, TGFBRII_EX4_T1, TGFBRII_EX4_T2, TGFBRII_EX5_T1), and optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63), which targets the .beta.2M locus.

[0269] Anti-CD19 CAR T cells that comprise both a disrupted TGFBRII gene and a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, optionally TRAC and/or .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves at least a sgRNA targeting TGFBRII (e.g., those listed in Table 39) and a sgRNA targeting Reg1 (e.g., those listed in Table 22), optionally TA-1 sgRNA (SEQ ID NO: 59), which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63), which targets the .beta.2M locus.

[0270] The anti-CD19 CAR T cells are composed of an anti-CD19 single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 120), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3.zeta. signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-CD19 CAR T cells comprises the amino acid sequence of SEQ ID NO: 118.

[0271] In some embodiments, at least 30% of a population of anti-CD19 CAR T cells express a detectable level of the anti-CD19 CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells express a detectable level of the anti-CD19 CAR.

[0272] In some embodiments, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of .beta.2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells may not express a detectable level of .beta.2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of .beta.2M surface protein.

[0273] Alternatively or in addition, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD19 CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-CD19 CAR T cells do not express a detectable TRAC surface protein.

[0274] In some embodiments, a substantial percentage of the population of anti-CD19 CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein.

[0275] For example, at least 50% of a population of anti-CD19 CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of .beta.2M and TRAC proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the anti-CD19 CAR T cells do not express a detectable level of TRAC and .beta.2M surface proteins. In another example, at least 50% of a population of the anti-CD19 CAR T cells do not express a detectable level of TRAC and .beta.2M surface proteins.

[0276] In some embodiments, the population of anti-CD19 CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, 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: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD19 CAR (e.g., SEQ ID NO: 117). Alternatively or in addition, the population of anti-CD19 CAR T cells may comprise a disrupted .beta.2M gene via CRISPR/Cas9 technology using the gRNA of .beta.2M-1. Such anti-CD19 CAR T cells may comprise Indels in the .beta.2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-CD19 CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.50% .beta.2M.sup.+ cells, and .ltoreq.30% TCR.alpha..beta..sup.+ cells. In additional specific examples, anti-CD19 CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.30% .beta.2M.sup.+ cells, and .ltoreq.0.5% TCR.alpha..beta..sup.+ cells.

[0277] 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.

[0278] In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Reg1 gene may comprise any of the sequences provided in Tables 29-38 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% Reg1.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.- cells.

[0279] In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells.

[0280] In specific examples, the genetically engineered T cell population may be the anti-CD19 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Reg1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-CD19 CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells. Alternatively or in addition, the anti-CD19 CAR T cells may comprise at least 80% Reg1.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.- cells. In some examples, the anti-CD19 CAR T cells may comprise at least 60% Reg1.sup.-/TGPBRII.sup.- cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.-/TGFBRII.sup.- cells.

[0281] In some examples, such a population of genetically engineered T cells may comprise about 90-97% Reg1.sup.- cells, about 80-89% TGFBRII.sup.- cells, about 90-99% TCR.sup.- cells, and/or about 60-82% .beta.2M.sup.- cells. The cell population may also contain at least 50% (e.g., at least 60%) cells expressing the anti-CD19 CAR.

[0282] ii Anti-BCMA CAR-T Cells Having Reg1 and/or TGFBRII Gene Disruption

[0283] Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene and expressing an anti-BCMA CAR, e.g., those disclosed herein. In some examples, the anti-BCMA CAR T cells disclosed herein, which express any of the anti-BCMA CAR disclosed herein (e.g., the anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146), may also comprise a disrupted TRAC gene and/or a disrupted .beta.2M gene as also disclosed herein.

[0284] In some examples, the population of genetically engineered T cells are anti-BCMA CAR T cells that further comprise a disrupted Reg1 gene, a disrupted TGFBRII gene, or a combination thereof. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-BCMA CAR, e.g., those disclosed herein. In some examples anti-BCMA CAR T cells are anti-BCMA CAR T cells having disrupted TRAC gene and .beta.2M gene. The nucleic acid encoding the anti-BCMA CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-BCMA CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-BCMA CAR T cells may comprise the nucleotide sequence of SEQ ID NO: 145.

[0285] Anti-BCMA CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, and optionally TRAC and .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-CD19 CAR T cells.

[0286] Anti-BCMA CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, and optionally TRAC and .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-BCMA CAR T cells.

[0287] Anti-BCMA CAR T cells that comprise a disrupted Reg1 gene and a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, and optionally TRAC and .beta.2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-BCMA CAR construct. CRISPR-Cas9-mediated gene editing involves at least three guide RNAs (sgRNAs), as described above for anti-BCMA CAR T cells.

[0288] The anti-BCMA CAR T cells are composed of an anti-BCMA single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 148), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3.zeta. signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-BCMA CAR T cells comprises the amino acid sequence of SEQ ID NO: 146.

[0289] In some embodiments, at least 30% of a population of anti-BCMA CAR T cells express a detectable level of the anti-BCMA CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells express a detectable level of the anti-BCMA CAR.

[0290] In some embodiments, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of .beta.2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells may not express a detectable level of .beta.2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of .beta.2M surface protein.

[0291] Alternatively or in addition, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-BCMA CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-BCMA CAR T cells do not express a detectable TRAC surface protein.

[0292] In some embodiments, a substantial percentage of the population of anti-BCMA CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein.

[0293] For example, at least 50% of a population of anti-BCMA CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of .beta.2M and TRAC proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the anti-BCMA CAR T cells do not express a detectable level of TRAC and .beta.2M surface proteins. In another example, at least 50% of a population of anti-BCMA CAR T cells do not express a detectable level of TRAC and .beta.2M surface proteins.

[0294] In some embodiments, the population of anti-BCMA CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, 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-CD19 CAR T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-BCMA CAR (e.g., SEQ ID NO: 145). Alternatively or in addition, the population of anti-BCMA CAR T cells may comprise a disrupted .beta.2M gene via CRISPR/Cas9 technology using the gRNA of .beta.2M-1. Such anti-BCMA CAR T cells may comprise Indels in the .beta.2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-BCMA CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.50% .beta.2M.sup.+ cells, and .ltoreq.30% TCR.alpha..beta..sup.+ cells. In additional specific examples, anti-BCMA CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.30%.beta.2M.sup.+ cells, and .ltoreq.0.5% TCR.alpha..beta..sup.+ cells.

[0295] 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.

[0296] In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Regnase 1 (Reg1) gene may comprise any of the sequences provided in Tables 29-38 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% Reg1.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.- cells.

[0297] In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. In some examples, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells.

[0298] In specific examples, the genetically engineered T cell population may be the anti-BCMA CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Reg1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. In some examples, the anti-BCMA CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells. Alternatively or in addition, the anti-BCMA CAR T cells may comprise at least 80% Reg1.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.- cells. In some examples, the anti-BCMA CAR T cells may comprise at least 60% Reg1.sup.-/TGBBRII.sup.- cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.-/TGFBRII.sup.- cells.

[0299] iii. Anti-CD70 CAR-T Cells Having Reg1 and/or TGFBRII Gene Disruption

[0300] Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising a disrupted Reg1 gene, a disrupted TRFBRII gene, or a combination thereof, and expressing anti-CD70 CAR, e.g., those disclosed herein. In some instances, the population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising both a disrupted Reg1 gene and a disrupted TGFBRII gene, and expressing an anti-CD70 CAR, e.g., those disclosed herein. In some examples, the anti-CD70 CART cells disclosed herein, which express any of the anti-CD70 CAR disclosed herein (e.g., the anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138), may also comprise a disrupted TRAC gene, a disrupted .beta.2M gene, and/or a disrupted CD70 gene as also disclosed herein.

[0301] In some examples anti-CD70 CAR T cells are anti-CD70 CAR T cells having disrupted TRAC gene, a disrupted .beta.2M gene, and a disrupted CD70 gene. The nucleic acid encoding the anti-CD70 CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 69, which is replaced by the nucleic acid encoding the anti-CD70 CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the anti-CD70 CAR T cells may comprise the nucleotide sequence of SEQ ID NO: 139.

[0302] Anti-CD70 CAR T cells that comprise a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (Reg1, and optionally TRAC, .beta.2M and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the Reg1 gene as those disclosed herein (see, e.g., Table 22), and optionally an sgRNA (SEQ ID NO: 55) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63) which targets the .beta.2M locus.

[0303] Anti-CD70 CAR T cells that comprise a disrupted TGFBRII gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII, and optionally, TRAC, .beta.2M, and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the TGFBRII gene as those disclosed herein (see, e.g., Table 39), and optionally an sgRNA (SEQ ID NO: 43) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63) which targets the .beta.2M locus.

[0304] Anti-CD70 CAR T cells that comprise a disrupted TGFBRII gene and a disrupted Reg1 gene can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TGFBRII and Reg1, and optionally, TRAC, .beta.2M, and/or CD70 genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD70 CAR construct. CRISPR-Cas9-mediated gene editing involves at least an sgRNA targeting the TGFBRII gene as those disclosed herein (see, e.g., Table 39), and an sgRNA targeting the Reg1 gene as those disclosed herein (see, e.g., Table 22), and optionally an sgRNA (SEQ ID NO: 55) which targets the CD70 locus, TA-1 sgRNA (SEQ ID NO: 59) which targets the TRAC locus, and .beta.2M-1 sgRNA (SEQ ID NO: 63) which targets the .beta.2M locus.

[0305] The anti-CD70 CAR T cells are composed of an anti-CD70 CAR single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 138), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 97) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 101) and a CD3.zeta. signaling domain (e.g., SEQ ID NO: 103). In specific examples, the anti-CD70 CAR T cells comprise the amino acid sequence of SEQ ID NO: 138.

[0306] In some embodiments, at least 30% of a population of anti-CD70 CAR T cells express a detectable level of the anti-CD70 CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells express a detectable level of the anti-CD70 CAR.

[0307] In some embodiments, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of .beta.2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells may not express a detectable level of .beta.2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of .beta.2M surface protein.

[0308] Alternatively or in addition, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of TRAC surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the anti-CD70 CAR T cells may not express a detectable level of TRAC surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the anti-CD70 CAR T cells do not express a detectable TRAC surface protein.

[0309] In some embodiments, at least 50% of a population of the anti-CD70 CAR T cells may not express a detectable level of CD70 surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the engineered T cells of a population may not express a detectable level of CD70 surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, or 95%-100% of the engineered T cells of a population does not express a detectable level of CD70 surface protein.

[0310] In some embodiments, a substantial percentage of the population of anti-CD70 CAR T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein.

[0311] For example, at least 50% of a population of anti-CD70 CAR T cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of .beta.2M and TRAC proteins, .beta.2M and CD70 proteins, or TRAC and CD70 proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of two surface proteins. In another example, at least 50% of a population of the CTX130 cells may not express a detectable level of all of the three target surface proteins .beta.2M, TRAC, and CD70 proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of .beta.2M, TRAC, and CD70 surface proteins.

[0312] In some embodiments, the population of anti-CD70 CAR T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, 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-CD70 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: 69) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD70 CAR (e.g., SEQ ID NO: 139). Alternatively or in addition, the population of anti-CD70 CAR T cells may comprise a disrupted .beta.2M gene via CRISPR/Cas9 technology using the gRNA of .beta.2M-1. Such anti-CD70 CAR T cells may comprise indels in the .beta.2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 83-88. In specific examples, anti-CD70 CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.50% .beta.2M.sup.+ cells, and .ltoreq.30% TCR.alpha..beta..sup.+ cells. In additional specific examples, anti-CD70 CAR T cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.30% .beta.2M.sup.+ cells, and .ltoreq.0.5% TCR.alpha..beta..sup.+ cells.

[0313] 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.

[0314] In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted Reg1 gene. The disrupted Regnase 1 gene may comprise any of the sequences provided in Tables 22-31 below. Such a genetically engineered T cells may have .gtoreq.30% CAR+ T cells, .ltoreq.0.4% TCR.sup.+ T cells, .ltoreq.30% .beta.2M.sup.+ T cells, and .ltoreq.2% CD70.sup.+ T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% Reg1.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.- cells.

[0315] In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene. Such a genetically engineered T cells may have .gtoreq.30% CAR+ T cells, .ltoreq.0.4% TCR.sup.+ T cells, .ltoreq.30% .beta.2M.sup.+ T cells, and .ltoreq.2% CD70.sup.+ T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells.

[0316] In specific examples, the genetically engineered T cell population may be the anti-CD70 CAR T cells disclosed herein that further comprise a disrupted TGFBRII gene and a disrupted Reg1 gene. The disrupted Regnase 1 gene may comprise any of the sequences provided in Tables 29-38 below. Alternatively or in addition, the disrupted TGFBRII gene may comprise a nucleotide sequence selected from those listed in Tables 40-48 below. Such a genetically engineered T cells may have .gtoreq.30% CAR+ T cells, .ltoreq.0.4% TCR.sup.+ T cells, .ltoreq.30% .beta.2M.sup.+ T cells, and .ltoreq.2% CD70+ T cells. In some examples, the anti-CD70 CAR T cells may comprise at least 80% TGFBRII.sup.- cells, for example, at least 85%, at least 90%, at least 95%, at least 98% or above TGFBRII.sup.- cells. In some examples, the anti-CD70 CAR T cells may comprise at least 60% Reg1.sup.-/TGFBRII.sup.- cells, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or above Reg1.sup.-/TGFBRII.sup.- cells.

III. Therapeutic Applications

[0317] The therapeutic T cells generated using the genetically engineered T cells disclosed herein would be expected to maintain T cell health enabled by the disruption of the Reg1 gene, the disruption of the TGFBRII gene, the disruption of the CD70 gene, or a combination thereof. For example, maintaining T cell health may extend expansion during manufacturing, thereby increasing yield and consistency. In another example, maintaining T cell health may rescue exhausted/unhealthy T cells, thereby enabling potentially lower doses in patients and more robust responses. Further, the disruption of the Reg1 gene and the TGFBRII gene showed synergistic effects in enhancing CAR-T cell potency and in vivo expansion.

[0318] The therapeutic T cells disclosed herein can be administered to a subject for therapeutic purposes, for example, treatment of a solid tumor targeted by the CAR construct expressed by the therapeutic T cells.

[0319] 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 life time 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] Because of the enhanced persistence and efficacy of the therapeutic T cells disclosed herein, the dose of the therapeutic T cells provided herein would be lower than the standard dose of CAR-T cells prepared by conventional approaches (e.g., using T cells that do not have one or more of the genetic editing events disclosed herein, including a disrupted Reg1 gene and/or a disrupted CD70 gene). In some examples, the effective amount of the therapeutic T cells disclosed herein may be at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, at least 50-fold lower, or at least 100-fold lower than a standard dose of a CAR-T therapy. In some examples, an effective amount of the therapeutic T cells disclosed herein may be less than 10.sup.6 cells, e.g., 10.sup.5 cells, 5.times.10.sup.4 cells, 10.sup.4 cells, 5.times.10.sup.3 cells, or 10.sup.3 cells. In some examples described herein, the cells are expanded in culture prior to administration to a subject in need thereof.

[0326] 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.

[0327] 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.

IV. Kits

[0328] The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses,

[0329] In some embodiments, a kit provided herein may comprise components for performing genetic edit of one or more of Reg1 gene, TGFBRII gene, and CD70 gene, and optionally a population of immune cells to which the genetic editing will be performed (e.g., a leukopak). 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 a Reg1 gene, a TGFBRII gene, and/or a CD70 gene. Any of the gRNAs specific to these target genes can be included in the kit. Such a kit may further comprise components for further gene editing, for example, gRNAs and optionally additional endonucleases for editing other target genes such as .beta.2M and/or TRAC.

[0330] 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.

[0331] The kit may further comprise gRNAs specific to a TRAC gene for inserting the CAR-coding sequence into the TRAC gene. In other examples, the kit may further comprise gRNAs specific to a .beta.2M gene for inserting the CAR-coding sequence into the .beta.2M gene. In other examples, the kit may further comprise gRNAs specific to a CD70 gene for inserting the CAR-coding sequence into the CD70 gene. In yet other examples, the kit may further comprise gRNAs specific to a Reg1 gene for inserting the CAR-coding sequence into the Reg1 gene. In still other examples, the kit may further comprise gRNAs specific to a TGFBRII gene for inserting the CAR-coding sequence into the TGFBRII gene.

[0332] In yet other embodiments, the kit disclosed herein may comprise a population of therapeutic T cells as disclosed for the intended therapeutic purposes.

[0333] 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., Reg1, TGFBRII, 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.

[0334] Alternatively, 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. 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.

[0335] 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.

[0336] 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.

General Techniques

[0337] 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.).

[0338] 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 Reg1 Targeting Site by CRISPR/Cas-Mediated Gene Editing

[0339] (A) Efficient Disruption of Reg1 by Cas9:sgRNA RNPs in T Cells

[0340] The Reg1 gene was efficiently edited in primary human T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segments of the Reg1 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 Reg1, leading to out of frame/loss of function allele(s) (referred to as "Reg1 knockout (KO)" alleles or "Reg1 disrupted alleles"). All ten (10) in silico-identified gRNA spacer sequences targeting the Reg1 gene were synthesized, and the gRNAs were specifically modified, as indicated in Table 1. While the gRNAs used in this example were modified with 2'-O-methyl phosphorothioate modifications, unmodified gRNAs, or gRNAs with other modifications, may be used. The target sequences and gRNA sequences of the Reg1 guides Z01-Z10 are provided in Table 22 below.

TABLE-US-00002 TABLE 1 Indel Rate of Reg1 Gene by Ten gRNAs Indel Efficiency Guide Name (TIDE) REG1-Z01 98.3% REG1-Z02 97.2% REG1-Z03 96.8% REG1-Z04 92.7% REG1-Z05 98.5% REG1-Z06 .sup. 95% REG1-Z07 94.8% REG1-Z08 .sup. 71% REG1-Z09 88.2% REG1-Z10 94.9%

[0341] Primary human T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the Reg1 gene (sequences in Table 22) or controls (no Cas9, no gRNA). Four (4) days post transfection, cells were subjected to a TIDE analysis to assess indel frequency.

[0342] Ten (10) gRNAs yielded measurable data by TIDE analysis, as indicated in Table 1. Eight (8) gRNA sequences yielded indel percentages (editing frequencies) above 90%, indicating highly efficient gene editing.

[0343] Four gRNAs which target either exon 2 or 4 were selected for subsequent studies (REG1-Z03, REG1-Z05, REG1-Z06 and REG1-Z10, which showed 96.8%, 98.5%, 95% and 94.9% editing rate of Reg1, respectively as shown in (Table 1).

(B) On-Target and Off-Target Editing of REG1 Guide RNAs

[0344] On-target and off-target editing efficiencies of various REG1-targeting gRNAs noted above were examined following the method disclosed in the above section. Briefly, activated T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the Reg1 gene (sequences in Table 22 below) or controls (no Cas9, no gRNA).

[0345] 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 (termed 1 and 2). Cells were gene edited with each of the ten guides noted above, and then collected 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.

[0346] (i) Analysis of On-Target Indel Profiles in T Cells

[0347] The data used to quantify off-target editing were also used to quantify and summarize the most frequent on-target indels for all Reg1 guides listed in Table 22. This data was generated from hybrid capture of the Reg1 locus combined with next-generation sequencing in two donors (termed Donor 1 and Donor 2).

[0348] Following gene editing, hybrid capture analysis of the Reg1 locus in a population of T cells following CRISPR/Cas9 gene editing to produce Reg1 KO T cells results in specific indel frequencies and edited gene sequences at the Reg1 locus (Tables 29-38; deletions as dashes and insertions in bold).

[0349] For the purposes of individual sequence quantification from hybrid capture data, sequence reads aligning across the Regnase 1 on-target site, 20 bp upstream and downstream of the cut site, were selected and considered for indel sequence quantification. From the selected reads, the sequence within 10 bp upstream and downstream of each putative cut site (.about.3 bp upstream of the PAM (Jinek, et al., Science 2012) was quantified as a representative region of on-target non-homologous end joining (NHEJ) editing.

[0350] Table 2 below shows the on and off target editing results (from two donors) of exemplary Reg1 gRNAs obtained by the hybrid capture assay disclosed herein.

TABLE-US-00003 TABLE 2 On and Off Target Results by Hybrid Capture Number of On-target predicted off mean target sites editing Guide tested hyb cap Detected off-targets REG1-Z01 35 97.0% 1 0.75% off-target; 1 0.25% off-target REG1-Z02 27 97.7% No off-target editing detected REG1-Z03 52 99.0% 1 5.0% off-target; 1 0.6% off-target; 1 0.4% off-target; 1 0.3% off-target; 1 0.2% off-target REG1-Z04 6 97.0% No off-target editing detected REG1-Z05 14 98.6% No off-target editing detected REG1-Z06 1 94.2% No off-target editing detected REG1-Z07 16 94.2% 1 0.2% off-target REG1-Z08 6 53.8% No off-target editing detected REG1-Z)9 6 86.2% No off-target editing detected REG1-Z10 14 98.2% No off-target editing detected

[0351] On-target gene edited sequences by the exemplary Reg1 gRNAs are presented in Tables 29-38 below, with the frequencies of these sequences representing the percent of all sequences spanning the on-target site within 20 bp upstream and downstream of each cut site. The indels for each guide are shown relative to an on-target reference sequence in Tables 29-38. The reference sequence is centered on the cleavage site with 10 bp in either direction, ending 4 bp 3' of the PAM.

Example 2: Regnase/Disruption Improves CAR-T Cell Expansion

[0352] Using T cells expressing an anti-CD70 CAR disclosed herein as an example, this study demonstrated that knocking out Reg1 in the CAR-T cells resulted in enhanced in vitro CAR-T cell culture expansion.

[0353] Allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene, CD70 gene, and Regnase-1 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. Briefly, activated human T cells were first isolated and then Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA) were delivered to the activated human T cells by electroporation, followed by incubation with recombinant adeno-associated adenoviral vectors (AAVs), serotype 6 (AAV6) (MOI 50,000). The nucleofection mix contained the Nucleofector.TM. Solution, 5.times.10.sup.6 cells, 1 .mu.M Cas9, and 5 .mu.M gRNA (as described in Hendel et al., Nat Biotechnol. 2015; 33(9):985-989, PMID: 26121415). The RNP complex comprised Cas9 and sgRNA targeting the TRAC, B2M, and CD70 (shown in Table 23) and optionally Regnase-1 genes (using the REG1-Z01 to REG1-Z10 sgRNAs shown in Table 22). The rAAV vector included the nucleotide sequence encoding an anti-CD70 CAR (the donor template in SEQ ID NO: 169, encoding an anti-CD70 CAR amino acid sequence of SEQ ID NO: 138).

[0354] To assess the ability of anti-CD70 CAR T cells to expand in cytokine containing media (IL-2+IL-7), anti-CD70 CAR T cells were utilized. Specifically, 2.5 to 3.8.times.10.sup.6 total anti-CD70 CAR T cells comprising a quadruple disruption (TRAC-/.beta.2M-/CD70-/Reg1-) were generated and compared to anti-CD70 CAR T cells with unedited Reg1 (TRAC-/.beta.2M-/CD70-).

[0355] Cells were plated and allowed to grow in flasks with cytokine containing media. Every 3-4 days the total number of cells were enumerated and re-plated as needed. This process was repeated each week for a total of 21 days. Allogeneic anti-CD70 CAR-T cells containing a disruption in the Reg1 gene show an increase in cell expansion after 21 days (FIG. 1A). Reg1 guides REG1-Z01, REG1-Z03, REG1-Z07, REG1-Z09, and REG1-Z10 appear to have a greater effect on cell expansion than cells made using Reg1 guides REG1-Z02 or REG1-Z08.

[0356] In a second experiment, Reg1 guide REG1-Z10 was used in CAR T cells made from a different T cell donor in replicates by two operators (labelled A and B). The effect of increased cell culture expansion was demonstrated again. The increase in cell expansion can be seen as early as day 13 and continues throughout the experiment to day 52 (FIG. 1B). Furthermore, anti-CD70 CAR-T cells containing a Reg1 disruption are maintained over a longer time in culture (at least up to day 52) as compared to anti-CD70 CAR-T cells with an unedited Regnase 1 gene, one of which was no longer viable on day 26. Collectively, these data show that disruption of the Reg1 gene results in greater cell culture yields and longer cell maintenance in culture as compared to CAR T cells with an unedited Reg1 gene.

Example 3: Cell Killing Function of Anti-CD70 CAR T Cells with Reg1 Disruption

[0357] Allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. The edited CAR T cells further comprised knock out of Reg1 gene. As in the examples above, activated human T cells we electroporated with Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000).

[0358] Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54), and optionally Reg1 (e.g., REG1-Z03, Z05, Z06, and Z10; see Table 22 and FIGS. 2A to 2E).

[0359] At time points of one week and one month post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells that lack Reg1 (using four gRNAs REG1-Z03, Z05, Z06, Z10) expressed nearly equivalent amount of CAR on their surface at day 7 (85.6% and 81.8%, 80%, 84.4%, 85.6%) and day 32 (97.6% and 90.7%, 91.5%, 92.6%, 93.2%) post HDR.

Cell Killing Function of Anti-CD70 CAR T Cells with Regnase-1 (Reg1) Disruption

[0360] A cell killing assay was used to assess the ability of the TRAC-/.beta.2M-/CD70-/Reg1-/anti-CD70 CAR+ cells to kill CD70+ adherent renal cell carcinoma (RCC)-derived cell lines (ACHN, Caki-1, and/or 769P cell lines). Adherent cells were seeded in 96-well plates at 50,000 cells per well and incubated overnight at 37.degree. C. 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 1:1, 2:1 or 1.5:1 CAR T:Target cell ratios. After 24 hours co-culture, CART cells were removed from the culture by aspiration and 100 .mu.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.

[0361] Cells with Reg1 disruption exhibited a more potent cell killing of RCC-derived cells following 24-hour co-incubation. The anti-CD70 CAR T cells at day 12 post HDR (FIGS. 2A and 2B) demonstrated slightly higher potency when Reg1 was knocked out, and much higher potency at day 27 post HDR (FIGS. 2C, 2D, and 2E). This suggests that knocking-out the Reg1 gene gives a maintained/persistent higher cell kill potency to anti-CD70 CAR+ T cells over time post HDR. This finding was consistent across the three tumor lines from Renal cell carcinoma tumor lines. CD70 CAR+ T cells with Reg1 disruption using gRNAs REG1-Z03, REG1-Z05, REG1-Z10 gave a higher persistent potency than when using gRNA REG1-Z06. CAR-T cells with Reg1 disruption demonstrated a visible increased in potency after 24 h co-culture with caki-1 (FIGS. 2A, 2B, and 2C) and ACHN (FIG. 2D), and after 6 hours co-culture with 769P (difference not visible anymore after 24 h) (FIG. 2E).

[0362] While CAR-T cells with or without the Regnase KO show similar efficacy at Day 13 post HDR, efficacy appears to be diminished in older cells (Day 19 and Day 26) without the Regnase KO. Surprisingly, TRAC-/.beta.2M-/CD70-/Reg1-/anti-CD70 CAR+ cells still retain the ability to kill with similar activity ACHN and Caki-1 cells in culture (FIGS. 6A and 6B).

[0363] This suggests that disrupting the Reg1 gene gives a persistent activity and higher cell kill potency to CAR+ T cells over a longer period of time post HDR editing.

Example 4. Effect of Regnase-1 (Reg1) Disruption on Exhaustion Marker Expression

[0364] The levels of the T cell exhaustion markers were assessed on TRAC-/.beta.2M-/CD70-/anti-CD70 CAR+ and TRAC-/.beta.2M-/CD70-/Reg1-/anti-CD70 CAR+ cells. CD4+ and CD8+ T cells were assessed for PD-1 expression (FIGS. 3A and 3B) and TIM3 expression (FIGS. 3C and 3D) by flow cytometry at Day 13 (FIGS. 3A and 3C) and Day 26 (FIGS. 3B and 3D) post HDR.

[0365] The data demonstrate that Reg1 KO (using Z10 guide as an example) reduces exhaustion marker expression in CAR T cells at all time points measured. The data demonstrate that knocking out Reg1 could reduce the potential exhaustion of CD8+ and CD4+ gene edited populations of CAR+ T cells leading to better therapeutics.

Example 5. Regnase-1 (Reg1) Disruption Increases the Proportion of Central Memory Cells in CAR T Cells Population

[0366] Upon activation of antigen peptides presented by antigen-presenting cells, native T cells differentiate to various types of T cells in the order of T stem cell memory (T.sub.SCM), T central memory cell (T.sub.CM), T effector memory cell (.sub.TEM), and T effector cell (T.sub.EFF). Exemplary surface markers of T cells at different differentiation stages are provided below. T.sub.CM cells have been associated with T cell long term persistence in vivo: CD8+ clones isolated from T.sub.CM cells were shown to persist long term in vivo during adoptive T cell transfer in non-human primates while clones isolated from effector cells did not. (Berger et al., J. Clin. Investig. (2008) 118:294-305). Representative cell surface markers of the various types of T cells are provided in Table 3 below.

TABLE-US-00004 TABLE 3 Representative Cell Surface Markers of Various Types of T Cells Stem Central Central Effector Naive Memory Memory Memory CD27 + + + - CD45RO - - + + CD45RA + + - - CD62L + + + - CD95 - + + +

[0367] The levels of CD27 and CD45 RO T central memory T cell markers were assessed on TRAC-/.beta.2M-/CD70-/anti-CD70 CAR+ and TRAC-/.beta.2M-/CD70-/Reg1-/anti-CD70 CAR+ cells. Cells were stained using commercial antibodies for CD27 (Biolegend, clone M-T271) and CD45 RO (Biolegend, clone UCHL1) and analyzed by flow cytometry.

[0368] CAR-T cells with Reg1 knock out were more likely to exhibit central memory T cell identity (double positive for CD27 and CD45 RO) and less likely to exhibit effector memory cell identity (identified as CD27- and CD45 RO+), as shown in Table 4.

TABLE-US-00005 TABLE 4 Central memory and effector memory T cell markers in cells with and without Reg1 KO CD27+/CD45 CD27-/CD45 Exper- RO+ Central RO+ Effector iment Cells memory cells memory cells 1 TRAC-/.beta.2M-/anti-CD70 62.3% .sup. 30% CAR+ TRAC-/.beta.2M-/CD70-/ 82.3% 15.1% Reg1-/anti-CD70 CAR+ 2 TRAC-/.beta.2M-/anti-CD70 61.8% 27.5% CAR+ TRAC-/.beta.2M-/CD70-/ 74.3% 22.2% Reg1-/anti-CD70 CAR+

[0369] The results obtained from this study indicate that Reg1 disruption led to an enhanced level of T.sub.CM cells in the total T cell population compared to the Reg1 WT counterparts, indicating that Reg1 disruption could increase T cell long term persistence in vivo, which would benefit CAR-T therapy.

Example 6. Reg1 Disruption does not Affect Cytokine Dependency of CAR T Cells

[0370] To determine whether the gene editing resulted in unwanted off-target editing that could generate cells with adverse properties, such as uncontrolled cell growth, the ability of TRAC-/.beta.2M-/anti-CD19 CAR+ and TRAC-/.beta.2M-/Reg1-/anti-CD19 CAR+ cells to grow in the absence of cytokines and/or serum was assessed. 5.times.10.sup.6 cells were plated approximately 2 weeks post cell production (Day 0) in 10 mL of full media containing IL2, IL7 and human serum, or in serum-containing media lacking cytokines (IL-2 and IL-7). Fresh full media or media lacking cytokines were added to the respective cultures once per week. The volume of media added allowed for the cultures to maintain a density of approximately 1-2 million cells/mL. If the cell density was below 1 million cells/mL, media was not added to the cultures. The number of viable cells were enumerated twice weekly until 40 days post plating. TRAC-/.beta.2M-/anti-CD19 CAR+ or TRAC-/.beta.2M-/Reg1-/anti-CD19 CAR+ were no longer detectable at 40 days in the cultures that lacked cytokines, indicating that any potential off-target effects due to genome editing did not induce growth factor independent growth/proliferation to the cells (FIG. 4). The cells only proliferated in the presence of cytokines (full media that contains cytokines) and did not proliferate in the presence of serum alone. Thus, genome editing did not induce any adverse events that allow the cells to grow in the absence of cytokine, growth factor or antigen stimulation.

Example 7: In Vivo Effect of Reg1 KO on Allogeneic CAR T Cells in the Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia Tumor Xenograft Model

[0371] A disseminated mouse model was utilized to further assess the in vivo efficacy of allogeneic CAR T cells lacking .beta.2M and TRAC, as well as Reg1. The intravenous disseminated model (disseminated model) utilized CD19+B-ALL derived Nalm-6 Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice to demonstrate the efficacy of TRAC-/.beta.2M-/anti-CD19 CAR+ T cells (anti-CD19 CAR T cells) with or without editing of the Reg1 locus. The Reg1 gene was edited via CRISPR/Cas-mediated gene editing using REG1-Z10 guide RNA (see Table 22). The anti-CD19 CAR T cells express an anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 118. See also the sequence Tables 27 and 28 below, and WO2019/097305, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0372] Efficacy of the anti-CD19 CAR T cells was evaluated in the disseminated model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 25, 5-8 week old female CIEA NOG (NOD.Cg-PrkdcscidI12rgtm1Sug/JicTac) 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 5 treatment groups as shown in Table 5. The mice were inoculated with Nalm6-Fluc-GFP (Nalm6-Fluc-Neo/eGFP--Puro) cells intravenously to model disseminated disease. On Day 1, all mice received an intravenous injection of 0.5.times.10.sup.6 Nalm6 cells/mouse. On Day 4, Groups 2-5 received an intravenous injection of CAR T cells (4.times.10.sup.6 CAR+ cells/mouse) as indicated in Table 5.

TABLE-US-00006 TABLE 5 Treatment groups for intravenous disseminated disease study Nalm6 tumor cells CAR T cells (i.v.) Group 0.5 .times. 10.sup.6 cells/mouse 4 .times. 10.sup.6 cells/mouse N 1 X NA 5 2 X anti-CD19 CAR/TRAC-/ 5 .beta.2M- (4e6 CAR+) 3 X anti-CD19 CAR/TRAC-/ 5 .beta.2M- (8e6 CAR+) 4 X anti-CD19 CAR/TRAC-/ 5 .beta.2M-/Reg1- (4e6 CAR+) 5 X anti-CD19 CAR/TRAC-/ 5 .beta.2M-/Reg1- (8e6 CAR+)

[0373] During the course of the study, the mice were monitored daily and body weight was measured two times weekly. Bioluminescence (BLI; total ROI, photon/s) was measured twice weekly beginning on Day 4 of the study. A significant endpoint was 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: [0374] Loss of body weight of 20% or greater sustained for a period of greater than 1 week; [0375] Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate; [0376] Prolonged, excessive diarrhea leading to excessive weight loss (>20%); or [0377] Persistent wheezing and respiratory distress. [0378] 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.

[0379] In Vivo Survival Rate

[0380] Mice in groups receiving TRAC-/.beta.2M-/anti-CD19 CAR+ T cells with or without an additional Reg1 disruption exhibited an increase in survival relative to mice in the untreated group (Group 1). Mice receiving either dose of TRAC-/.beta.2M-/Reg1-/anti-CD19 CAR+ T cells exhibited increased survival in comparison to TRAC-/.beta.2M-/anti-CD19 CAR+ T cells at each respective dose (FIGS. 5A and 5B). In addition, mice receiving either dose of TRAC-/.beta.2M-/Reg1-/anti-CD19 CAR+ T cells had reduced leukemia burdens as indicated by diminished bioluminescence signal in comparison to TRAC-/.beta.2M-/anti-CD19 CAR+ T cells at each respective dose (FIGS. 5C and 5D).

[0381] These data demonstrate that the Reg1 disruption in CAR T cells increases efficacy of CAR T cells in vivo, decreasing tumor burden and increasing survival.

Example 8: Efficient Disruption of TGFBRII by Cas9:sgRNA RNPs in T Cells

[0382] This example describes efficient editing of the TGFBRII gene in primary human T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segments of the TGFBRII gene containing the first five (5) protein coding exons were used as input in gRNA design software. The genomic segments also included flanking splice site acceptor/donor sequences. Desired gRNAs were those that would lead to insertions or deletions in the coding sequence, disrupting the amino acid sequence of TFBRII, leading to out of frame/loss of function allele(s) (referred to as "TGFBRII knockout alleles" or "TGFBRII disrupted alleles"). Eight (8) in silico-identified gRNA spacer sequences targeting the CD70 gene were synthesized, and the gRNAs were specifically modified, as indicated in Table 39 and FIGS. 7A and 7B. While the modified gRNAs in Table 39 were modified with 2'-O-methyl phosphorothioate modifications, unmodified gRNAs, or gRNAs with other modifications, can be used.

[0383] Primary human T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the TGFBRII gene (sequences in Table 39) or controls (no Cas9, no gRNA). Four to six (4-6) days post transfection, cells were: (1) subjected to a TIDE analysis to assess indel frequency, and (2) processed by western blot (primary antibody: anti-human TGFBRII antibody, clone #16H2L4) to assess TGFBRII expression levels at the cell surface (FIG. 7B).

[0384] Eight (8) gRNAs yielded measurable data by TIDE analysis, as indicated in FIG. 7A. Seven (7) gRNA sequences yielded indel percentages (editing frequencies) above 80% indicating highly efficient gene editing (FIG. 7A). The level of TGFBRII protein expression was assessed by western blot to confirm the TIDE analysis data and GAPDH was used as a loading control. Seven (7) of the gRNAs showed nearly complete knock out of TGFBRII on the T cells (FIG. 7B).

On-Target and Off-Target Editing of TGFBRII Guide RNAs

[0385] On-target and off-target editing efficiencies of various TGFBRII-targeting gRNAs noted above were examined following the method disclosed in the above section. Briefly, activated T cells were transfected (electroporated) with a ribonucleoprotein particle (RNP) containing Cas9 nuclease and a synthetic modified sgRNA targeting the TGFBRII gene (sequences in Table 39 below) or controls (no Cas9, no gRNA).

[0386] 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 nine guides noted in Table 39 and then collected 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.

[0387] Five (5) gRNAs showed no off-target effect with an on-target editing rate greater than 85%, which includes TGFBRII_Ex1_T1, TGFBRII-Ex1-T2, TGFBRII_Ex1_T3, TGFBRII_Ex2_T1 and TGBBRII_Ex5_T1 as shown in Table 6 below.

TABLE-US-00007 TABLE 6 On-Targeting Editing Efficiency and Off-Target Effects of Anti-TGFBRII gRNAs Number of predicted off- On-target target sites mean editing Detected Guide gRNA target sequence + (PAM) tested hyb cap off-targets TGFBRII- CCGACTTCTGAACGTGCGGT 7 86.80% None Ex1-T1 (GGG) (SEQ ID NO: 2) TGFBRII- TGCTGGCGATACGCGTCCAC 8 98.30% None Ex1-T2 (AGG) (SEQ ID NO: 3) TGFBRII- TCGGTCTATGACGAGCAGCG 7 99.60% None Ex1-T3 (GGG) (SEQ ID NO: 4) TGFBRII- ATGGGCAGTCCTATTACAGC 82 96.00% None Ex2-T1 (TGG) (SEQ ID NO: 5) TGFBRII- ATTGTTCACTTGTTAGCCCC 83 98.50% One <1% Ex3-T1 (AGG) (SEQ ID NO: 6) off-target TGFBRII- GCTGAAGAACTGCCTCTATA 133 98.10% One Ex3-T2 (TGG) (SEQ ID NO: 7) 1-10% off-target TGFBRII- GCAGGATTTCTGGTTGTCAC 222 98.80% One <1% Ex4-T1 (AGG) (SEQ ID NO: 8) off-target TGFBRII- CTCCATCTGTGAGAAGCCAC 255 99.40% Four <1% Ex4-T2 (AGG) (SEQ ID NO: 9) off-targets TGFBRII- CCCCTACCATGACTTTATTC 85 94.20% None Ex5-T1 (TGG) (SEQ ID NO: 10)

[0388] Tables 29-38 list potential indel sequences that may be generated by the gRNAs disclosed herein (deletions as dashes and insertions in bold).

Example 9: Generation of Genetically Modified T Cells that Lack TGFBRII Expression and are Resistant to TGF-.beta.

[0389] This example describes the production of CAR T cells that lack expression of TGFBRII and the assessment of the effect of TGF-.beta. on CAR T cell expansion with TGFBRII KO cells grown in complete media (X-Vivo 15 supplemented with IL-2 and IL-7).

[0390] Briefly, human T cells were first isolated and Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA) were delivered to activated human T cells by electroporation, followed by incubation with the recombinant adeno-associated adenoviral vectors (AAVs), serotype 6 (AAV6) (MOI 50,000). The nucleofection mix contained the Nucleofector.TM. Solution, 5.times.10.sup.6 cells, 1 .mu.M Cas9, and 5 .mu.M gRNA (as described in Hendel et al., Nat Biotechnol. 2015; 33(9):985-989, PMID: 26121415). The RNP complex comprised Cas9 and sgRNA targeting the TRAC, B2M, CD70, and optionally TGFBRII genes (sgRNA sequences are shown in Table 23 and Tables 39, SEQ ID NOs: 58, 62, 54, and 301, respectively). The rAAV vector included the nucleotide sequence encoding an anti-CD70 CAR (the donor template in SEQ ID NO: 169 and the anti-CD70 CAR amino acid sequence of SEQ ID NO: 138.

[0391] About one week post-electroporation, CAR T cells with an intact (i.e.: wild-type or non-engineered counterpart) TGFBRII gene were exposed to varying amounts recombinant human TGF-.beta. (10, 20, 50 and 100 ng/ml) and cell expansion was recorded over time. TGF-.beta. significantly inhibited CAR T expansion, a concentration as low as 10 ng/ml was sufficient to reduce CAR T expansion in cells with an intact TGFBRII gene (FIG. 8A).

[0392] In another study, anti-CD70 CAR T cells with TGFBRII disruption were incubated with or without 50 ng/ml of recombinant human TGF-.beta., and the T cell expansion was monitored at day 2 and day 8 post-incubation with TGF-.beta. and compared to mock cells. Mock cells (FIG. 8B) were anti-CD70 CAR T cells that did not have a disrupted TGFBRII gene. As shown in FIGS. 8C-8K, T cells with TGFBRII knocked-out were protected against the inhibitory effect of TGF-.beta. on T cell expansion. The extent of protection varied with the sgRNA used to disrupt the TGFBRII gene. T cells that were transfected with gRNA targeting exon 1, 4 and 5 (TGFBRII_EX1_T2, TGFBRII_EX4_T1, TGFBRII_EX4_T2, TGFBRII_EX5_T1) showed the most resistance against a TGF-.beta. inhibitory effect. Sequences of these gRNAs are provided in Table 39 below.

Example 10: Cell Killing Function of Anti-CD70 CAR T Cells with TGFBRII Disruption

[0393] This example describes the production of allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70. The edited CAR T cells further comprised knock out of the TGFBRII gene. As in the examples above, activated human T cells were electroporated with Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000).

[0394] Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54) and TGFBRII (SEQ ID NO: 301).

[0395] About one week post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells lacking TGFBRII expressed nearly equivalent amount of CAR on their surface (71.5% CAR.sup.+ cells versus 73.7% CARP cells).

[0396] A cell killing assay was used to assess the ability of the TRAC-/.beta.2M-/CD70-/TGFBRII-/anti-CD70 CAR+ cells to kill a CD70+ adherent renal cell carcinoma (RCC)-derived cell line (A498 cells). Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37.degree. C. The next day edited anti-CD70 CAR T cells were added to the wells containing target cells at 0.05:1 or 0.1:1 CAR T:T cell (E:T) ratios. After the indicated incubation period, CAR T cells were removed from the culture by aspiration and 100 .mu.L Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted per well was then quantified using a plate reader. Cells with TGFBRII knock out exhibited a more potent cell killing of RCC-derived cells following 24-hour co-incubation. The anti-CD70 CAR T cells demonstrated higher potency when TGFBRII was knocked out, which is clearly visible at two T cell: A498 ratios (0.05:1 and 0.1:1) (FIG. 9). This suggests that knocking-out the TGFBRII gene gives a higher cell kill potency to anti-CD70 CAR+ T cells. This finding was consistent across a wide panel of tumor lines from different tissues as shown in FIGS. 10A-10E. Knocking-out the TGFBRII gene enhances the cell killing capacity of anti-CD70 CAR T cells against 786-O and CAM-1 (Renal cell carcinoma tumor lines), H1975 (Non-small cell lung cancer), Hs-766T (Pancreatic carcinoma) and SK-OV3 (Ovarian cancer) (FIGS. 10A-10E).

[0397] In another study, anti-CD70 CAR T was incubated with 50 ng/ml of recombinant human TGF-.beta. for 24 hours and the expression of CD25 (IL-2R) on cell surface was assessed by flow cytometry. As shown in FIG. 11, anti-CD70 CAR T cells are susceptible to the inhibitory effect of TGF-.beta. that causes downregulation of CD25. CD25 is an activation marker and involved in T cell proliferation. When the TGFBRII gene was knocked out, these cells become resistant to TGF-.beta. and the CAR T cells retain activity and CD25 expression.

[0398] Also, when the cell kill of target cells (A498) was repeated in presence of 1, 10 and 50 ng/ml of recombinant human TGF-.beta.. Anti-CD70 CAR T cells were adversely affected by presence of TGF-.beta. as demonstrated by reduction in the cell kill capacity by CAR T cells with an intact TGFBRII gene (FIG. 12). However, anti-CD70 CAR T cells with a TGFBRII KO (anti-CD70 CAR+TGFBRII_EX4_T1) did not exhibit reduced cell killing ability in the presence of TGF-.beta. (FIG. 12). In addition, T cell proliferation upon exposure to target antigen and effector cytokines production (IFN-.gamma. and IL-2) were reduced in the presence of TGF-.beta. (FIGS. 13A-13C). However, when the cells lacked the expression of TGFBRII, they we were completely protected against TGF-.beta. inhibitory effects, also shown in FIGS. 13A-13C. This suggests that knocking out TGFBRII on the surface of CAR T cells protects the CAR T cells from the adverse effect of TGF-.beta. in the tumor microenvironment.

Example 11: Generation of Anti-CD70 CAR T Cells that Lack TGFBRII Expression and are Resistant to the Inhibitory Effect of Fibroblasts

[0399] This example describes the production of allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene and CD70 gene, and express a chimeric antigen receptor (CAR) targeting CD70 and how they are susceptible to the inhibitory effect of fibroblasts, which are a major component of solid tumor microenvironment (TME). The edited CAR T cells further comprised knock out of the TGFBRII gene. As in the examples above, activated human T cells we electroporated with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000), and Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA).

[0400] Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), CD70 (SEQ ID NO: 54) and TGFBRII (SEQ ID NO: 301).

[0401] A cell killing assay was used to assess the inhibitory effect of fibroblast on anti-CD70 CAR T cells to kill CD70+ adherent tumor cell lines: H1975 (Non-small cell lung cancer), Hs-766T (Pancreatic carcinoma), or SK-OV3 (Ovarian cancer). The cell kill assay was performed as described in example 3. Briefly, Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37.degree. C. and the fibroblast cells (LL 86 (LeSa) ATCC.RTM. CCL-190.TM.) were added to the top chamber of a transwell plate without direct contact with target cells. The next day edited anti-CD70 CAR T cells were added to the wells containing target cells. After the indicated incubation period, CAR T cells were removed from the culture by aspiration and 100 .mu.L Cell titer-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted per well was then quantified using a plate reader. As shown in FIG. 14, the presence of the fibroblast cells on the top chamber led to a decrease of the cell kill capacity of anti-CD70 CAR T cells against the target cells which might suggest that these fibroblast secreted a factor that decrease anti-CD70 CAR T killing effect.

[0402] This finding was confirmed when this experiment was repeated with the presence of conditioned media from the fibroblast instead on the cells and similar inhibition was observed. Briefly, 1.times.10.sup.6 CCL-190 fibroblast cells we seeded/0.5 ml in a 24 well plate and incubated overnight and supernatants were collected. A cell kill assay as previously described was carried out with anti-CD70 CAR T cells and tumors cells at a ratio of 0.1:1 effector to target cell ratio, in the presence or absence of fibroblast supernatant and incubated overnight. Cell kill was measured using the CellTiter-Glo.RTM. Luminescent Cell Viability Assay. This experiment confirms that fibroblasts secrete a factor that causes a reduction in the killing capacity of anti-CD70 CAR T cells. Disruption of the TGFBRII gene on the surface of anti-CD70 CAR T protected these cells against this inhibitory effect. The TGFBRII KO improved the cell killing ability of anti-CD70 CAR T cells against pancreatic tumor cells, Hs-766T (FIG. 15A), kidney tumor cells, A498 (FIG. 15B), and lung tumor cells, H1975 (FIG. 15C) in the presence of fibroblasts. These data suggest that fibroblasts are contributing to the TGF-.beta. production in TME and reduce the cell kill capacity of anti-CD70 CAR T cells and this could be avoided by disrupting TGFBRII on the surface of the CAR T cell.

Example 12: Generation of CAR T Cells with Disrupted TGFBRII and Regnase-1 Genes

[0403] Allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene, CD70 gene, TGFBRII gene and Regnase-1 gene, and express a chimeric antigen receptor (CAR) targeting CD70 were produced. Activated human T cells were electroporated with Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000).

[0404] Recombinant AAV comprised the nucleotide sequence of SEQ ID NO: 169 (encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 138). The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), CD70 (SEQ ID NO:54), TGFBRII (SEQ ID NO: 313) and REG-1 (SEQ ID NO: 51). The sgRNAs, which form RNPs with the Cas9 enzyme, can be introduced into the T cells in a single electroporation event to produce the resulting modified cell populations shown in Table 7 below. Alternatively, they can be introduced into the T cells in two sequential electroporation events to produce the resulting cell populations. After the electroporation, the cells were transduced with the recombinant AAVs to introduce the donor template encoding for the anti-CD70 CAR.

TABLE-US-00008 TABLE 7 Genetically Engineered CAR-T Cell Populations Population Edits Anti-CD70 CAR T cells anti-CD70 CAR+/TRAC-/ B2M-/CD70- Anti-CD70 CAR T + Reg KO anti-CD70 CAR+/TRAC-/ cells B2M-/CD70-/Reg- Anti-CD70 CAR T + TGFBRII anti-CD70 CAR+/TRAC-/ KO cells B2M-/CD70-/TGFBRII- Anti-CD70 CAR T + Reg KO + anti-CD70 CAR+/TRAC-/ TGFBRII KO cells B2M-/CD70-/Reg-/ TGFBRII-

[0405] At 7 days post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-CD70 CAR T cells and anti-CD70 CAR T cells that lack Regnase expressed nearly equivalent amount of CAR on their surface at day 7 post HDR. The results are provided in Table 7A below.

TABLE-US-00009 TABLE 7A CAR Expression Levels in Genetically Engineered Anti-CD70 CAR T Cells Population Edits CAR % Anti-CD70 CAR T cells anti-CD70 CAR+/TRAC-/ 82.2 B2M-/CD70- Anti-CD70 CAR T + Reg KO anti-CD70 CAR+/TRAC-/ 83.1 cells B2M-/CD70-/Reg- Anti-CD70 CAR T + TGFBRII anti-CD70 CAR+/TRAC-/ 79.7 KO cells B2M-/CD70-/TGFBRII- Anti-CD70 CAR T + Reg KO + anti-CD70 CAR+/TRAC-/ 81.8 TGFBRII KO cells B2M-/CD70-/Reg-/ TGFBRII-

Example 13: Disruption of Regnase-1 and TGFBRII Increases CAR T Cell Killing Upon Serial Rechallenge In Vitro

[0406] The anti-CD70 CARP T cells generated above were serially rechallenged with CD70+ kidney cancer cell line, ACHN, and evaluated for their ability to kill the CD70+ kidney cancer cell line ACHN.

[0407] The anti-CD70 CAR.sup.+ T cells used in this experiment contained the following edits: [0408] Anti-CD70 CAR T cells: anti-CD70 CAR+/TRAC-/B2M-/CD70- [0409] Anti-CD70 CAR T+Reg KO cells: anti-CD70 CAR+/TRAC-/B2M-/CD70-/Reg- [0410] Anti-CD70 CAR T+TGFBRII KO cells: anti-CD70 CAR+/TRAC-/B2M-/CD70-/TGFBRII- [0411] Anti-CD70 CAR T+Reg KO+TGFBRII KO cells: anti-CD70 CAR+/TRAC-/B2M-/CD70-/Reg-/TGFBRII-

[0412] In a 96-well plate format, CAR T cells were first co-cultured with ACHN cells (4,000 CAR T cells, 16,000 tumor cells) on D0 and re-challenged with tumor cells as follows: 16,000 tumor cells on D2 and D4; 40,000 cells on D7; 50,000 cells on D9; 50,000 cells on D11).

[0413] Analysis of tumor cell and CAR T cell number was performed at D1, D3, D6, D8, D10 and D12 using flow cytometry (method adapted from Wang et al., JoVE 2019). The following antibodies in Table 8 were used at 1:100 dilution.

TABLE-US-00010 TABLE 8 Antibody Information Antibody Flour cat # Dilution Vendor CD4 BV510 344718 1:100 Biolegend CD8 PacBlue 300546 1:100 Biolegend CD70 FITC 355106 1:100 Biolegend CD62L BV605 304833 1:100 Biolegend human CD45 BV785 304048 1:100 Biolegend PD1 APC/Cy7 329922 1:100 Biolegend CD45RO PE/Cy7 304230 1:100 Biolegend Streptavidin APC 405207 1:100 Biolegend Tim3 PE 345006 1:100 Biolegend Live/Dead 7AAD BDB559925 1:500 BD

[0414] The results demonstrate that disrupting both the TGFBRII gene and the Regnase gene improved potency (FIG. 16A) and CAR+ T cell expansion (FIG. 16B) when CAR T cells are repeatedly challenged with CD70+ positive target cells. Potency and expansion is improved compared to CAR T cells that have neither, or only one (i.e.: TGFBRII or Regnase), of the genes disrupted.

Example 14: Treatment Efficacy of Anti-CD70 CART Cells with Multiple Gene Disruptions in the Subcutaneous Renal Cell Carcinoma Tumor Xenograft Model

Treatment in the Renal Cell Carcinoma Tumor Model

[0415] The ability of T cells expressing a CD70 CAR with TGFBRII and/or Regnase gene edits to eliminate renal cell carcinoma cells that express medium levels of CD70 was evaluated in vivo using a subcutaneous renal cell carcinoma (CAKI-1) tumor xenograft mouse model. Anti-CD70 CAR+ T cells were produced as described above. See, e.g., Example 13.

[0416] The ability of these anti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+ renal carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, Ariz.). In brief, 20, 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. Mice received a subcutaneous inoculation of 5.times.10.sup.6 Caki-1 renal cell carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of -70 mm.sup.3, the mice were further divided into 5 treatment groups as shown in Table 9. On Day 1, treatment four groups received a single 200 .mu.l intravenous dose of 1.times.10.sup.7 anti-CD70 CAR+ T cells according to Table 9.

TABLE-US-00011 TABLE 9 Treatment groups CAR-T cell treatment Group CAR-T Caki-1 cells (i.v.) N 1 None 5 .times. 10.sup.6 None 4 cells/mouse 2 Anti-CD70 CAR T cells: 5 .times. 10.sup.6 1 .times. 10.sup.7 4 anti-CD70 CAR+/TRAC-/ cells/mouse cells/mouse B2M-/CD70- 3 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 1 .times. 10.sup.7 4 KO cells: anti-CD70 CAR+/ cells/mouse cells/mouse TRAC-/B2M-/CD70-/ Reg- 4 Anti-CD70 CAR T + 5 .times. 10.sup.6 1 .times. 10.sup.7 4 TGFBRII KO cells: anti- cells/mouse cells/mouse CD70 CAR+/TRAC-/ B2M-/CD70-/TGFBRII- 5 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 1 .times. 10.sup.7 4 KO + TGFBRII KO cells: cells/mouse cells/mouse anti-CD70 CAR+/TRAC-/ B2M-/CD70-/Reg-/ TGFBRII-

[0417] Tumor volume was measured 2 times weekly (.about.every 3-4 days) from day of treatment initiation. By day 11 post-injection, anti-CD70 CAR T cells with both TGFBRII and Regnase genes KO began to show a significant effect on reducing tumor volume compared to other treatment groups. Approximately one month later the anti-CD70 CAR T+Reg KO+TGFBRII KO cells had completely eliminated tumor growth in the subcutaneous CAM-1 model (FIG. 17A).

[0418] These results demonstrated that disrupting both the TGFBRII and Regnase genes in CAR T cells increased the potency of the CAR T Cells and effectively cleared tumors in the subcutaneous CAM-1 renal cell carcinoma tumor xenograft model.

Treatment in the Non-Small Cell Lung Carcinoma (NSCLC) Tumor Model

[0419] The ability of T cells expressing a CD70 CAR with TGFBRII and/or Regnase gene edits to eliminate lung adenocarcinoma cells that express moderate levels of CD70 was evaluated in vivo using a subcutaneous lung carcinoma (NCI-H1975) tumor xenograft mouse model. Anti-CD70 CAR+ T cells were produced as described herein. See, e.g., Example 13.

[0420] The ability of these anti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+ lung carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, Ariz.). In brief, 20, 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. Mice received a subcutaneous inoculation of 5.times.10.sup.6 NCI-H1975 lung carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of .about.85 mm.sup.3, the mice were further divided into 5 treatment groups as shown in Table 10. On Day 1, treatment four groups received a single 200 .mu.l intravenous dose of 1.times.10.sup.7 anti-CD70 CAR+ T cells according to Table 10.

TABLE-US-00012 TABLE 10 Treatment groups CAR+ T cell NCI-H1975 treatment Group CAR-T cells (i.v.) N 1 None 5 .times. 10.sup.6 None 4 cells/mouse 2 Anti-CD70 CAR T cells: 5 .times. 10.sup.6 1 .times. 10.sup.7 4 anti-CD70 CAR+/TRAC-/ cells/mouse cells/mouse B2M-/CD70- 3 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 1 .times. 10.sup.7 4 KO cells: anti-CD70 CAR+/ cells/mouse cells/mouse TRAC-/B2M-/CD70-/ Reg- 4 Anti-CD70 CAR T + 5 .times. 10.sup.6 1 .times. 10.sup.7 4 TGFBRII KO cells: anti- cells/mouse cells/mouse CD70 CAR+/TRAC-/ B2M-/CD70-/TGFBRII- 5 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 1 .times. 10.sup.7 4 KO + TGFBRII KO cells: cells/mouse cells/mouse anti-CD70 CAR+/TRAC-/ B2M-/CD70-/Reg-/ TGFBRII-

[0421] Tumor volume was measured 2 times weekly from day of treatment initiation. By day 12 post-injection, animal treated with anti-CD70 CAR T cells having the TGFBRII edit exhibited attenuated tumor growth. Tumors treated with anti-CAR T cells with both TGFBRII and Regnase genes disrupted began to show a decrease in tumor volume by day 8 post-injection and cleared tumors by day 29 in 4 mice out of 4. This complete regression of tumors in treated animals continued through day 53 post injection. Treatment with anti-CD70 CAR+/TRAC-/B2M-/CD70-/Reg-/TGFBRII- T cells resulted in potent activity against established H1975 lung cancer xenografts through 53 days post injection (FIG. 17B). These data demonstrate that disrupting TGFBRII alone or TGFBRII and Regnase-1 in CAR T cells have potent activity against human CD70+ lung cancer tumors in vivo.

Example 15: Tumor Re-Challenge Model Renal Cell Carcinoma Large Tumor Xenograft Model

[0422] The efficacy of anti-CD70 CAR T cells having TGFBRII and/or Regnase-1 genes disrupted (see, e.g., Example 10) were tested in a subcutaneous A498 xenograft model with an ACHN re-challenge. In brief, five million A498 cells were injected subcutaneously in the right flank of NSG mice. Tumors were allowed to grow to an average size of approximately 425 mm.sup.3 after which the tumor-bearing mice were randomized in five groups (N=5/group). Group 1 was left untreated (no treatment) while Groups 2-5 received one of the anti-CD70 CAR T cell treatments shown Table 11.

TABLE-US-00013 TABLE 11 Treatment Conditions CAR+ T cell treatment Group CAR-T A498 cells (i.v.) N 2 Anti-CD70 CAR T cells: 5 .times. 10.sup.6 8 .times. 10.sup.6 5 anti-CD70 CAR+/TRAC-/ cells/mouse cells/mouse B2M-/CD70- 3 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 8 .times. 10.sup.6 5 KO cells: anti-CD70 CAR+/ cells/mouse cells/mouse TRAC-/B2M-/CD70-/ Reg- 4 Anti-CD70 CAR T + 5 .times. 10.sup.6 8 .times. 10.sup.6 5 TGFBRII KO cells: anti- cells/mouse cells/mouse CD70 CAR+/TRAC-/ B2M-/CD70-/TGFBRII- 5 Anti-CD70 CAR T + Reg 5 .times. 10.sup.6 8 .times. 10.sup.6 5 KO + TGFBRII KO cells: cells/mouse cells/mouse anti-CD70 CAR+/TRAC-/ B2M-/CD70-/Reg-/ TGFBRII-

[0423] On Day 56, a tumor re-challenge was initiated whereby 1.times.10.sup.7 ACHN cells were injected into the left flank of treated mice and into a new control group (no treatment).

[0424] As shown in FIG. 18A, all mice treated with all CAR T cell populations having a disrupted TGFBRII and/or Regnase gene showed complete clearance of the A498 tumor by day 50. However, when mice were rechallenged with a new RCC tumor cell (ACHN) only CAR T Cells with both Regnase and TGFBRII edits were able to clear the tumor compared to cells with either Regnase-1 or TGFBRII disruptions alone (FIG. 18B).

Example 16: Analysis of T Cell Fraction in Renal Cell Carcinoma (CAKI-1) Tumor Xenograft Model

[0425] Blood samples were taken from mice with CAKI-1 RCC tumors, 44 days after CAR T administration. Briefly, 100 ul of mouse whole blood was collected via submandibular vein. Red blood cell lysis buffer was used to achieve optimal lysis of erythrocytes with minimal effect on lymphocytes. Human CD45 and mouse CD45 were used as a biomarker to separate human and mouse cells by FACS. The blood samples were evaluated by flow cytometry looking for absolute CAR T counts as well as memory T cell subsets. An anti-CD70 CAR anti-idiotype antibody was used to detect CAR T cells and CD45RO+CD27+ to define central memory T cells. See U.S. Patent Application No. 63/069,889, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0426] The results demonstrate that the addition of the TGFBRII and Regnase-1 gene edit significantly enhanced the population of central memory T cells compared to the edit of either TFGBRII or Regnase-1 alone, which correlates with massive expansion of CAR T cells (FIG. 19A) seen in these animals. And the TGFBRII edit further promoted the potential of CAR T cell proliferation in vivo, suggesting a robust synergistic effect along with the Regnase edit (FIG. 19B).

Example 17: Assessment of Anti-CD19 CAR-T Cells Having TGFBRII and/or Regnase-1 Gene Disruptions in an Intravenous Disseminated Models in NOG Mice

[0427] Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia Tumor Xenograft Model

[0428] The Intravenous Disseminated Model (Disseminated Model) using the Nalm-6 Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice was used to further demonstrate the efficacy of anti-CD19 CAR T cells with TGFBRII and/or Regnase-1 gene edits. Efficacy of various anti-CD19 CAR T populations were evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-Prkdc.sup.scidI12rg.sup.tm1Sug/JicTac) 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 5 treatment groups as shown in Table 12. On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5.times.10.sup.6 Nalm6 cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the Nalm6 cells), treatment Groups 2-4 received a single 200 .mu.l intravenous dose of CAR+ T cells per Table 12.

TABLE-US-00014 TABLE 12 Treatment groups. anti-CD19 CAR T Nalm6 Cells Treatment Group CAR T (i.v.) (i.v.) N 1 Untreated 0.5 .times. 10.sup.6 None 5 cells/mouse 2 Anti-CD19 CAR T cells: 0.5 .times. 10.sup.6 4 .times. 10.sup.6 5 anti-CD19 CAR+/TRAC-/ cells/mouse CAR-T B2M- positive cells/mouse 3 Anti-CD19 CAR T + Reg 0.5 .times. 10.sup.6 4 .times. 10.sup.6 5 KO cells: anti-CD19 CAR+/ cells/mouse CAR-T TRAC-/B2M-/Reg- positive cells/mouse 4 Anti-CD19 CAR T + 0.5 .times. 10.sup.6 4 .times. 10.sup.6 5 TGFBRII KO cells: cells/mouse CAR-T anti-CD19 CAR+/TRAC-/ positive B2M-/TGFBRII- cells/mouse 5 Anti-CD19 CAR T + Reg 0.5 .times. 10.sup.6 4 .times. 10.sup.6 5 KO + TGFBRII KO cells: cells/mouse CAR-T anti-CD19 CAR+/TRAC-/ positive B2M-/Reg- cells/mouse

[0429] During the course of the study mice were monitored daily and body weight was measured two times weekly as described above.

[0430] TGFBRII gene editing combined with Regnase editing induced a maintained NALM6 tumor regression at an early time point (day 18) post tumor inoculation, compared to either edit alone. This reduction in tumor size was maintain (FIG. 20A). The sharp decline in tumor size in the TGFBRII KO group at day 74 post tumor inoculation represents only 5 of 15 mince. Ten of the 15 mice in TGFBRIIKO group had already reached the tumor BLI endpoint.

[0431] While disruption of either TGFBRII or Regnase showed some survival advantage in the Nalm6 Model mice treated with anti-CD19 CAR+ cells, having both TGFBRII and Regnase gene disruptions exhibited the greatest survival advantage (FIG. 20B).

Intravenous Disseminated JeKo-1 Tumor Xenograft Model

[0432] The Intravenous Disseminated Model (Disseminated Model) using the JeKo-1 Human Mantle cell lymphoma (MCL) tumor cell line in NOG mice was used to further demonstrate the efficacy of anti-CD19 CAR T cells with TGFBRII and/or Regnase gene edits. Efficacy of various anti-CD19 CAR T populations were evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-Prkdc.sup.scidI12rg.sup.tm1Sug/JicTac) 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 5 treatment groups as shown in Table 13. On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5.times.10.sup.6 JeKo-1 cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the JeKo-1 cells), treatment Groups 2-4 received a single 200 .mu.l intravenous dose of CAR T cells per Table 13.

TABLE-US-00015 TABLE 13 Treatment groups. anti-CD19 CAR+ T cell JeKo-1 Cells Treatment Group CAR T (i.v.) (i.v.) N 1 Untreated 5 .times. 10.sup.6 None 5 cells/mouse 2 Anti-CD19 CAR T cells: 5 .times. 10.sup.6 4 .times. 10.sup.6 5 anti-CD19 CAR+/TRAC-/ cells/mouse cells/mouse* B2M- 3 Anti-CD19 CAR T + Reg 5 .times. 10.sup.6 4 .times. 10.sup.6 5 KO cells: anti-CD19 CAR+/ cells/mouse cells/mouse* TRAC-/B2M-/Reg- 4 Anti-CD19 CAR T + 5 .times. 10.sup.6 4 .times. 10.sup.6 5 TGFBRII KO cells: cells/mouse cells/mouse* anti-CD19 CAR+/TRAC-/ B2M-/TGFBRII- 5 Anti-CD19 CAR T + Reg 5 .times. 10.sup.6 4 .times. 10.sup.6 5 KO + TGFBRII KO cells: cells/mouse cells/mouse* anti-CD19 CAR+/TRAC-/ B2M-/Reg-/TGFBRII-

[0433] During the course of the study mice were monitored daily and body weight was measured two times weekly as described above.

[0434] While either TGFBRII or Regnase showed some survival advantage in the JeKo-1 Model, mice treated with anti-CD19 CAR+ cells having both TGFBRII and Regnase gene edits exhibited the greatest survival advantage FIG. 21.

[0435] CAR T Cell Expansion In Vivo

[0436] CAR T cell expansion was assessed by measuring the CAR copy number by ddPCR of DNA isolated from blood samples collected throughout the Jeko-1 and Nalm-6 studies as described above.

[0437] DNA was isolated from mouse tissue using the Qiagen Dneasy blood and tissue kit (Qiagen, Venlo, Netherlands). Total mass of nucleic acid from RBC-lysed samples was quantitated using either Nanodrop (Thermo Fisher Scientific) or DropSense96 (trinean, Gentbrugge, Belgium) machines. Primers and 6-carboxyfluorescein (FAM)-labeled probe sets (provided in Table 14 below) were designed to quantitate the levels of the integrated CAR construct into the human TRAC locus by droplet digital PCR (ddPCR). ddPCR was performed using the Bio-Rad Automated Droplet Generator, Bio-Rad T100 Thermal Cycler, and Bio-Rad QX200 Droplet Reader machine(s) (Bio-rad Laboratories, Hercules, Calif.). QuantaSoft Version 1.7.4.0917 (Bio-rad Laboratories) software was used to calculate the absolute number of integrated CAR copies per sample. Finally, the number of detected CAR alleles was divided by the input total DNA amount to compute the absolute number of CAR copies per mass of input sample. The ddPCR assay detects the number of copies of integrated CAR transgene per mass of genomic DNA (gDNA) by amplifying an 866 bp amplicon spanning endogenous TRAC sequence and the CAR expression cassette promotor (EF-1.alpha.). In brief, qualification of the assay yielded linear data (R.sup.2>0.95) within the range tested (2 to 300,000 copies per ug of gDNA) as well as generated a % relative error (% RE) and % coefficient of variation (% CV) within normal ranges (% RE.ltoreq.100% and % CV.ltoreq.20%) for conditions .gtoreq.LLOQ. The LLOD and LLOQ were calculated based on the available data and the LLOD was set to 5 copies per 0.2 .mu.g of gDNA and the LLOQ was set to 40 copies per 0.2

TABLE-US-00016 TABLE 14 Primers and probes used for ddPCR CAR primers and probe CTX110-20-30_dd_1 GGCACCATATTCATTTTGCAGGTGAA Forward (SEQ ID NO: 11) CTX110-20-30_dd_1 ATGTGCGCTCTGCCCACTGACGGGC Reverse (SEQ ID NO: 12) CTX110-20-30_dd_1 AGACATGAGGTCTATGGACTTCAGGCTCC Probe (FAM) (SEQ ID NO: 13)

[0438] These analysis demonstrate that the addition of either TGFBRII or Regnase-1 KO to allogeneic CAR T cells (TRAC.sup.-/B2M.sup.-; Group C-10) allowed the T cells to expand to larger levels in the blood of treated mice (e.g., Groups C10-TG, C10-R, C10-TG/R) compared to groups treated with the allogeneic CART cells without those KOs (e.g., Group C10) (FIG. 22A). This expansion was apparent at day 14 of the Jeko-1 study. Loss of both TGFBRII and Regnase-1 (FIG. 22A, C10-TG/R) led to a more uniform expansion relative to TGFBRII (FIG. 22A, C10-TG) or Regnase-1 (FIG. 22A, C10-R) single KOs. In the Nalm-6 study, disruption of both TGFBRII and Regnase-1 had a synergistic effect on CAR T cell expansion at day 28 as shown in FIG. 22B

[0439] In sum, all groups with loss of either TGFBRII or Regnase-1 had expanded CAR-T cells in the peripheral blood.

Example 18: Generation of CAR T Cells with Multiple Gene Editing and Verification of Gene Edits

[0440] Activated primary human T cells were electroporated with Cas9/sgRNA RNP complexes (200 pmol Cas9, 1000 pmol gRNA) to generate cells edited for TRAC-/.beta.2M-, TRAC-/.beta.2M-/Regnase-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Regnase-1-/TGFBRII-. Sequence encoding anti-BCMA CAR was inserted into the TRAC locus using recombinant AAV6 carrying the DNA sequence for anti-BCMA CAR (SEQ ID NO: 170). The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), Reg-1 (SEQ ID NO: 51; REG1-Z10) and TGFBRII (SEQ ID NO: 313).

[0441] Flowcytometry was used to verify the editing for TRAC, .beta.2M and the insertion and expression of anti-BCMA CAR. Briefly, about one week post electroporation, cells were stained with anti-human TCR, anti-human .beta.2M and recombinant biotinylated human BCMA/streptavidin-APC to assess the levels of editing for TRAC and .beta.2M, and insertion of the nucleotide sequence encoding anti-BCMA CAR.

[0442] TRAC-/.beta.2M-, TRAC-/.beta.2M-/Reg-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells show consistent rates of TCR and .beta.2M disruptions at >90% and >60% rates, respectively as determined by flow cytometry (FIGS. 23A and 23B). Anti-BCMA CAR expression was measured flow cytometrically by determining the percentage of cells that bound recombinant biotinylated BCMA/streptavidin-APC conjugate. All the conditions including TRAC-/.beta.2M-, TRAC-/.beta.2M-/Reg-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells show consistent rates of CAR insertion (>70%), while the unedited RNP- T-cells have no detectable staining for anti-BCMA CAR (FIG. 23C). The ratio of CD4/CD8 T cells as assessed by flow cytometry in the TRAC-/.beta.2M-, TRAC-/.beta.2M-/Reg-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells were found to be consistent in the range of 55-60%/40-45% across all the samples (FIG. 23D).

[0443] TIDE analysis was performed for the verification of editing rates for Reg-1 and TGFBRII genes. Briefly, about one week post electroporation, two million cells from TRAC-/.beta.2M-, TRAC-/.beta.2M-/Reg-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells and two million unedited T-cells from the same donor were removed from culture and transferred to 1.5 mL microcentrifuge tubes. Cells were spun down in a tabletop microcentrifuge at 300 g for 10 minutes and the resulting supernatant was discarded. The cells were washed twice with 1000 uL 1.times.PBS and the cell pellets were frozen at -80.degree. C. The frozen cell pellets were then used for the extraction of genomic DNA using QIAamp DNA Blood Mini Kit (Qiagen, catalog #51106). Gene-specific primers were used to amplify the region flanking the cut sites of Reg-1 and TGFBRII (Invitrogen.TM. Platinum.TM. SuperFi.TM. II Green PCR Master Mix; catalog #12369050) and the PCR amplicons derived were subsequently sequenced and analyzed by TIDE to determine the indel patterns/frequencies (editing frequencies).

[0444] The analyzed indel frequencies were found to be within the expected range of 65-80% for TGF sgRNA and >80% for the Regnase-1 sgRNA, respectively (FIGS. 24A and 24B).

Example 19: Cytotoxicity of Anti-BCMA CAR T Cells with Multiple Gene Edits

[0445] A cytotoxicity (cell kill) assay was used to assess the ability of the TRAC-/.beta.2M-, TRAC-/.beta.2M-/Reg-1-, TRAC-/.beta.2M-/TGFBRII- and TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells (produced by the methods disclosed herein, see, e.g., Example 18) to cause cell lysis in two target cell lines, MM.1S (multiple myeloma cell line) and JeKo-1 (mantle cell lymphoma cell line). Unedited RNP- cells without CAR were used as a negative control to determine the specific lysis by CAR+ T cells. Briefly, the target cell lines were stained with eBioscience.TM. Cell Proliferation Dye eFluor.TM. 670 (Thermofisher Scientific; catalog #65-0840-85) per manufacturer's instructions and seeded into 96-well plates at 50,000 cells per well. Next, CAR T-cells or RNP- T cells were added to the wells containing target cells at ratios of 0, 0.5:1, 1:1, 2:1, or 4:1 (T cell: target cell) and incubated further for approximately 4 hours for MM.1S and 24 hours for JeKo-1. After the respective incubation period, the 96-well plates were spun down at 300 g for 10 minutes and 100 .mu.L of supernatant was removed for cytokine quantification. Cells were then washed once with 1.times.PBS and stained with 150 ul of 1.times.PBS supplemented with 0.5% BSA and 5 .mu.g/mL DAPI (Invitrogen; catalog #D3571) and incubated for 15 minutes in dark. Post-incubation, cells were washed-off DAPI, resuspended in 150 .mu.l of 1.times.PBS supplemented with 0.5% BSA, and acquired and analyzed using a flow cytometer. Target cells were identified via eFluor-based fluorescence and then divided into live and dead cells based on their DAPI fluorescence.

[0446] The TRAC-/.beta.2M-/Reg-1-/TGFBRII- anti-BCMA CAR+ T-cells exhibited greater cytotoxicity towards the MM.1S (FIG. 25A) and JeKo-1 cell lines (FIG. 25C) compared to TRAC-/.beta.2M-, TRAC-/.beta.2M-/Regnase-1- or TRAC-/.beta.2M-/TGFBRII- anti-BCMA CAR+ T-cells. Comparative data from K562 cells (as controls) are provided in FIG. 25B and FIG. 25D.

Example 20: In Vivo Effects of TGFBRII+Regnase-1 Disruption on Allogeneic CAR T Cells in the Subcutaneous RPMI-8226 Xenograft Tumor Model

[0447] A subcutaneous tumor mouse model was utilized to assess the in vivo efficacy of allogeneic anti-BCMA CARs with the following gene disruptions: 1) .beta.2M and TRAC, 2) .beta.2M, TRAC, and TGFBRII, 3) .beta.2M, TRAC, and Reg-1, and 4) .beta.2M, TRAC, TGFBRII, and Reg-1. The subcutaneous tumor mouse model utilized the BCMA+ multiple myeloma derived RPMI-8226 tumor cell line in NSG mice. The TGFBRII gene was edited via CRISPR/Cas-mediated gene editing using the TGFBRII Ex5_T1 guide (SEQ ID NO. 313). The Reg-1 gene was edited via CRISPR/Cas-mediated gene editing using the Z10 guide (SEQ ID NO. 51). The anti-BCMA CAR T cells express an anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146). See also the sequence Tables 22, 23, 27, and 39 below.

[0448] Efficacy of the anti-BCMA CAR T cells was evaluated in the subcutaneous xenograft model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 25 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. On day 1, mice received a subcutaneous inoculation of 1.times.10.sup.7 RPMI-8226 cells/mouse in the right hind flank. Nine days later (Day 10), the tumor inoculation sites were inspected to determine if the tumors were palpable. After confirming palpability, the mice were further divided into 5 treatment groups as shown in Table 1. All treatment groups received a single 200 ul intravenous dose of 1e6 anti-BCMA CAR+ T cells.

TABLE-US-00017 TABLE 15 Treatment Groups for the RMPI-8226 Xenograft Study Group CAR T cells (i.v.) N 1 NA 5 2 anti-BCMA CAR/TRAC-/.beta.2M- 5 3 anti-BCMA CAR/TRAC-/.beta.2M-/TGFBRII- 5 4 anti-BCMA CAR/TRAC-/.beta.2M-/TGFBRII-/ 5 Regnase- 5 anti-BCMA CAR/TRAC-/.beta.2M-/TGFBRII-/ 5 Regnase-

[0449] Throughout the course of the study, the mice were subjected to gross observations daily, while tumor volume and body weight were measured twice weekly (.about.every 3-4 days) starting on Day 10. A significant endpoint was 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: [0450] Loss of body weight of 20% or greater sustained for a period of greater than 1 week; [0451] Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate; [0452] Prolonged, excessive diarrhea leading to excessive weight loss (>20%); or [0453] Persistent wheezing and respiratory distress.

[0454] 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.

[0455] Mice in groups receiving TRAC-/.beta.2M-/TGFBRII-/Reg-1- anti-BCMA CAR+ T-cells saw an increase in survival relative to untreated mice; mice treated TRAC-/.beta.2M- anti-BCMA CAR+ T-cells, TRAC-/.beta.2M-/TGFBRII anti-BCMA CAR+ T-cells, or TRAC-/.beta.2M-/Reg-1- anti-BCMA CAR+ T-cells (FIG. 26B). Mice receiving TRAC-/B2M-/TGFBRII-/Regnase- anti-BCMA CAR+ T cells showed significant tumor regression, while none of the other conditions tested showed significant inhibition of tumor growth (FIG. 26A). These data demonstrate that disruption of TGFBRII and Reg-1 in CAR T cells increases efficacy of CAR T cells in a mouse xenograft tumor model.

[0456] Next, small amounts of blood were taken from each mouse for FACS analysis to characterize circulating CAR-T cells and determine drug pharmacokinetics. Approximately 75 uL of blood was drawn 2 weeks post CAR-T dosing via submandibular bleeds. The blood was then transferred into K2 EDTA tubes and shipped overnight to CRISPR Therapeutics on 4 C cold packs. The following day, blood samples were processed with RBC (Red Blood Cell) Lysis Buffer (BioLegend.RTM., catalog #420301) per manufacturer's instructions. The samples then underwent anti-mouse CD16/32 blocking via anti-mouse Trustain FcX.TM. (BioLegend.RTM., catalog #101320) per manufacturer's instructions. The samples were then processed via flow cytometry to determine prevalence of human CD45 expressing cells, which would represent the circulating CAR-T cells. Blood from mice that had received TRAC-/.beta.2M-/TGFBRII-/Regnase- anti-BCMA CAR+ T-cells showed a high amount of circulating human CD45+ cells, which was not seen in any other treatment groups (FIG. 26C). This indicates that the TGFBRII and Reg-1 knockouts confer superior expansion of CAR-T cells in a multiple myeloma mouse xenograft model.

Example 21: In Vivo Synergistic Effects of TGFBRII+Regnase-1 Disruptions on Allogeneic CAR T Cells in the Subcutaneous JeKo-1 Xenograft Tumor Model

[0457] A subcutaneous tumor mouse model was utilized to further assess the in vivo efficacy of TRAC-/.beta.2M- anti-BCMA CAR+ T-cells and TRAC-/.beta.2M-/TGFBRII-/Reg-1/anti-BCMA CAR+ T-cells. The subcutaneous tumor mouse model utilized the low BCMA expressing mantle cell lymphoma derived JeKo-1 tumor cell line in NSG mice. The TGFBRII gene was edited via CRISPR/Cas-mediated gene editing using TGFBRII Ex5_T1 guide (SEQ ID NO: 313). The Reg-1 gene was edited via CRISPR/Cas-mediated gene editing using the Z10 guide (SEQ ID NO: 51). The anti-BCMA CAR T cells express an anti-BCMA CAR comprising the amino acid sequence of SEQ ID NO: 146. See also the sequence Tables 22, 23, 27, and 39 below.

[0458] Efficacy of the anti-BCMA CAR T cells was evaluated in the subcutaneous xenograft model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 15 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. On day 1, mice received a subcutaneous inoculation of 5.times.10.sup.6 JeKo-1 cells/mouse in the right hind flank. Tumors were then periodically sized in via calipers. Once average tumor size reached an average of 150 mm.sup.3 (with an acceptable range of 125-175 mm.sup.3), the mice were further divided into 3 treatment groups as shown in Table 1. All treatment groups received a single 200 ul intravenous dose of 10e6 anti-BCMA CAR+ T cells. The day of T-cell injection was marked as Day 1.

TABLE-US-00018 TABLE 16 Treatment Groups for the RMPI-8226 Xenograft Study Group CAR T cells (i.v.) N 1 NA 5 2 anti-BCMA CAR/TRAC-/.beta.2M- 5 5 anti-BCMA CAR/TRAC-/.beta.2M-/TGFBRII-/ 5 Regnase-

[0459] Throughout the course of the study, the mice were subjected to gross observations daily, while tumor volume and body weight were measured twice weekly (every 3-4 days) starting on Day 1. A significant endpoint was 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: [0460] Loss of body weight of 20% or greater sustained for a period of greater than 1 week; [0461] Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate; [0462] Prolonged, excessive diarrhea leading to excessive weight loss (>20%); or [0463] Persistent wheezing and respiratory distress.

[0464] 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.

[0465] Mice in groups receiving TRAC-/.beta.2M-/TGFBRII-/Reg-1- anti-BCMA CAR+ T-cells saw a significant increase in survival relative to both untreated mice and mice treated TRAC-/.beta.2M- anti-BCMA CAR+ T-cells (FIG. 27B). Mice receiving TRAC-/B2M-/TGFBRII-/Reg-1- anti-BCMA CAR+ T cells arrested tumor growth, while TRAC-/.beta.2M- anti-BCMA CAR+ T-cells did not significant inhibit tumor growth (FIG. 27A). These data demonstrate that disruption of TGFBRII and Reg-1 genes in CAR T cells increases efficacy of CAR-T cells in a mouse xenograft tumor model.

[0466] Next, small amounts of blood were taken from each mouse for FACS analysis to characterize circulating CAR-T cells and determine drug pharmacokinetics. Approximately 75 uL of blood was drawn 2 and 3 weeks post CAR-T dosing via submandibular bleeds. The blood was then transferred into K2 EDTA tubes and shipped overnight to CRISPR Therapeutics on 4 C cold packs. The following day, blood samples were processed with RBC (Red Blood Cell) Lysis Buffer (BioLegend.RTM., catalog #420301) per manufacturer's instructions. The samples then underwent anti-mouse CD16/32 blocking via anti-mouse Trustain FcX.TM. (BioLegend.RTM., catalog #101320) per manufacturer's instructions. To quantify the number of circulating T-cells, the sum of cells positive for human CD4 and CD8 was determined. At the two week timepoint, blood from mice that had received TRAC-/.beta.2M-/TGFBRII-/Reg-1- anti-BCMA CAR+ T-cells showed significantly higher concentrations of human CD4 and human CD8+ expressing cells relative to blood from mice that received TRAC-/.beta.2M- anti-BCMA CAR+ T-cells (FIG. 27C).

[0467] Furthermore, the TRAC-/.beta.2M- TGFBRII-/Reg-1- anti-BCMA CAR+ T-cells showed lower expression of the T-cell exhaustion markers Lag3 and PD1 relative to the TRAC-/.beta.2M- anti-BCMA CAR+ T-cells (FIG. 27D). At the three week timepoint, the overall level of hCD45+ cells in circulation had equalized between groups (FIG. 27E), but the expression of Lag3 and PD1 remained lower in mice treated with TRAC-/.beta.2M- TGFBRII-/Reg-1- anti-BCMA CAR+ T-cells (FIG. 27F). This indicates that CAR-T cells containing the TGFBRII and Regnase knockouts have a superior ability to expand when compared to CAR-T cells lacking those edits while also reducing the expression of T-cell exhaustion markers PD-1 and Lag3.

Example 22: Generation of Anti-PTK7 CAR T Cells with Disrupted TGFBRII and Regnase-1 Genes

[0468] Allogeneic human T cells that lack expression of the TRAC gene, .beta.2M gene, TGFBRII gene and Reg-1 gene, and express a chimeric antigen receptor (CAR) targeting PTK7 were produced. Activated human T cells were electroporated with Cas9:sgRNA RNPs (1 .mu.M Cas9, 5 .mu.M gRNA), followed by incubation with a recombinant adeno-associated adenoviral vectors, serotype 6 (AAV6) (MOI 50,000).

[0469] Recombinant AAV comprised a nucleotide sequence encoding an anti-PTK7 CAR comprising the amino acid sequence of SEQ ID NO: 349. The following sgRNAs were used: TRAC (SEQ ID NO: 58), .beta.2M (SEQ ID NO: 62), TGFBRII (SEQ ID NO: 313) and REGNASE-1 (SEQ ID NO: 51). The sgRNAs, which form RNPs with the Cas9 enzyme, can be introduced into the T cells in a single electroporation event to produce the resulting modified cell populations shown in Table 17 below. After the electroporation, the cells were transduced with the recombinant AAV to introduce the donor template encoding for the anti-PTK7 CAR.

TABLE-US-00019 TABLE 17 Genetically Engineered CAR-T Cell Populations Population Edits Anti-PTK7 CAR T cells anti-PTK7 CAR+/TRAC-/B2M- Anti-PTK7 CAR T + TGFBRII anti-PTK7 CAR+/TRAC-/B2M-/ KO cells TGFBRII- Anti-PTK7 CAR T + TGFBRII anti-PTK7 CAR+/TRAC-/B2M-/ KO + Reg KO cells TGFBRII-/Reg-

[0470] At 7 days post-electroporation, T cells were checked for CAR expression by flow cytometry. Both anti-PTK7 CAR T cells and anti-PTK7 CAR T cells that lack TGFBRII and anti-PTK7 CAR T cells that lack TGFBRII and Regnase expressed nearly equivalent amount of CAR on their surface at day 7 post HDR. The results are provided in Table 18 below.

TABLE-US-00020 TABLE 18 Percentage of CAR, TCR, and b2M Expression on Day 7 Post HDR CAR+ TCR+ .beta.2M+ Treatment % % % No RNP 3.33 92 93.7 No AAV 5.16 2.63 3.87 Anti-Ptk7 CAR 82.2 1.24 2.49 Anti-Ptk7 CAR & TGFBRII KO 83.2 0.82 2.1 Anti-Ptk7 CAR & TGFBRII/Reg-1 KO 81.7 0.77 2

[0471] Efficient editing of TGFBRII and/or Regnase was achieved in the engineered anti-Ptk7 CAR T cell (Table 19 below) and show an increase in cell proliferation with TGFBRII and Reg-1 disruption (FIG. 28), while cell viability and CD4+/CD8+ T cells ratios remain unchanged.

TABLE-US-00021 TABLE 19 Indel Percentage in TGFBRII and Regnase-1 on Day 7 Post HDR TGFBRII Reg-1 Treatment Indel % Indel % No RNP 1.8 1.6 No AAV 97.75 88.8 Anti-Ptk7 CAR 1.45 2.2 Anti-Ptk7 CAR & TGFBRII KO 97.15 3 Anti-Ptk7 CAR & TGFBRII/Reg-1 KO 97.7 92.2

[0472] In summary, the data presented in this example demonstrated that TGFBRII and/or Reg-1 disruption in anti-Ptk7 CAR T cells (e.g., anti-PTK7 CAR+/TRAC-/B2M-/TGFBRII- or anti-PTK7 CAR+/TRAC-/B2M-/TGFBRII-/Reg-1-), can increase cell proliferation, while not affecting cell viability or CD4/CD8 cell ratios.

Example 23: Disruption of TGFBRII Alone Increases CAR T Cell Killing Upon Serial Rechallenge In Vitro

[0473] The anti-PTK7 CAR+ T cells generated above were serially rechallenged with PTK7+ osteosarcoma cancer cell line, Saos2, and evaluated for their ability to kill the PTK7+ osteosarcoma cancer cell line Saos2.

[0474] The anti-PTK7 CAR+ T cells used in this experiment contained the following edits: [0475] Anti-PTK7 CAR T cells: anti-PTK7 CAR+/TRAC-/B2M- [0476] Anti-PTK7 CAR T+TGFBRII KO cells: anti-PTK7 CAR+/TRAC-/B2M-/TGFBRII- [0477] Anti-PTK7 CAR T+TGFBRII KO+Reg KO cells: anti-PTK7 CAR+/TRAC-/B2M-/TGFBRII-/Reg-

[0478] In a 96-well plate format, CAR T cells were first co-cultured with Saos2 cells (6,250 CAR T cells, 50,000 tumor cells) on D0 and re-challenged with 50,000 tumor cells on D2, D4, D6, D8, D10, D12 and D14.

[0479] Analysis of tumor cell and CAR T cell number was performed at D1, D3, D5, D7, D9, D11 and D13 using flow cytometry (method adapted from Wang et al., JoVE 2019). The following antibodies in Table 20 were used at 1:100 dilution.

TABLE-US-00022 TABLE 20 Antibody Information Antibody Flour cat # Dilution Vendor CD4 BV510 300546 1:100 Biolegend CD8 FITC 344704 1:100 Biolegend PTK7 PE 130-091-364 1:50 Miltenyi CD62L BV605 304833 1:100 Biolegend human CD45 BV785 304048 1:100 Biolegend PD1 APC/Cy7 329922 1:100 Biolegend CD45RO PE/Cy7 304230 1:100 Biolegend Streptavidin APC 405207 1:100 Biolegend Tim3 BV421 345008 1:100 Biolegend Live/Dead 7AAD BDB559925 1:500 BD

[0480] The results demonstrate that disrupting the TGFBRII gene improved potency (FIG. 29A) and CAR T cell expansion (FIG. 29B) as measured by hum CD45 staining, when CAR T cells are repeatedly challenged with PTK7+ positive target cells. The addition of Regnase gene disruption does not provide an added advantage in potency over TGFBRII deletion alone. Potency and expansion is improved compared to CAR T cells that have neither, or both (i.e.: TGFBRII and Regnase), of the genes disrupted. In addition, the results demonstrate that cytotoxic CD8+ CAR T cells persist longer during serial rechallenge (FIG. 29C) with tumor cells if the TGFBRII gene is disrupted compared to anti-PTK7 CAR T cells that have neither or both (i.e.: TGFBRII and Regnase) of the genes disrupted. CD4+ CAR T cells remain consistent regardless of whether TGFBRII and/or Regnase genes are disrupted (FIG. 29D).

Example 24: Treatment Efficacy of Anti-PTK7 CART Cells with Multiple Gene Disruptions in the Subcutaneous Pancreatic Cell Carcinoma Tumor Xenograft Model

Treatment in the Pancreatic Cell Carcinoma Tumor Model

[0481] The ability of T cells expressing a PTK7 CAR with TGFBRII and/or Reg-1 gene edits to eliminate pancreatic cell carcinoma cells that express medium levels of PTK7 was evaluated in vivo using a subcutaneous renal cell carcinoma (Hs766T) tumor xenograft mouse model. Anti-PTK7 CAR+ T cells were produced as described above. See, e.g., Example 22.

[0482] The ability of these anti-PTK7 CAR+ T cells to ameliorate disease caused by a PTK7+ pancreatic carcinoma cell line was evaluated in NSG mice using methods employed by Translational Drug Development, LLC (Scottsdale, Ariz.). In brief, 20, 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. Mice received a subcutaneous inoculation of 5.times.10.sup.6 Hs766T pancreatic cell carcinoma cells/mouse in the right hind flank. When mean tumor size reached target of .about.50 mm.sup.3, the mice were further divided into 3 treatment groups as shown in Table 21. On Day 1, treatment four groups received a single 200 .mu.l intravenous dose of 0.5.times.10.sup.7 anti-PTK7 CAR+ T cells according to Table 21.

TABLE-US-00023 TABLE 21 Treatment groups CAR-T cell treatment Group CAR-T Hs766T cells (i.v.) N 1 None 5 .times. 10.sup.6 None 5 cells/mouse 2 Anti-PTK7 CAR T cells: 5 .times. 10.sup.6 0.5 .times. 10.sup.7 5 anti-PTK7 CAR+/TRAC-/ cells/mouse cells/mouse B2M- 3 Anti-PTK7 CAR T + 5 .times. 10.sup.6 0.5 .times. 10.sup.7 5 TGFBRII KO cells: cells/mouse cells/mouse anti-PTK7 CAR+/TRAC-/ B2M-/TGFBRII-

[0483] Tumor volume was measured 2 times weekly (.about.every 3-4 days) from day of treatment initiation. By day 11 post-injection, anti-PTK7 CAR T cells with and without TGFBRII gene KO began to show a significant effect on reducing tumor volume compared to no treatment group 1. Approximately one month later the anti-PTK7CAR T with and without TGFBRII KO cells had completely eliminated tumor growth in the subcutaneous Hs766T model (FIG. 30A).

[0484] These results demonstrated that disrupting the TGFBRII gene in CAR T cells effectively cleared tumors in the subcutaneous Hs766T renal cell carcinoma tumor xenograft model. No clinical signs of GvHD were observed in anti-PTK7 CAR T cells with and without TGFBRII KO cells (FIG. 30B).

Example 25: Analysis of T Cell Fraction in Pancreatic Cell Carcinoma (Hs766T) Tumor Xenograft Model

[0485] Blood samples were taken from mice with Hs766T tumors, 47 days after CAR T administration. Briefly, 100 ul of mouse whole blood was collected via submandibular vein. Red blood cell lysis buffer was used to achieve optimal lysis of erythrocytes with minimal effect on lymphocytes. Human CD45 and mouse CD45 were used as a biomarker to separate human and mouse cells by FACS. The blood samples were evaluated by flow cytometry looking for absolute human CD45+ counts as well as memory T cell subsets. Staining for CD45RO+CD27+ was used to define central memory T cells.

[0486] The results demonstrate that the addition of the TGFBRII gene edit significantly enhanced the population of central memory T cells (FIG. 31B) compared to anti-PTK7 CAR T cells without TGFBRII KO which correlates with massive expansion of CAR T cells (FIG. 31A) seen in these animals. And the TGFBRII edit further promoted the potential of CAR T cell proliferation in vivo (FIG. 31B).

[0487] Sequence Tables

[0488] The following tables provide details for the various nucleotide and amino acid sequences disclosed herein.

TABLE-US-00024 TABLE 22 sgRNA Sequences and Target Gene Sequences for Reg1 Name Unmodified Sequence Modified Sequence Target Sequences (PAM) REG1-Z01 GGUCAUCGAUGGGAGCAA G*G*U*CAUCGAUGGGAGCAAC GGTCATCGATGGGAGCAACG sgRNA CGguuuuagagcuagaaa Gguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX2_T1) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 171) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac GGTCATCGATGGGAGCAACG gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 318) cggugcUUUU (SEQ ID NO: 15) (SEQ ID NO: 14) REG1-Z01 GGUCAUCGAUGGGAGCAA G*G*U*CAUCGAUGGGAGCAAC sgRNA CG G (SEQ ID NO: 17) (EX2_T1) (SEQ ID NO: 16) spacer REG1-Z02 CACCACCCCGCGGGACUA C*A*C*CACCCCGCGGGACUAG CACCACCCCGCGGGACTAGA sgRNA GAguuuuagagcuagaaa Aguuuuagagcuagaaauagca (GGG) (SEQ ID NO: (EX2_T2) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 172) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac CACCACCCCGCGGGACTAGA gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 319) cggugcUUUU (SEQ ID NO: 19) (SEQ ID NO: 18) REG1-Z02 CACCACCCCGCGGGACUA mC*mA*mC*CACCCCGCGGGAC sgRNA GA UAGA (SEQ ID NO: 21) (EX2_T2) (SEQ ID NO: 20) spacer REG1-Z03 GGUCUGGCGCUCCCGCUC G*G*U*CUGGCGCUCCCGCUCG GGTCTGGCGCTCCCGCTCGG sgRNA GGguuuuagagcuagaaa Gguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX2_T3) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 173) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac GGTCTGGCGCTCCCGCTCGG gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 320) cggugcUUUU (SEQ ID (SEQ ID NO: 23) NO: 22) REG1-Z03 GGUCUGGCGCUCCCGCUC mG*mG*mU*CUGGCGCUCCCGC sgRNA GG (SEQ ID NO: 24) UCGG (SEQ ID NO: 25) (EX2_T3) spacer REG1-Z04 UUCACACCAUCACGACGC U*U*C*ACACCAUCACGACGCG TTCACACCATCACGACGCGT sgRNA GUguuuuagagcuagaaa Uguuuuagagcuagaaauagca (GGG) (SEQ ID NO: (EX4_T1) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 174) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac TTCACACCATCACGACGCGT gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 321) cggugcUUUU (SEQ ID NO: 27) (SEQ ID NO: 26) REG1-Z04 UUCACACCAUCACGACGC U*U*C*ACACCAUCACGACGCG sgRNA GU (SEQ ID NO: 28) U (EX4_T1) (SEQ ID NO: 29) spacer REG1-Z05 ACACCAUCACGACGCGUG A*C*A*CCAUCACGACGCGUGG ACACCATCACGACGCGTGGG sgRNA GGguuuuagagcuagaaa Gguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX4_T2) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 175) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac ACACCATCACGACGCGTGGG gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 322) cggugcUUUU (SEQ ID NO: 31) (SEQ ID NO: 30) REG1-Z05 ACACCAUCACGACGCGUG A*C*A*CCAUCACGACGCGUGG sgRNA GG (SEQ ID NO: 32) G (SEQ ID NO: 33) (EX4_T2) spacer REG1-Z06 CUACGAGUCUGACGGGAU C*U*A*CGAGUCUGACGGGAUC CTACGAGTCTGACGGGATCG sgRNA CGguuuuagagcuagaaa Gguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX4_T3) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 176) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac CTACGAGTCTGACGGGATCG gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 323) cggugcUUUU (SEQ ID NO: 35) (SEQ ID NO: 34) REG1-Z06 CUACGAGUCUGACGGGAU C*U*A*CGAGUCUGACGGGAUC sgRNA CG (SEQ ID NO: 36) G (SEQ ID NO: 37) (EX4_T3) spacer REG1-Z07 UUGCCACCCACGCGUCGU U*U*G*CCACCCACGCGUCGUG TTGCCACCCACGCGTCGTGA sgRNA GAguuuuagagcuagaaa Aguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX4_T4) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 177) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac TTGCCACCCACGCGTCGTGA gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ (SEQ ID NO: 324) cggugcUUUU (SEQ ID ID NO: 39) NO: 38) REG1-Z07 UUGCCACCCACGCGUCGU U*U*G*CCACCCACGCGUCGUG sgRNA GA (SEQ ID NO: 40) A (SEQ ID NO: 41) (EX4_T4) spacer REG1-Z08 GUUCACACCAUCACGACG G*U*U*CACACCAUCACGACGC GTTCACACCATCACGACGCG sgRNA CGguuuuagagcuagaaa Gguuuuagagcuagaaauagca (TGG) (SEQ ID NO: (EX4_T5) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 178) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac GTTCACACCATCACGACGCG gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 325) cggugcUUUU (SEQ ID NO: 43) (SEQ ID NO: 42) REG1-Z08 GUUCACACCAUCACGACG G*U*U*CACACCAUCACGACGC sgRNA CG (SEQ ID NO: 44) G (SEQ ID NO: 45) (EX4_T5) spacer REG1-Z09 CACGAUCCCGUCAGACUC C*A*C*GAUCCCGUCAGACUCG CACGATCCCGTCAGACTCGT sgRNA GUguuuuagagcuagaaa Uguuuuagagcuagaaauagca (AGG) (SEQ ID NO: (EX4_T6) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 179) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac CACGATCCCGTCAGACTCGT gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 326) cggugcUUUU (SEQ ID (SEQ ID NO: 47) NO: 46) REG1-Z09 CACGAUCCCGUCAGACUC C*A*C*GAUCCCGUCAGACUCG sgRNA GU (SEQ ID NO: 48) U (SEQ ID NO: 49) (EX4_T6) spacer REG1-Z10 ACGACGCGUGGGUGGCAA A*C*G*ACGCGUGGGUGGCAAG ACGACGCGTGGGTGGCAAGC sgRNA GCguuuuagagcuagaaa Cguuuuagagcuagaaauagca (GGG) (SEQ ID NO: (EX4_T7) uagcaaguuaaaauaagg aguuaaaauaaggcuaguccgu 180) cuaguccguuaucaacuu uaucaacuugaaaaaguggcac ACGACGCGTGGGTGGCAAGC gaaaaaguggcaccgagu cgagucggugcU*U*U*U (SEQ ID NO: 327) cggugcUUUU (SEQ ID (SEQ ID NO: 51) NO: 50) REG1-Z10 ACGACGCGUGGGTGGCAA A*C*G*ACGCGUGGGUGGCAAG sgRNA GC (SEQ ID NO: 52) C (SEQ ID NO: 53) (EX4_T7) spacer *indicates a nucleotide with a 2'-O-methyl phosphorothioate modification.

TABLE-US-00025 TABLE 23 sgRNA Sequences and Target Gene Sequences for TRAC, .beta.2M, and CD70 SEQ ID sgRNA Sequences NO: CD70 sgRNA Modified G*C*U*UUGGUCCCAUUGGUCGCguuuuagagcuagaaauagc 54 (CD70-7) aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggc accgagucggugcU*U*U*U Unmodified GCUUUGGUCCCAUUGGUCGCguuuuagagcuagaaauagcaag 55 uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcUUUU CD70 sgRNA Modified G*C*U*UUGGUCCCAUUGGUCGC 56 spacer Unmodified GCUUUGGUCCCAUUGGUCGC 57 TRAC sgRNA Modified A*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaauagc 58 (TA-1) aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggc accgagucggugcU*U*U*U Unmodified AGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaag 59 uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcUUUU TRAC sgRNA Modified A*G*A*GCAACAGUGCUGUGGCC 60 spacer Unmodified AGAGCAACAGUGCUGUGGCC 61 .beta.2M sgRNA Modified G*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaauagc 62 (.beta.2M-1) aaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggc accgagucggugcU*U*U*U Unmodified GCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaag 63 uuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugcUUUU .beta.2M sgRNA Modified G*C*U*ACUCUCUCUUUCUGGCC 64 spacer Unmodified GCUACUCUCUCUUUCUGGCC 65 Target Sequences (PAM) CD70 target GCTTTGGTCCCATTGGTCGC (GGG) 66 sequence with (PAM) CD70 target GCTTTGGTCCCATTGGTCGC 67 sequence TRAC target AGAGCAACAGTGCTGTGGCC (TGG) 68 sequence with (PAM) TRAC target AGAGCAACAGTGCTGTGGCC 69 sequence .beta.2M target GCTACTCTCTCTTTCTGGCC (TGG) 70 sequence with (PAM) .beta.2M target GCTACTCTCTCTTTCTGGCC 71 sequence Exemplary sgRNA Formulas sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcu 72 sequence aguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcu 73 sequence aguccguuaucaacuugaaaaaguggcaccgagucggugc sgRNA n(17-30)guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguua 74 sequence ucaacuugaaaaaguggcaccgagucggugcu(1-8) *indicates a nucleotide with a 2'-O-methyl phosphorothioate modification. "n" refers to the spacer sequence at the 5' end.

TABLE-US-00026 SEQUENCE TABLE 24 Edited TRAC Gene Sequence. Sequence (Deletions indicated by dashes SEQ ID Description (-); insertions indicated by bold) NO: TRAC gene edit AA---------------------GAGCAACAAATCTGACT 75 TRAC gene edit AAGAGCAACAGTGCTGT-GCCTGGAGCAACAAATCTGACT 76 TRAC gene edit AAGAGCAACAGTG-------CTGGAGCAACAAATCTGACT 77 TRAC gene edit AAGAGCAACAGT------GCCTGGAGCAACAAATCTGACT 78 TRAC gene edit AAGAGCAACAGTG---------------------CTGACT 79 TRAC gene edit AAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGACT 80 TRAC gene edit AAGAGCAACAGTGC--TGGCCTGGAGCAACAAATCTGACT 81 TRAC gene edit AAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGACT 82

TABLE-US-00027 SEQUENCE TABLE 25 Edited .beta.2M Gene Sequence. Sequence (Deletions indicated by SEQ dashes (-); insertions indicated ID Description by bold) NO: .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTC 83 TTTCT-GCCTGGAGGCTATCCAGCGTGAGTCT CTCCTACCCTCCCGCT .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTC 84 TTTC--GCCTGGAGGCTATCCAGCGTGAGTCT CTCCTACCCTCCCGCT .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTC 85 TTT-----CTGGAGGCTATCCAGCGTGAGTCT CTCCTACCCTCCCGCT .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTC 86 TTTCTGGATAGCCTGGAGGCTATCCAGCGTGA GTCTCTCCTACCCTCCCGCT .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGC----------- 87 --------------GCTATCCAGCGTGAGTCT CTCCTACCCTCCCGCT .beta.2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTC 88 TTTCTGTGGCCTGGAGGCTATCCAGCGTGAGT CTCTCCTACCCTCCCGCT

TABLE-US-00028 SEQUENCE TABLE 26 Edited CD70 Gene Sequence. Sequence (Deletions indicated SEQ by dashes (-); insertions ID Description indicated by bold) NO: CD70 CACACCACGAGGCAGATCACCAAGCCCGCG-- 89 gene-edit CAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGCGAA 90 gene-edit CCAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATC------------AC 91 gene-edit CAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGCG-C 92 gene-edit CAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCAAGCCCGC-AC 93 gene-edit CAATGGGACCAAAGCAGCCCGCAGGACG CD70 CACACCACGAGGCAGATCACCA---------- 94 gene-edit ---------------AGCCCGCAGGACG

TABLE-US-00029 SEQUENCE TABLE 27 Chimeric Antigen Receptor Sequences SEQ ID NO Description Sequence 95 signal peptide MLLLVTSLLLCELPHPAFLLIP 96 signal peptide MALPVTALLLPLALLLHAARP 97 CD8a IYIWAPLAGTCGVLLLSLVITLY transmembrane domain 98 4-1BB nucleotide AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA sequence GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC AGAAGAAGAAGAAGGAGGATGTGAACTG 99 4-1BB amino acid KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL sequence 100 CD28 nucleotide TCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTC sequence GCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACG AGACTTCGCTGCGTACAGGTCC 101 CD28 amino acid SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS sequence 102 CD3-zeta CGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGAC nucleotide sequence AGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGA CGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCC CGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATA AGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACG GGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAA GATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA 103 CD3-zeta amino RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP acid sequence RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR 105 anti-CD19 VL RASQDISKYLN CDR1 (Kabat) 106 anti-CD19 VL HTSRLHS CDR2 (Kabat) 107 anti-CD19 VL QQGNTLPYT CDR3 (Kabat) 108 anti-CD19 VH DYGVS CDR1 (Kabat) 109 anti-CD19 VH VIWGSETTYYNSALKS CDR2 (Kabat) 110 anti-CD19 VH HYYYGGSYAMDY CDR3 (Kabat) 111 anti-CD19 VL RASQDISKYLN CDR1 (Chothia) 112 anti-CD19 VL HTSRLHS CDR2 (Chothia) 113 anti-CD19 VL QQGNTLPYT CDR3 (Chothia) 114 anti-CD19 VH GVSLPDY CDR1 (Chothia) 115 anti-CD19 VH WGSET CDR2 (Chothia) 116 anti-CD19 VH HYYYGGSYAMDY CDR3 (Chothia) 117 Anti-CD19 CAR ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAG FMC63-28Z CGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTT (FMC63-CD8[tm]- GTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAA CD28[co- GACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGG stimulatory TAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTC domain]-CD3z) ACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCA AACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATA CCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTC CACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGC GAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAA GCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGG CGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGG GTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTC GCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAAT GAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACAT TATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTT CTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGC CAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACC ATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCG CCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTA CATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTC GTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGT TGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGAC AAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTAC AGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGC AAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGA GTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGT AAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGA AGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACG ACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCA ACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA 118 Anti-CD19 CAR MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQ FMC63-28Z DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTIS (FMC63-CD8[tm]- NLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKG CD28[co- EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG stimulatory VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH domain]-CD3z) YYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPT Amino Acid IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL with signal peptide VITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 353 Anti-CD19 CAR DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY FMC63-28Z HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF (FMC63-CD8[tm]- GGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTC CD28 [co- TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK stimulatory DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS domain]-CD3z) AAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH Amino Acid TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDY without signal MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQL peptide YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 119 Anti-CD19 scFv GATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAG coding sequence ACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCT CAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTAT CATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTG GGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGA CATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTC GGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGA AGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGA GAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGC ACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGC AGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGA GACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAA GATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTG ACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAG TTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGT 120 Anti-CD19 scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY amino acid HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF sequence GGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTC Linker underlined TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIK DNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 121 CD8a extracellular + GCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTC CD8a CCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCT transmembrane + 5' TAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCAT Linker (underlined) ACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGG CGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTG TAATCACAGGAATCGC 122 CD8a extracellular + TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGC CD8a transmembrane GCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCG (without linker) CCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGC TTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGT GCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAG GAATCGC 123 CD8a extracellular + FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG CD8a transmembrane LDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR 124 Anti-CD19 VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSS 125 Anti-CD19 VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEIT 126 CD19 linker GSTSGSGKPGSGEGSTKG 127 CD70 VL CDR1 RASKSVSTSGYSFMH (Kabat) 128 CD70 VL CDR1 SKSVSTSGYSF (Chothia) 129 CD70 VL CDR2 LASNLES (Kabat) N/A CD70 VL CDR2 LAS (Chothia) 130 CD70 VL CDR3 QHSREVPWT (Kabat) 131 CD70 VL CDR3 SREVPW (Chothia) 132 CD70 VH CDR1 NYGMN (Kabat) 133 CD70 VH CDR1 GYTFTNYGMN (Chothia) 134 CD70 VH CDR2 WINTYTGEPTYADAFKG (Kabat) 135 CD70 VH CDR2 NTYTGE (Chothia) 136 CD70 VH CDR3 DYGDYGMDY (Kabat) 137 CD70 VH CDR3 CARDYGDYGMDYWG (Chothia) 138 CD70 CAR amino MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYT acid sequence FTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSIS (CD70B scFv with TAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGGGGSGGG 41BB) GSGGGGSGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQ With signal peptide QKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQHSREVPWTFGQGTKVEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 354 CD70 CAR amino QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMG acid sequence WINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (CD70B scFv with DYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMTQSPDSLAV 41BB) SLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGV Without signal PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK peptide SAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 139 Anti-CD70A GATATAGTTATGACCCAATCACCCGATAGTCTTGCGGTAAGCCTGGGGG scFv nucleotide AGCGAGCAACAATAAACTGTCGGGCATCAAAATCCGTCAGTACAAGCGG sequence GTATTCATTCATGCACTGGTATCAACAGAAACCCGGTCAGCCACCCAAG CTCCTGATTTATCTTGCGTCTAATCTTGAGTCCGGCGTCCCAGACCGGT TTTCCGGCTCCGGGAGCGGCACGGATTTTACTCTTACTATTTCTAGCCT TCAGGCCGAAGATGTGGCGGTATACTACTGCCAGCATTCAAGGGAAGTT CCTTGGACGTTCGGTCAGGGCACGAAAGTGGAAATTAAAGGCGGGGGGG GATCCGGCGGGGGAGGGTCTGGAGGAGGTGGCAGTGGTCAGGTCCAACT

GGTGCAGTCCGGGGCAGAGGTAAAAAAACCCGGCGCGTCTGTTAAGGTT TCATGCAAGGCCAGTGGATATACTTTCACCAATTACGGAATGAACTGGG TGAGGCAGGCCCCTGGTCAAGGCCTGAAATGGATGGGATGGATAAACAC GTACACCGGTGAACCTACCTATGCCGATGCCTTTAAGGGTCGGGTTACG ATGACGAGAGACACCTCCATATCAACAGCCTACATGGAGCTCAGCAGAT TGAGGAGTGACGATACGGCAGTCTATTACTGTGCAAGAGACTACGGCGA TTATGGCATGGATTACTGGGGCCAGGGCACTACAGTAACCGTTTCCAGC 140 Anti-CD70A DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPK scFv amino acid LLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREV sequence PWTFGQGTKVEIKGGGGSGGGGSGGGGSGQVQLVQSGAEVKKPGASVKV (linker underlined) SCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVT MTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS 141 Anti-CD7OB CAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTT scFv nucleotide CCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGG sequence GATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGG TGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAG GGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGA GCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGG GACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAA CAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGG GGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTT TCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTT CAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACA ACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTG CCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGA TCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAG TAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAA 142 Anti-CD70B QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMG scFv amino acid WINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR sequence DYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMTQSPDSLAV (linker underlined) SLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK 143 Anti-CD70 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMG WINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR DYGDYGMDYWGQGTTVTVSS 144 Anti-CD70 VL DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPK LLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREV PWTFGQGTKVEIK 145 BCMA CAR ATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCC nucleotide sequence ACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAA GAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACC CTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGC TGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAG CCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCC ACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGT ACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCA GGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGC GGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCA CACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAG CCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAG CAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACA GGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGA CTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTAT TACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCA AGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGC CAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACC ATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCG CCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTA CATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTC GTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGA AACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTAC TCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCAT ATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCG CGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATG GGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAAC TCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGG CGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGT ACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTC CCAGA 146 BCMA CAR amino MALPVTALLLPLALLLHAARPQVQLVQSGAELKKPGASVKVSCKASGNT acid sequence LTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRDKSAS With signal peptide TAYMELSSLRSEDTAVYYCTRWDWDGFFDPWGQGTTVTVSSGGGGSGGG GSGGGGSEIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQ QRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVY YCSQTSHIPYTFGGGTKLEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR 355 BCMA CAR amino QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMG acid sequence YILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTR Without signal WDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVS peptide PGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEV PARESGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIK SAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 147 BCMA CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCT scFv nucleotide CCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGT sequence GATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGC TACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGG GCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGA GCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGG TGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGA CCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGG CGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGC CCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGC ACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACA GGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTG CCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCA TCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGAC CAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAA 148 BCMA QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMG scFv amino acid YILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTR sequence WDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVS (linker underlined) PGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEV PARESGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIK 149 BCMA VH QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMG YILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYYCTR WDWDGFFDPWGQGTTVTVSS 150 BCMA VL EIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAP RLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSH IPYTFGGGTKLEIK 151 BCMA VL CDR1 RASQSLVHSNGNTHLH (Kabat & Chothia) 152 BCMA VL CDR2 SVSNRFS (Kabat & Chothia) 153 BCMA VL CDR3 SQTSHIPYT (Kabat) 154 BCMA VL CDR3 SQTSHIPYT (Chothia) 155 BCMA VH CDR1 NYVIH (Kabat) 156 BCMA VH CDR1 GNTLTNY (Chothia) 157 BCMA VH CDR2 YILPYNDLTKYSQKFQG (Kabat) 158 BCMA VH CDR2 LPYNDL (Chothia) 159 BCMA VH CDR3 WDWDGFFDP (Kabat) 160 BCMA VH CDR3 WDWDGFFDP (Chothia) 328 anti-CD33 antibody SYYIH VH CDR1 (Kabat) 329 anti-CD33 antibody VIYPGNDDISYNQKFQG VH CDR2 (Kabat) 330 anti-CD33 antibody EVRLRYFDV VH CDR3 (Kabat) 331 anti-CD33 antibody KSSQSVFFSSSQKNYLA VL CDR1 (Kabat) 332 anti-CD33 antibody WASTRES VL CDR2 (Kabat) 333 anti-CD33 antibody HQYLSSRT VL CDR3 (Kabat) 334 anti-CD33 antibody QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVG VH VIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAR EVRLRYFDVWGQGTTVTVSS 335 anti-CD33 antibody EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQS VL PRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYL SSRTFGQGTKLEIK 336 Anti-CD33 and EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQS anti-CD33b PRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYL scFv SSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVK Linker underlined MSCKASGYTFTSYYTHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKA TLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVS 337 Anti-CD33 and GAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGG anti-CD33b AACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAG scFv TAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCT CCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGG ATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAG CTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTG AGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAG GGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACA GTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAG ATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATT GGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATA CCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCG ACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTT CCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCG CCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCA AGC 338 Anti-CD33 MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQS CAR VFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDF CD28 costim. TLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKGGGGGSGGGGSGGG With signal peptide GSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEW VGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYC AREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR 356 Anti-CD33 EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQS CAR PRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYL CD28 costim. SSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVK Without signal MSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKA peptide TLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVS SSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 339 Anti-CD33b MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQS CAR VFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDF 41BB costim. TLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKGGGGGSGGGGSGGG With signal peptide GSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEW VGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYC AREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS LVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR 357 Anti-CD33b EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQS CAR PRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYL 41BB costim. SSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASVK Without signal MSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKA peptide TLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVS SSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 340 Anti-PTK7 SYGMH VH CDR1 341 Anti-PTK7 VIWDDGSNKYYVDSVKG VH CDR2 342 Anti-PTK7 DDYYGSGSFNSYYGTDV VH CDR3 343 Anti-PTK7 RASQSVSIYLA VL CDR1 344 Anti-PTK7 DASNRAT VL CDR2 345 Anti-PTK7 QQRSNWPPFT VL CDR3 346 Anti-PTK7 V.sub.H QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA VIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DDYYGSGSFNSYYGTDVWGQGTTVTVSS 347 Anti-PTK7 V.sub.L EIVLTQSPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFT FGPGTKVDIK 348 Anti-PTK7 scFv QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA (linker underlined) VIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQ SPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRA TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTK VDIK 349 Anti-PTK7 CAR MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFT CD28 co-stim FSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKN With signal peptide TLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSS GGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLA WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDF AVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR 358 Anti-PTK7 CAR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA CD28 co-stim VIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Without signal DDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQ peptide SPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRA TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTK VDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 350 Anti-PTK7 CAR MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFT 41BB co-stim FSSYGMHWVRQAPGKGLEWVAVIWDDGSNKYYVDSVKGRFTISRDNSKN With signal peptide TLYLQMNSLRAEDTAVYYCARDDYYGSGSFNSYYGTDVWGQGTTVTVSS GGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSIYLA WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDF AVYYCQQRSNWPPFTFGPGTKVDIKSAAAFVPVFLPAKPTTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR 359 Anti-PTK7 CAR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVA 41BB co-stim VIWDDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR Without signal DDYYGSGSFNSYYGTDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQ peptide SPATLSLSPGERATLSCRASQSVSIYLAWYQQKPGQAPRLLIYDASNRA TGIPARESGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTK VDIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

TABLE-US-00030 TABLE 28 AAV Donor Template Sequences 161 Left ITR (5' ITR) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAC CAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA GCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 162 Left ITR (5' ITR) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTC (alternate) GGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCT 163 Right ITR (3' ITR) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC GCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 164 Right ITR (3' ITR) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTC (alternate) GCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCC CGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 165 TRAC-LHA GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA (800 bp) CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAA GTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGG CCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGT TTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAG CCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAAC GTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTC CCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCC TTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGG AAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATG AGGTCTATGGACTTCA 166 TRAC-RHA TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCA (800 bp) TTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGG TGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCA GAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCC TTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTT GTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAG GTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTC CTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAG GCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTG TCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCAC TCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGT GTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAA GCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAA TAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTAC CTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAA GATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGA CAGGAGCTCAATGAGAAAGG 167 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 168 CD19 GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA LHA to RHA 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 169 CD70 GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA LHA to RHA CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC (CD70B scFV with CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC 41BB) 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 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 170 BCMA GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA RHA to LHA 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 351 Anti-CD33 CAR GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA Donor CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC LHA to RHA CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC CD28 costim. 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 CCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTC ACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTA TGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAA CTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTT ATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGG GATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCC TGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACT TTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTG GTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCC AGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAA GCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAA CTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGA TGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCC GATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCG AAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTT TGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCT GCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCC CGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCT TCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGG GGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTA CGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCA CAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAAT ATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATG CCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCG AAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAAC GAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCC GGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCA AGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTAC TCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATG GCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACT GCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCG AAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTT TGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTC TTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCC TTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTC TAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAAC CCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAAT GACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTG GCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTC AGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTC TCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCC AGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACT GATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAA AAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGA GCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTG TTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGG AAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGG CAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG 352 Anti-CD33b CAR GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAA Donor CGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAAC LHA to RHA CTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAAC 41BB costim. 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 CCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTC ACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTA TGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAA CTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTT ATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGG GATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCC TGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACT TTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTG GTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCC AGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAA GCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAA CTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGA TGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCC GATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCG AAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTT

TGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCT GCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCC CGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCT TCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGG GGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTA CGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCA CAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCT GCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTT TTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTG TATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATA AACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAA TCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAG GCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTC ACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGA TGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTAT CCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATC TGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGAC ACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCT GTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAAT GATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCAC CAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCA GAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGG CACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCT TTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGC CCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATC TTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCA ATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGA ATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTT GGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGG AATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAG TCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTAC CAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGA GAAAGG

TABLE-US-00031 TABLE 29 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z01 gRNA. Reference on-target sequence.sup.a: GATGGGAGCAACG(TGG)CCAT (SEQ ID NO: 104) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 181 GATGGGAGCAAACGTGGCCAT 46.1 43.9 45.0 1.6 ------------GTGGCCAT 6.5 4.3 5.4 1.6 182 GATGGGAGC-ACGTGGCCAT 4.1 4.9 4.5 0.6 GA-----------TGGCCAT 3.5 3.9 3.7 0.3 -------------------- 3.3 3.7 3.5 0.3 183 GATGGG---AACGTGGCCAT 2.6 3.6 3.1 0.7 184 GATGGGA--------GCCAT 3.6 2.1 2.8 1.1 -----------------CAT 2.4 1.8 2.1 0.4 -----------CGTGGCCAT 1.4 1.2 1.3 0.1 185 GATG----------GGCCAT 1.1 1.3 1.2 0.1 GAT----------------- 0.9 1.1 1.0 0.1 GATGG--------------- 0.7 1.2 1.0 0.4 186 ----------ACGTGGCCAT 1.1 0.5 0.8 0.4 .sup.aOn-target the sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00032 TABLE 30 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z02 gRNA. Reference on-target sequence.sup.a: CCGCGGGACTAGA(GGG)AGCT (SEQ ID NO: 268) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 187 CCGCGGGACTTAGAGGGAGCT 49.2 39.4 44.3 6.9 188 ---------CCGCGGGAGCT 11.9 11.5 11.7 0.3 -------------------- 2.6 4.6 3.6 1.4 CCGCGGG------------- 2.1 3.4 2.8 0.9 -------------------T 2.1 2.0 2.0 0.1 189 CCGCGGGA-TAGAGGGAGCT 1.7 1.8 1.8 0.1 190 CCGCGGGACT---------- 1.8 1.3 1.6 0.4 191 CCGCGGG--TAGAGGGAGCT 1.0 1.6 1.3 0.4 192 CCGCGGG--------GAGCT 1.1 1.3 1.2 0.1 193 CCGCGGGAC-AGAGGGAGCT 1.0 1.2 1.1 0.1 194 CCGCGGGACT-GAGGGAGCT 1.3 0.9 1.1 0.3 195 CCG---------AGGGAGCT 1.2 0.9 1.0 0.2 196 CCGCGGGA-----GGGAGCT 0.8 1.1 1.0 0.2 197 CCG------TAGAGGGAGCT 0.3 1.1 0.7 0.6 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00033 TABLE 31 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z03 gRNA. Reference on-target sequence.sup.a: CGCTCCCGCTCGG(TGG)CTGT(SEQ ID NO: 274) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 198 CGCTCCCGCTTCGGTGGCTGT 41.3 38.6 40.0 1.9 C----------------TGT 7.9 7.8 7.8 0.1 CGCTCCCG------------ 7.9 7.5 7.7 0.3 199 CGCTCCCGC-CGGTGGCTGT 3.3 3.7 3.5 0.3 -------------------- 2.7 3.7 3.2 0.7 200 CGCTCCCG-TCGGTGGCTGT 2.8 3.7 3.2 0.6 201 CGCTCCCGC--GGTGGCTGT 2.3 2.8 2.6 0.4 -------------------T 1.7 3.0 2.4 0.9 202 CGCTCCCGCT-GGTGGCTGT 2.2 2.4 2.3 0.1 ---------------GCTGT 2.3 1.7 2.0 0.4 203 CGCTCCC--TCGGTGGCTGT 1.6 1.8 1.7 0.1 204 CGCTCCCGCTTTCGGTGGCTGT 1.1 1.4 1.2 0.2 205 CGCTCCCG----GTGGCTGT 1.3 0.8 1.0 0.4 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00034 TABLE 32 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell donor for the REG1-Z04 gRNA. Reference on-target sequence.sup.a: CATCACGACGCGT(GGG)TGGC (SEQ ID NO: 280) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 206 CATCACGA--CGTGGGTGGC 34.0 32.9 33.4 0.8 207 CATCA-----CGTGGGTGGC 7.7 6.2 7.0 1.1 -------------------- 2.9 3.8 3.4 0.6 208 CATCACGACGCCGTGGGTGGC 2.5 4.2 3.4 1.2 209 CATCACGAC------GTGGC 3.1 3.6 3.4 0.4 210 CATCACGACGGCGTGGGTGGC 2.3 3.4 2.8 0.8 CATCACGA------------ 2.3 2.4 2.3 0.1 211 ----------CGTGGGTGGC 1.5 1.7 1.6 0.1 212 CATCACGACG---TGGTGGC 1.8 1.2 1.5 0.4 213 CATCACGACGTCGTGGGTGGC 1.5 1.2 1.4 0.2 CATCACGAC----------- 1.7 1.1 1.4 0.4 -------------------C 1.5 1.2 1.4 0.2 --------------GGTGGC 1.1 1.3 1.2 0.1 ----------------TGGC 1.1 1.0 1.0 0.1 214 CATCACGAC----GGGTGGC 0.7 1.3 1.0 0.4 CATCA--------------- 0.9 1.1 1.0 0.1 215 CATCACGAC-----GGTGGC 1.1 0.7 0.9 0.3 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00035 TABLE 33 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z05 gRNA. Reference on-target sequence.sup.a: CACGACGCGTGGG(TGG)CAAG (SEQ ID NO: 286) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 216 CACGACGCGTTGGGTGGCAAG 58.4 50.0 54.2 5.9 CACGAC-------------G 5.5 7.8 6.6 1.6 217 CACGACGC--GGGTGGCAAG 1.7 3.7 2.7 1.4 218 CACGAC---------GCAAG 2.2 2.8 2.5 0.4 219 CACGACGC----GTGGCAAG 2.4 1.5 2.0 0.6 220 CACGACGCG-GGGTGGCAAG 1.6 1.9 1.8 0.2 -------------------- 1.4 1.5 1.4 0.1 CACGA--------------- 1.0 1.4 1.2 0.3 CACGACGC------------ 0.9 1.3 1.1 0.3 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00036 TABLE 34 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z06 gRNA. Reference on-target sequence .sup.a: TCTGACGGGATCG (TGG) TTTC (SEQ ID NO: 292) SEQ Donor Donor Std. ID Gene Edited 1 2 Mean Dev. NO: Sequence .sup.b (%) (%) (%) (%) 221 TCTGACGGGAATCGTGGTTTC 28.1 21.9 25.0 4.4 222 TCTGACG-------GGTTTC 7.0 7.4 7.2 0.3 223 TCTGA------CGTGGTTTC 7.3 7.2 7.2 0.1 224 TCTGACGGGATTCGTGGTTTC 5.4 2.6 4.0 2.0 225 TCTGACGGGA-CGTGGTTTC 4.2 2.8 3.5 1.0 226 TCTG------TCGTGGTTTC 3.5 3.1 3.3 0.3 TCTG---------------- 2.3 3.4 2.8 0.8 -------------------- 2.4 3.1 2.8 0.5 ------------------TC 2.9 2.2 2.6 0.5 227 TCTGAC--------GGTTTC 2.0 2.0 2.0 0.0 TCT----------------- 1.5 2.3 1.9 0.6 228 TCTGACGGG-TCGTGGTTTC 1.7 2.1 1.9 0.3 229 TCTGACGGGAGTCGTGGTTTC 2.4 1.3 1.8 0.8 230 TCTGACGGGACTCGTGGTTTC 1.5 1.8 1.6 0.2 231 ----------TCGTGGTTTC 1.3 1.6 1.5 0.2 -------------------C 1.0 1.5 1.2 0.4 232 TCTGACGG--TCGTGGTTTC 0.6 1.4 1.0 0.6 233 TCTGACGGGA--GTGGTTTC 1.2 0.5 0.8 0.5 .sup.a On-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.b Deletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00037 TABLE 35 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z07 gRNA. Reference on-target sequence .sup.a: CCACGCGTCGTGA (TGG) TGTG (SEQ ID NO: 298) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence .sup.b (%) (%) (%) (%) 234 CCACGCGTCGGTGATGGTGTG 15.1 12.9 14.0 1.6 235 CCACGCGTCGTTGATGGTGTG 12.3 8.5 10.4 2.7 -------------------- 4.4 5.1 4.8 0.5 236 CCACGCGT---------GTG 4.9 4.4 4.6 0.4 CCACGCGT-----------G 3.6 3.0 3.3 0.4 237 CCACGCGTCGATGATGGTGTG 2.9 1.4 2.2 1.1 CCACGCGTC----------- 1.9 2.5 2.2 0.4 238 CCACGCGTCG--ATGGTGTG 2.2 2.1 2.2 0.1 239 CCACGCGTC-TGATGGTGTG 2.0 2.2 2.1 0.1 CCAC---------------- 1.9 2.2 2.0 0.2 C------------------- 2.2 1.9 2.0 0.2 240 CCACGCGTCGCTGATGGTGTG 1.9 1.6 1.8 0.2 241 CCACGCGTCG---------- 2.0 1.7 1.8 0.2 242 CCACGCGTCG-----GTGTG 1.7 1.7 1.7 0.0 243 CCACGCGTGG-----GTGTG 1.8 1.5 1.6 0.2 244 CCACGCGT---GATGGTGTG 1.4 1.3 1.4 0.1 CCA----------------- 1.1 1.7 1.4 0.4 245 CCACGCGTCGTG------TG 1.4 1.1 1.2 0.2 246 CCACGCGTCGTGA------- 1.2 1.1 1.2 0.1 CCACGC-------------- 0.8 1.5 1.2 0.5 CCACGCG------------- 1.1 0.9 1.0 0.1 CCACG-----------TGTG 0.8 1.2 1.0 0.3 247 CCACGCGTGG-------GTG 1.1 0.7 0.9 0.3 .sup.a On-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.b Deletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00038 TABLE 36 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z08 gRNA. Reference on-target sequence .sup.a: CCATCACGACGCG (TGG) GTGG (SEQ ID NO: 304) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence .sup.b (%) (%) (%) (%) 248 CCATCACGACCGCGTGGGTGG 28 15.4 21.7 8.9 249 CCATCA-----CGTGGGTGG 8.5 3.4 6 3.6 250 CCATC---ACGCGTGGGTGG 4.4 2.4 3.4 1.4 -------------------- 2.3 1.8 2 0.4 ---------------GGTGG 1.5 0.7 1.1 0.6 251 CCATCACGACAGCGTGGGTGG 1.3 0.2 0.8 0.8 .sup.a On-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.b Deletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00039 TABLE 37 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z09 gRNA. Reference on-target sequence .sup.a: CCGTCAGACTCGT (AGG) ccag (SEQ ID NO: 310) SEQ Donor Donor Std. ID Gene Edited 1 2 Mean Dev. NO: Sequence .sup.b (%) (%) (%) (%} CCGTCAG------------- 13.5 9.9 11.7 2.5 252 CCGTCAGACTTCGTAGGCCAG 11.3 8.5 9.9 2.0 253 CCGT---------AGGCCAG 7.5 8.3 7.9 0.6 254 CCGTCAGACT---------- 6.9 6.1 6.5 0.6 255 CCGTCAGAC--------CAG 4.2 4.3 4.2 0.1 -------------------- 3.9 4.2 4.0 0.2 CCGTCA-------------- 3.6 2.3 3.0 0.9 256 CCGTCAGAC--GTAGGCCAG 2.5 2.4 2.4 0.1 257 CCGTCAG--------GCCAG 1.9 2.4 2.2 0.4 CCG-------------CCAG 1.2 2.2 1.7 0.7 258 CCGTCAGAC-CGTAGGCCAG 1.7 1.4 1.5 0.2 ------------TAGGCCAG 1.0 1.4 1.2 0.3 259 CCGTCAGACT-GTAGGCCAG 1.5 1.0 1.2 0.4 CCGTCAGA------------ 1.6 0.7 1.2 0.6 CCGTCAGAC----------- 1.2 0.6 0.9 0.4 .sup.a On-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.b Deletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00040 TABLE 38 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the REG1-Z10 gRNA. Reference on-target sequence .sup.a: GTGGGTGGCAAGC (GGG) TGGT (SEQ ID NO: 316) SEQ Donor Donor Std. ID Gene Edited 1 2 Mean Dev. NO: Sequence .sup.b (%) (%) (%) (%) 260 GTGGGTGGCAAAGCGGGTGGT 23.8 21.7 22.8 1.5 GT-----------GGGTGGT 20.7 22.9 21.8 1.6 -----------GCGGGTGGT 10.4 7.7 9.0 1.9 261 GTGGGTGGC-AGCGGGTGGT 7.0 6.5 6.8 0.4 ---------------GTGGT 3.3 4.3 3.8 0.7 GTG--------------GGT 2.8 4.0 3.4 0.8 ------------CGGGTGGT 2.6 3.3 3.0 0.5 2.0 3.5 2.8 1.1 GTGGGTGGC----------- 2.4 1.8 2.1 0.4 262 GTGGGTGGCATAGCGGGTGGT 1.8 1.8 1.8 0.0 GTGGGTG------------- 1.6 1.5 1.6 0.1 GTGG---------------- 1.5 1.8 1.6 0.2 263 GTGGGTGG--AGCGGGTGGT 0.9 1.1 1.0 0.1 .sup.a On-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-t&rgct sequence is underlined and the PAM is indicated by parenthesis. .sup.b Deletions indicated by dashes (-); insertions indicated by bold

TABLE-US-00041 TABLE 39 TGFBRII gRNA Sequences/Target Sequences Unmodified Modified Target Sequence Name Sequence Sequence (PAM) TGFBRII CCGACUUCUGAACGUGCG C*C*G*ACUUCUGAACGUG CCGACTTCTGAACGTGCGGT sgRNA GUguuuuagagcuagaaa CGGUGGGguuuuagagcua (GGG) (SEQ ID NO: 2) (EX1_T1) uagcaaguuaaaauaagg gaaauagcaaguuaaaaua CCGACTTCTGAACGTGCGGT cuaguccguuaucaacuu aggcuaguccguuaucaac (SEQ ID NO: 269) gaaaaaguggcaccgagu uugaaaaaguggcaccgag cggugcmUUUU ucggugcU*U*U*U (SEQ ID NO: 264) (SEQ ID NO: 265) TGFBRII CCGACUUCUGAACGUGCG C *C *C *GACUUCUGAACGU sgRNA GU GCGGU (SEQ ID NO: (EX1_T1) (SEQ ID NO: 266) 267) spacer TGFBRII UGCUGGCGAUACGCGUCC U*G*C*UGGCGAUACGCGU TGCTGGCGATACGCGTCCAC sgRNA ACguuuuagagcuagaaa CCACguuuuagagcuagaa (AGG) (EX1_T2) uagcaaguuaaaauaagg auagcaaguuaaaauaagg (SEQ ID NO: 3) cuaguccguuaucaacuu cuaguccguuaucaacuug TGCTGGCGATACGCGTCCAC gaaaaaguggcaccgagu aaaaaguggcaccgagucg (SEQ ID NO: 275) cggugcmUUUU gugcmU*U*U*U (SEQ ID NO: 270) (SEQ ID NO: 271) TGFBRII UGCUGGCGAUACGCGUCC U*G*C*UGGCGAUACGCGU sgRNA AC CCAC (EX1.T2) (SEQ ID NO: 272) (SEQ ID NO: 273) spacer TGFBRII UCGGUCUAUGACGAGCAG U*C*G*GUCUAUGACGAGC TCGGTCTATGACGAGCAGCG sgRNA CGguuuuagagcuagaaa AGCGguuuuagagcuagaa GGG) (SEQ ID NO: 4) (EX1_T3) uagcaaguuaaaauaagg auagcaaguuaaaauaagg TCGGTCTATGACGAGCAGCG cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 281) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcUUUU gugcU*U*U*U (SEQ ID NO: 276) (SEQ ID NO: 277) TGFBRII UCGGUCUAUGACGAGCAG U *C*G*GUCUAUGACGAGC sgRNA CG (SEQ ID NO: AGCG (SEQ ID NO: (EX1_T3) 278) 279) spacer TGFBRII AUGGGCAGUCCUAUUACA A*U*G*GGCAGUCCUAUUA ATGGGCAGTCCTATTACAGC sgRNA GCguuuuagagcuagaaa CAGCguuuuagagcuagaa (TGG) (SEQ ID NO: 5) (EX2_T1) uagcaaguuaaaauaagg auagcaaguuaaaauaagg ATGGGCAGTCCTATTACAGC cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 287) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcmUUUU gugcU*U*U*U (SEQ ID NO: 282) (SEQ ID NO: 283) TGFBRII AUGGGCAGUCCUAUUACA A*U*G*GGCAGUCCUAUUA sgRNA GC (SEQ ID NO: CAGC (SEQ ID NO: (EX2.T1) 284) 285) spacer TGFBRII AUUGUUCACUUGUUAGCC A*U*U*GUUCACUUGUUAG ATTGTTCACTTGTTAGCCCC sgRNA CCguuuuagagcuagaaa CCCCAGGguuuuagagcua (AGG) (SEQ ID NO: 6) (EX3.T1) uagcaaguuaaaauaagg gaaauagcaaguuaaaaua ATTGTTCACTTGTTAGCCCC cuaguccguuaucaacuu aggcuaguccguuaucaac (SEQ ID NO: 293) gaaaaaguggcaccgagu uugaaaaaguggcaccgag cggugcUUUU ucggugcU*U*U*U (SEQ ID NO: 288) (SEQ ID NO: 289) TGFBRII AUUGUUCACUUGUUAGCC A*U*U*GUUCACUUGUUAG sgRNA CC (SEQ ID NO: CCCC (SEQ ID NO: (EX3.T1) 290) 291) spacer TGFBRII GCUGAAGAACUGCCUCUA G*C*U*GAAGAACUGCCUC GCTGAAGAACTGCCTCTATA sgRNA UAguuuuagagcuagaaa UAUAguuuuagagcuagaa (TGG) (SEQ ID NO: 7) (EX3_T2) uagcaaguuaaaauaagg auagcaaguuaaaauaagg GCTGAAGAACTGCCTCTATA cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 299) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcUUUU gugcU*U*U*U (SEQ ID NO: 294) (SEQ ID NO: 295) TGFBRII GCUGAAGAACUGCCUCUA g*c*u*gaagaacugccuc sgRNA UA (SEQ ID NO: UAUA (SEQ ID NO: (EX3_T2) 296) 297) spacer TGFBRII GCAGGAUUUCUGGUUGUC G*C*A*GGAUUUCUGGUUG GCAGGATTTCTGGTTGTCAC sgRNA ACguuuuagagcuagaaa UCACguuuuagagcuagaa (AGG) (SEQ ID NO: 8) (EX4_T1) uagcaaguuaaaauaagg auagcaaguuaaaauaagg GCAGGATTTCTGGTTGTCAC cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 305) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcUUUU gugcU*U*U*U (SEQ ID NO: 300) (SEQ ID NO: 301) TGFBRII GCAGGAUUUCUGGUUGUC G*C*A*GGAUUUCUGGUUG sgRNA AC (SEQ ID NO: UCAC (SEQ ID NO: (EX4_T1) 302) 303) spacer TGFBRII CUCCAUCUGUGAGAAGCC c*U*C*CAUCUGUGAGAAG CTCCATCTGTGAGAAGCCAC sgRNA ACguuuuagagcuagaaa CCACguuuuagagcuagaa (AGG) (SEQ ID NO: 9) (EX4.T2) uagcaaguuaaaauaagg auagcaaguuaaaauaagg CTCCATCTGTGAGAAGCCAC cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 311) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcUUUU gugcU*U*U*U (SEQ ID NO: 306) (SEQ ID NO: 307) TGFBRII CUCCAUCUGUGAGAAGCC C*U*C*CAUCUGUGAGAAG sgRNA AC (SEQ ID NO: CCAC (SEQ ID NO: (EX4_T2) 308) 309) spacer TGFBRII CCCCUACCAUGACUUUAU C*C*C*CUAC CAUGACUUU CCCCTACCATGACTTTATTC sgRNA UCguuuuagagcuagaaa AUUCguuuuagagcuagaa (TGG) (SEQ ID NO: 10) (EX5.T1) uagcaaguuaaaauaagg auagcaaguuaaaauaagg CCCCTACCATGACTTTATTC cuaguccguuaucaacuu cuaguccguuaucaacuug (SEQ ID NO: 317) gaaaaaguggcaccgagu aaaaaguggcaccgagucg cggugcUUUU gugcU*U*U*U (SEQ ID NO: 312) (SEQ ID NO: 313) TGFBRII CCCCUACCAUGACUUUAU C*C*C*CUACCAUGACUUU sgRNA UCUGG (SEQ ID NO: AUUC (SEQ ID NO: (EX5.T1) 314) 315) spacer *: 2'-O-methyl phosphorothioate residue

TABLE-US-00042 TABLE 40 On-Target Gene Edited Sequences > 1% Frequency in At Least One Gene Edited T Cell Donor fur the TGFBRII-Ex1-T1 gRNA. Reference on-target sequence.sup.a: CTGAACGTGCGGT (GGG) ATCG (SEQ ID NO: 360) SEQ Donor Donor Std. ID Gene Edited 1 2 Mean Dev. NO: Sequence.sup.b (%) (%) (%) (%) 361 CTGAACGTGC---------- 28.7 29.8 29.2 0.8 362 CTGAACGTG-----GGATCG 10.7 12 11.4 0.9 CTGA-------------TCG 9.8 9.3 9.6 0.4 3.7 1.3 2.5 1.7 363 CTGAACGTGCCGGTGGGATCG 1.2 3.2 2.2 1.4 CTG----------------- 2.8 1.1 2 1.2 364 CTGAACGTG-GGTGGGATCG 0.8 2.1 1.5 0.9 365 ----------GGTGGGATCG 2.2 0.8 1.5 1 366 CTGAACGTG--GTGGGATCG 1 1.6 1.3 0.4 367 CTGAACG----GTGGGATCG 1.5 0.8 1.2 0.5 CTGAACG------------- 1.3 1 1.2 0.2 368 CTG--------GTGGGATCG 1.3 0.4 0.8 0.6 369 CTGAACGTGCAGGTGGGATCG 1.3 0.3 0.8 0.7 370 CTGAACGTGCGT--GGATCG 0 1.1 0.6 0.8 -----------------TCG 0 1.1 0.6 0.8 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00043 TABLE 41 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex1-T2 gRNA. Reference on-target sequence.sup.a: GATACGCGTCCAC (AGG) ACGA (SEQ ID NO: 371) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 372 GATACGCGTC-ACAGGACGA 15.2 15.3 15.2 0.1 GAT----------------- 8.5 10.3 9.4 1.3 GATACGC------------- 6.7 5.9 6.3 0.6 373 GATACGCGTCCCACAGGACGA 3.7 6.1 4.9 1.7 GATACG-------------A 4.3 5.6 4.9 0.9 -------------------- 5.4 3.5 4.4 1.3 ----------------ACGA 3.4 3.9 3.6 0.4 -------------AGGACGA 3.7 2.2 3 1.1 374 GATACGCGTCCA--GGACGA 2.2 3.2 2.7 0.7 375 GATACGC----ACAGGACGA 2.3 2.8 2.6 0.4 376 GATAC------ACAGGACGA 2.8 1.7 2.2 0.8 -----------ACAGGACGA 1.4 2.5 2 0.8 GATACGCG-----------A 2.5 1.4 2 0.8 377 GATACGCGTCC-------GA 1.9 1.7 1.8 0.1 378 GATACGCGTC--------GA 1.1 2 1.6 0.6 379 GATACGCGTC---AGGACGA 1.9 1.1 1.5 0.6 380 GATAC--------AGGACGA 1.2 1.5 1.4 0.2 381 GATACGC---CACAGGACGA 1.5 0.8 1.2 0.5 382 GATACGCGTC---------- 1 1.3 1.2 0.2 383 GATACGCGTCACACAGGACGA 1.4 0.8 1.1 0.4 384 GATACGC-TGCACAGGACGA 1.1 0.8 1 0.2 385 GATACGC------AGGACGA 0.8 1.3 1 0.4 GATACGCG------------ 0.6 1.1 0.8 0.4 GATACGCGT----------- 0.6 1.1 0.8 0.4 -------------------A 1.1 0.3 0.7 0.6 386 ---ACGC----ACAGGACGA 1.2 0 0.6 0.8 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00044 TABLE 42 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex1-T3 gRNA. Reference on-target sequence.sup.a: ATGACGAGCAGCG (GGG) TCTG (SEQ ID NO: 387) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 388 ATGACGAGCAAGCGGGGTCTG 66.7 65.9 66.3 0.6 389 ATGACG---AGCGGGGTCTG 4.5 5.8 5.2 0.9 --------------GGTCTG 2.2 2.5 2.4 0.2 390 ATGACGA--AGCGGGGTCTG 1.9 1.9 1.9 0 -------------------- 2.1 1.4 1.8 0.5 ------------GGGGTCTG 1 1.7 1.4 0.5 391 ATG------AGCGGGGTCTG 1.6 1.1 1.4 0.4 392 ATGACGAGCAAAGCGGGGT 1.8 0.6 1.2 0.8 CTG 393 ATGA-------CGGGGTCTG 0.7 1.5 1.1 0.6 A-----------------TG 1.2 0.5 0.8 0.5 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00045 TABLE 43 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex5-T1 gRNA. Reference on-target sequence.sup.a: (SEQ ID NO: 394) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) 395 CATGA-------CTGGAAGA 10.6 12.4 11.5 1.3 396 CATGAC----TTCTGGAAGA 8.8 8.9 8.9 0.1 397 CATGACT---TTCTGGAAGA 7 5.4 6.2 1.1 398 CATGACTTTATTTCTGGAAGA 5 6.2 5.6 0.8 399 CATGACTTTAATTCTGGAAGA 5.1 6.2 5.6 0.8 CA-----------TGGAAGA 3.7 3.8 3.8 0.1 400 CATGACTT--TTCTGGAAGA 3.6 3 3.3 0.4 CAT------------GAAGA 2.2 3.2 2.7 0.7 C------------------A 2.5 2.1 2.3 0.3 2.5 1.9 2.2 0.4 CATGA--------------- 2.6 1.8 2.2 0.6 CAT---------------GA 2 2 2 0 401 CA---------TCTGGAAGA 2 2.1 2 0.1 402 CATGACTTT-TTCTGGAAGA 1.6 2.3 2 0.5 403 CATGACTTTA-TCTGGAAGA 2.1 1.4 1.8 0.5 404 CATGACTTT-------AAGA 1.1 1 1 0.1 405 ----------TTCTGGAAGA 1.2 0.9 1 0.2 406 CATGACTTTA--CTGGAAGA 1.1 0.9 1 0.1 .sup.aOn-target sequence centered on cleavage site, with 10 bp in cither direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00046 TABLE 44 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex2-T1 gRNA. Reference on-target sequence.sup.a: GTCCTATTACAGC(TGG) GGCA (SEQ ID NO: 407) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence.sup.b (%) (%) (%) (%) G------------------ 18.4 17.4 17.9 0.7 408 GTCCTATTA-GCTGGGGCA 6.4 13 9.7 4.7 ----------------GCA 9.2 5.7 7.4 2.5 409 GTCCTATTA-AGCTGGGGCA 7.5 7.1 7.3 0.3 410 GTCCTAT---AGCTGGGGCA 6.8 7.5 7.2 0.5 411 GTCCTA----AGCTGGGGCA 7.3 4.6 5.9 1.9 412 GTCCTA-----GCTGGGGCA 7.5 4.2 5.8 2.3 -------------------- 2.8 2.2 2.5 0.4 413 GTCCTATTAC---TGGGGCA 2 1.7 1.8 0.2 G-----------CTGGGGCA 1 2 1.5 0.7 414 GTCC------AGCTGGGGCA 1 1.7 1.4 0.5 415 GTCCTATTACCAGCTGGGGCA 1.2 1.3 1.2 0.1 GTCCTAT------------- 1.4 0.8 1.1 0.4 416 GTCCTATT---GCTGGGGCA 1.1 1.1 1.1 0 417 GTCCTATTAC-GCTGGGGCA 0.7 1.2 1 0.4 418 GTCCT---------GGGGCA 1.6 0.3 1 0.9 GT------------------ 1.1 0.1 0.6 0.7 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00047 TABLE 45 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex3-T1 gRNA. Reference on-target sequence.sup.a: ACTTGTTAGCCCC (AGG) GCCA (SEQ ID NO: 419) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence (%) (%) (%) (%) 420 ACTTGTTAG--CCAGGGCCA 26.7 22.6 24.6 2.9 421 ACTTGTTAG-CCCAGGGCCA 5.1 9.1 7.1 2.8 -------------------- 6 4.1 5 1.3 422 ACTTGTTAG---CAGGGCCA 4.9 3.7 4.3 0.8 423 ACTTGTTA--------GCCA 4.6 3.1 3.8 1.1 ------------CAGGGCCA 4.1 2.7 3.4 1 424 ACTTGTT------AGGGCCA 2.1 3.3 2.7 0.8 ------------------CA 3.6 1.6 2.6 1.4 425 ACTTGTTAGCCCCCAGGGCCA 2 3.3 2.6 0.9 426 ACTTGTT---CCCAGGGCCA 1.3 3 2.2 1.2 427 ----------CCCAGGGCCA 2.3 1.7 2 0.4 428 ACTTGTTA--CCCAGGGCCA 2 1.9 0.1 429 ACTTG-----CCCAGGGCCA 2 1.8 0.2 ACT----------------- 1.3 1.3 0 430 ACTTGT----CCCAGGGCCA 0.8 1.5 1.2 0.5 431 A----------CCAGGGCCA 1.6 0.7 1.2 0.6 ---------------GGCCA 1.1 1.1 1.1 0 A-----------CAGGGCCA 0.5 1.1 0.8 0.4 432 ACTTG-------CAGGGCCA 0.2 1.2 0.7 0.7 433 ACTTGTTAGC-------CCA 0.3 1.1 0.7 0.6 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00048 TABLE 46 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex3-T2 gRNA. Reference on-target sequence.sup.a: AACTGCCTCTATA (TGG) TGTG (SEQ ID NO: 434) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence (%) (%) (%) (%) 435 AACTGCCTCTTATATGGTGTG 37.1 41.7 39.4 3.3 AAC----------------- 7 6 6.5 0.7 -------------------- 7.2 5 6.1 1.6 436 AACTGCCT--ATATGGTGTG 2.9 4.1 3.5 0.8 437 AACTGCCTCTAT--GGTGTG 3 3 3 0 AACTG--------------- 2.7 2.3 2.5 0.3 438 AACTGCCTC-ATATGGTGTG 2 2.4 2.2 0.3 439 AACTG----TATATGGTGTG 1.6 2.4 2 0.6 440 AACTGC---TATATGGTGTG 1.6 1.8 1.7 0.1 441 AACT------ATATGGTGTG 1.1 1.8 1.5 0.5 AACTGCC------------- 1.2 1.5 1.4 0.2 A------------------- 1.8 0.9 1.4 0.6 442 AACTGCCT-TATATGGTGTG 1.1 1.3 1.2 0.1 443 AACTGCCTCT---------- 1.5 1 1.2 0.4 444 ---------TATATGGTGTG 1.1 0.9 1 0.1 AACTG-------------TG 0.8 1.1 1 0.2 AACTGCCTC----------- 0.6 1.4 1 0.6 AACT---------------- 1.1 1 1 0.1 445 AACTGCCTCTA--------- 1.1 0.7 0.9 0.3 446 AACTGCCTCT-TATGGTGTG 0.7 1.1 0.9 0.3 447 AACTG----------GTGTG 1.1 0.7 0.9 0.3 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with the Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in the Reference Sequence

TABLE-US-00049 TABLE 47 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex4-T1 gRNA. Reference On-target sequence.sup.a: TTCTGGTTGTCAC (AGG) TGGA (SEQ ID NO: 448) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence (%) (%) (%) (%) 449 TTCTGGTTGTTCACAGGTGGA 31.3 33.1 32.2 1.3 450 TTCTGGT----------GGA 11.2 11.5 11.4 0.2 451 TTC----------AGGTGGA 5.2 4 4.6 0.8 ----------------TGGA 4.2 3.7 4 0.4 452 TTCTGGTT--CACAGGTGGA 3.5 3.5 3.5 0 453 TTCTGGTTGTTTCACAGGTG 2.1 2.7 2.4 0.4 GA 454 TTCTGGTTG---------GA 2.3 2.2 2.2 0.1 TTCTGG-------------A 1.9 1.6 1.8 0.2 455 TTCTGGTTGTCCACAGGTGGA 1.6 1.9 1.8 0.2 456 TTC-------CACAGGTGGA 1.4 2.1 1.8 0.5 457 TTCTGGTT-TCACAGGTGGA 1.4 2 1.7 0.4 -------------------- 2 1.1 1.6 0.6 458 TTCTGGTTG-CACAGGTGGA 1.1 1.4 1.2 0.2 459 TTCTGGTTGTACACAGGTGGA 1.1 1.2 1.2 0.1 TTCT---------------- 1.4 0.7 1 0.5 460 TTCTGGTTG----------A 1.1 1 1 0.1 461 TTCTGGTTGT-ACAGGTGGA 0.7 1.2 1 0.4 .sup.aOn-target sequence centered on cleavage site, with 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with die Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in die Reference Sequence

TABLE-US-00050 TABLE 48 On-Target Gene Edited Sequences >1% Frequency in At Least One Gene Edited T Cell Donor for the TGFBRII-Ex4-T2 gRNA. Reference on-target sequence.sup.a: TGTGAGAAGCCAC (AGG) AAGT (SEQ ID NO: 462) SEQ Donor Donor Std. ID 1 2 Mean Dev. NO: Gene Edited Sequence (%) (%) (%) (%) 463 TGTGA----------GAAGT 22.3 17.3 19.8 3.5 464 TGTGAGAAG-CACAGGAAGT 9.9 12.7 11.3 2 -------------------T 11.8 8.2 10 2.5 465 TGTGAGAAGCCCACAGGAAGT 4.8 8.1 6.4 2.3 466 TGTG---------AGGAAGT 3.1 3.5 3.3 0.3 467 TGTGAGAAGC--CAGGAAGT 3 3.1 3 0.1 468 TGTGAGA------AGGAAGT 3 2.8 2.9 0.1 469 ----------CACAGGAAGT 25 2.7 2.6 0.1 470 TGTGAGAAGCACACAGGAAGT 1.2 2.3 1.8 0.8 471 TGTGAGAAG---CAGGAAGT 1.6 1.6 1.6 0 ------------CAGGAAGT 1.3 1.8 1.6 0.4 472 TGTG------CACAGGAAGT 12 1.8 1.5 0.4 -------------------- 1.7 1 1.4 0.5 473 ---------CCACAGGAAGT 1.5 1.4 1.4 0.1 474 TGTGAGA---CACAGGAAGT 0.7 1.4 1 0.5 475 TGTGAG-----ACAGGAAGT 1.2 0.8 1 0.3 TGT------------GAAGT 12 0.7 1 0.4 476 TGTGAGAA--CACAGGAAGT 0.6 1.4 1 0.6 477 TGTGAGAAGC---------- 0.8 1.1 1 0.2 478 TGTGAGAAGCCACACAGGA 1.4 0.7 1 0.5 AGT .sup.aOn-target sequence centered on cleavage site, w ith 10 bp in either direction. For comparison, the portion of the gRNA target sequence aligning with die Reference on-target sequence is underlined and the PAM is indicated by parenthesis. .sup.bDeletions indicated by dashes (-); insertions indicated by bold .sup.cPositions of inserted bases in the gene edited sequence indicated by dashes (-) in die Reference Sequence

Other Embodiments

[0489] 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.

[0490] 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

[0491] 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.

[0492] 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.

[0493] 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.

[0494] 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."

[0495] 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.

[0496] 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.

[0497] 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.

[0498] 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.

[0499] 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.

Sequence CWU 1

1

47811368PRTArtificial SequenceSynthetic 1Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155 160Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310 315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550 555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu705 710 715 720His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790 795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys865 870 875 880Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365223DNAArtificial SequenceSynthetic 2ccgacttctg aacgtgcggt ggg 23323DNAArtificial SequenceSynthetic 3tgctggcgat acgcgtccac agg 23423DNAArtificial SequenceSynthetic 4tcggtctatg acgagcagcg ggg 23523DNAArtificial SequenceSynthetic 5atgggcagtc ctattacagc tgg 23623DNAArtificial SequenceSynthetic 6attgttcact tgttagcccc agg 23723DNAArtificial SequenceSynthetic 7gctgaagaac tgcctctata tgg 23823DNAArtificial SequenceSynthetic 8gcaggatttc tggttgtcac agg 23923DNAArtificial SequenceSynthetic 9ctccatctgt gagaagccac agg 231023DNAArtificial SequenceSynthetic 10cccctaccat gactttattc tgg 231126DNAArtificial SequenceSynthetic 11ggcaccatat tcattttgca ggtgaa 261225DNAArtificial SequenceSynthetic 12atgtgcgctc tgcccactga cgggc 251329DNAArtificial SequenceSynthetic 13agacatgagg tctatggact tcaggctcc 2914100RNAArtificial SequenceSynthetic 14ggucaucgau gggagcaacg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10015100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 15ggucaucgau gggagcaacg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1001620RNAArtificial SequenceSynthetic 16ggucaucgau gggagcaacg 201720RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 17ggucaucgau gggagcaacg 2018100RNAArtificial SequenceSynthetic 18caccaccccg cgggacuaga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10019100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 19caccaccccg cgggacuaga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002020RNAArtificial SequenceSynthetic 20caccaccccg cgggacuaga 202120RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 21caccaccccg cgggacuaga 2022100RNAArtificial SequenceSynthetic 22ggucuggcgc ucccgcucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10023100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 23ggucuggcgc ucccgcucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002420RNAArtificial SequenceSynthetic 24ggucuggcgc ucccgcucgg 202520RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 25ggucuggcgc ucccgcucgg 2026100RNAArtificial SequenceSynthetic 26uucacaccau cacgacgcgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10027100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 27uucacaccau cacgacgcgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002820RNAArtificial SequenceSynthetic 28uucacaccau cacgacgcgu 202920RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 29uucacaccau cacgacgcgu 2030100RNAArtificial SequenceSynthetic 30acaccaucac gacgcguggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10031100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 31acaccaucac gacgcguggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1003220RNAArtificial SequenceSynthetic 32acaccaucac gacgcguggg 203320RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 33acaccaucac gacgcguggg 2034100RNAArtificial SequenceSynthetic 34cuacgagucu gacgggaucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10035100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 35cuacgagucu gacgggaucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1003620RNAArtificial SequenceSynthetic 36cuacgagucu gacgggaucg 203720RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 37cuacgagucu gacgggaucg 2038100RNAArtificial SequenceSynthetic 38uugccaccca cgcgucguga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10039100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 39uugccaccca cgcgucguga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1004020RNAArtificial SequenceSynthetic 40uugccaccca cgcgucguga 204120RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 41uugccaccca cgcgucguga 2042100RNAArtificial SequenceSynthetic 42guucacacca ucacgacgcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10043100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 43guucacacca ucacgacgcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1004420RNAArtificial SequenceSynthetic 44guucacacca ucacgacgcg 204520RNAArtificial

SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 45guucacacca ucacgacgcg 2046100RNAArtificial SequenceSynthetic 46cacgaucccg ucagacucgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10047100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 47cacgaucccg ucagacucgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1004820RNAArtificial SequenceSynthetic 48cacgaucccg ucagacucgu 204920RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 49cacgaucccg ucagacucgu 2050100RNAArtificial SequenceSynthetic 50acgacgcgug gguggcaagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10051100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 51acgacgcgug gguggcaagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1005220DNAArtificial SequenceSynthetic 52acgacgcgug ggtggcaagc 205320RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 53acgacgcgug gguggcaagc 2054100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 54gcuuuggucc cauuggucgc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10055100RNAArtificial SequenceSynthetic 55gcuuuggucc cauuggucgc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1005620RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 56gcuuuggucc cauuggucgc 205720RNAArtificial SequenceSynthetic 57gcuuuggucc cauuggucgc 2058100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 58agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10059100RNAArtificial SequenceSynthetic 59agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1006020RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 60agagcaacag ugcuguggcc 206120RNAArtificial SequenceSynthetic 61agagcaacag ugcuguggcc 2062100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 62gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10063100RNAArtificial SequenceSynthetic 63gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1006420RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 64gcuacucucu cuuucuggcc 206520RNAArtificial SequenceSynthetic 65gcuacucucu cuuucuggcc 206623DNAArtificial SequenceSynthetic 66gctttggtcc cattggtcgc ggg 236720DNAArtificial SequenceSynthetic 67gctttggtcc cattggtcgc 206823DNAArtificial SequenceSynthetic 68agagcaacag tgctgtggcc tgg 236920DNAArtificial SequenceSynthetic 69agagcaacag tgctgtggcc 207023DNAArtificial SequenceSynthetic 70gctactctct ctttctggcc tgg 237120DNAArtificial SequenceSynthetic 71gctactctct ctttctggcc 2072100RNAArtificial SequenceSyntheticmisc_feature(1)..(20)n is a, c, g, or u 72nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1007396RNAArtificial SequenceSyntheticmisc_feature(1)..(20)n is a, c, g, or u 73nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugc 9674114RNAArtificial SequenceSyntheticmisc_feature(1)..(30)n is a, c, g, or umisc_feature(18)..(30)may be absentmisc_feature(108)..(114)may be absent 74nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau 60aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuuu 1147519DNAArtificial SequenceSynthetic 75aagagcaaca aatctgact 197639DNAArtificial SequenceSynthetic 76aagagcaaca gtgctgtgcc tggagcaaca aatctgact 397733DNAArtificial SequenceSynthetic 77aagagcaaca gtgctggagc aacaaatctg act 337834DNAArtificial SequenceSynthetic 78aagagcaaca gtgcctggag caacaaatct gact 347919DNAArtificial SequenceSynthetic 79aagagcaaca gtgctgact 198041DNAArtificial SequenceSynthetic 80aagagcaaca gtgctgtggg cctggagcaa caaatctgac t 418138DNAArtificial SequenceSynthetic 81aagagcaaca gtgctggcct ggagcaacaa atctgact 388241DNAArtificial SequenceSynthetic 82aagagcaaca gtgctgtgtg cctggagcaa caaatctgac t 418379DNAArtificial SequenceSynthetic 83cgtggcctta gctgtgctcg cgctactctc tctttctgcc tggaggctat ccagcgtgag 60tctctcctac cctcccgct 798478DNAArtificial SequenceSynthetic 84cgtggcctta gctgtgctcg cgctactctc tctttcgcct ggaggctatc cagcgtgagt 60ctctcctacc ctcccgct 788575DNAArtificial SequenceSynthetic 85cgtggcctta gctgtgctcg cgctactctc tctttctgga ggctatccag cgtgagtctc 60tcctaccctc ccgct 758684DNAArtificial SequenceSynthetic 86cgtggcctta gctgtgctcg cgctactctc tctttctgga tagcctggag gctatccagc 60gtgagtctct cctaccctcc cgct 848755DNAArtificial SequenceSynthetic 87cgtggcctta gctgtgctcg cgctatccag cgtgagtctc tcctaccctc ccgct 558882DNAArtificial SequenceSynthetic 88cgtggcctta gctgtgctcg cgctactctc tctttctgtg gcctggaggc tatccagcgt 60gagtctctcc taccctcccg ct 828958DNAArtificial SequenceSynthetic 89cacaccacga ggcagatcac caagcccgcg caatgggacc aaagcagccc gcaggacg 589061DNAArtificial SequenceSynthetic 90cacaccacga ggcagatcac caagcccgcg aaccaatggg accaaagcag cccgcaggac 60g 619148DNAArtificial SequenceSynthetic 91cacaccacga ggcagatcac caatgggacc aaagcagccc gcaggacg 489259DNAArtificial SequenceSynthetic 92cacaccacga ggcagatcac caagcccgcg ccaatgggac caaagcagcc cgcaggacg 599359DNAArtificial SequenceSynthetic 93cacaccacga ggcagatcac caagcccgca ccaatgggac caaagcagcc cgcaggacg 599435DNAArtificial SequenceSynthetic 94cacaccacga ggcagatcac caagcccgca ggacg 359522PRTArtificial SequenceSynthetic 95Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro 209621PRTArtificial SequenceSynthetic 96Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro 209723PRTArtificial SequenceSynthetic 97Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr Leu Tyr 2098126DNAArtificial SequenceSynthetic 98aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg 1269942PRTArtificial SequenceSynthetic 99Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40100120PRTArtificial SequenceSynthetic 100Thr Cys Ala Ala Ala Gly Cys Gly Gly Ala Gly Thr Ala Gly Gly Thr1 5 10 15Thr Gly Thr Thr Gly Cys Ala Thr Thr Cys Cys Gly Ala Thr Thr Ala 20 25 30Cys Ala Thr Gly Ala Ala Thr Ala Thr Gly Ala Cys Thr Cys Cys Thr 35 40 45Cys Gly Cys Cys Gly Gly Cys Cys Thr Gly Gly Gly Cys Cys Gly Ala 50 55 60Cys Ala Ala Gly Ala Ala Ala Ala Cys Ala Thr Thr Ala Cys Cys Ala65 70 75 80Ala Cys Cys Cys Thr Ala Thr Gly Cys Cys Cys Cys Cys Cys Cys Ala 85 90 95Cys Gly Ala Gly Ala Cys Thr Thr Cys Gly Cys Thr Gly Cys Gly Thr 100 105 110Ala Cys Ala Gly Gly Thr Cys Cys 115 12010140PRTArtificial SequenceSynthetic 101Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro1 5 10 15Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 20 25 30Arg Asp Phe Ala Ala Tyr Arg Ser 35 40102336DNAArtificial SequenceSynthetic 102cgagtgaagt tttcccgaag cgcagacgct ccggcatatc agcaaggaca gaatcagctg 60tataacgaac tgaatttggg acgccgcgag gagtatgacg tgcttgataa acgccggggg 120agagacccgg aaatgggggg taaaccccga agaaagaatc cccaagaagg actctacaat 180gaactccaga aggataagat ggcggaggcc tactcagaaa taggtatgaa gggcgaacga 240cgacggggaa aaggtcacga tggcctctac caagggttga gtacggcaac caaagatacg 300tacgatgcac tgcatatgca ggccctgcct cccaga 336103112PRTArtificial SequenceSynthetic 103Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 11010420DNAArtificial SequenceSynthetic 104gatgggagca acgtggccat 2010511PRTArtificial SequenceSynthetic 105Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn1 5 101067PRTArtificial SequenceSynthetic 106His Thr Ser Arg Leu His Ser1 51079PRTArtificial SequenceSynthetic 107Gln Gln Gly Asn Thr Leu Pro Tyr Thr1 51085PRTArtificial SequenceSynthetic 108Asp Tyr Gly Val Ser1 510916PRTArtificial SequenceSynthetic 109Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser1 5 10 1511012PRTArtificial SequenceSynthetic 110His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr1 5 1011111PRTArtificial SequenceSynthetic 111Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn1 5 101127PRTArtificial SequenceSynthetic 112His Thr Ser Arg Leu His Ser1 51139PRTArtificial SequenceSynthetic 113Gln Gln Gly Asn Thr Leu Pro Tyr Thr1 51147PRTArtificial SequenceSynthetic 114Gly Val Ser Leu Pro Asp Tyr1 51155PRTArtificial SequenceSynthetic 115Trp Gly Ser Glu Thr1 511612PRTArtificial SequenceSynthetic 116His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr1 5 101171518DNAArtificial SequenceSynthetic 117atgcttcttt tggttacgtc tctgttgctt tgcgaacttc ctcatccagc gttcttgctg 60atccccgata ttcagatgac tcagaccacc agtagcttgt ctgcctcact gggagaccga 120gtaacaatct cctgcagggc aagtcaagac attagcaaat acctcaattg gtaccagcag 180aagcccgacg gaacggtaaa actcctcatc tatcatacgt caaggttgca ttccggagta 240ccgtcacgat tttcaggttc tgggagcgga actgactatt ccttgactat ttcaaacctc 300gagcaggagg acattgcgac atatttttgt caacaaggta ataccctccc ttacactttc 360ggaggaggaa ccaaactcga aattaccggg tccaccagtg gctctgggaa gcctggcagt 420ggagaaggtt ccactaaagg cgaggtgaag ctccaggaga gcggccccgg tctcgttgcc 480cccagtcaaa gcctctctgt aacgtgcaca gtgagtggtg tatcattgcc tgattatggc 540gtctcctgga taaggcagcc cccgcgaaag ggtcttgaat ggcttggggt aatatggggc 600tcagagacaa cgtattataa ctccgctctc aaaagtcgct tgacgataat aaaagataac 660tccaagagtc aagttttcct taaaatgaac agtttgcaga ctgacgatac cgctatatat 720tattgtgcta aacattatta ctacggcggt agttacgcga tggattattg ggggcagggg 780acttctgtca cagtcagtag tgctgctgcc tttgtcccgg tatttctccc agccaaaccg 840accacgactc ccgccccgcg ccctccgaca cccgctccca ccatcgcctc tcaacctctt 900agtcttcgcc ccgaggcatg ccgacccgcc gccgggggtg ctgttcatac gaggggcttg 960gacttcgctt gtgatattta catttgggct ccgttggcgg gtacgtgcgg cgtccttttg 1020ttgtcactcg ttattacttt gtattgtaat cacaggaatc gctcaaagcg gagtaggttg 1080ttgcattccg attacatgaa tatgactcct cgccggcctg ggccgacaag aaaacattac 1140caaccctatg cccccccacg agacttcgct gcgtacaggt cccgagtgaa gttttcccga 1200agcgcagacg ctccggcata tcagcaagga cagaatcagc tgtataacga actgaatttg 1260ggacgccgcg aggagtatga cgtgcttgat aaacgccggg ggagagaccc ggaaatgggg 1320ggtaaacccc gaagaaagaa tccccaagaa ggactctaca atgaactcca gaaggataag 1380atggcggagg cctactcaga aataggtatg aagggcgaac gacgacgggg aaaaggtcac 1440gatggcctct accaagggtt gagtacggca accaaagata cgtacgatgc actgcatatg 1500caggccctgc ctcccaga 1518118506PRTArtificial SequenceSynthetic 118Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala145 150 155 160Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr225 230 235 240Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Phe Val 260 265 270Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 275 280 285Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 290 295 300Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu305 310 315 320Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

Ala Gly Thr Cys 325 330 335Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg 340 345 350Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 355 360 365Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 370 375 380Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg385 390 395 400Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 405 410 415Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 420 425 430Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 435 440 445Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 450 455 460Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His465 470 475 480Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 485 490 495Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505119735DNAArtificial SequenceSynthetic 119gatattcaga tgactcagac caccagtagc ttgtctgcct cactgggaga ccgagtaaca 60atctcctgca gggcaagtca agacattagc aaatacctca attggtacca gcagaagccc 120gacggaacgg taaaactcct catctatcat acgtcaaggt tgcattccgg agtaccgtca 180cgattttcag gttctgggag cggaactgac tattccttga ctatttcaaa cctcgagcag 240gaggacattg cgacatattt ttgtcaacaa ggtaataccc tcccttacac tttcggagga 300ggaaccaaac tcgaaattac cgggtccacc agtggctctg ggaagcctgg cagtggagaa 360ggttccacta aaggcgaggt gaagctccag gagagcggcc ccggtctcgt tgcccccagt 420caaagcctct ctgtaacgtg cacagtgagt ggtgtatcat tgcctgatta tggcgtctcc 480tggataaggc agcccccgcg aaagggtctt gaatggcttg gggtaatatg gggctcagag 540acaacgtatt ataactccgc tctcaaaagt cgcttgacga taataaaaga taactccaag 600agtcaagttt tccttaaaat gaacagtttg cagactgacg ataccgctat atattattgt 660gctaaacatt attactacgg cggtagttac gcgatggatt attgggggca ggggacttct 720gtcacagtca gtagt 735120245PRTArtificial SequenceSynthetic 120Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser145 150 155 160Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser225 230 235 240Val Thr Val Ser Ser 245121261DNAArtificial SequenceSynthetic 121gctgctgcct ttgtcccggt atttctccca gccaaaccga ccacgactcc cgccccgcgc 60cctccgacac ccgctcccac catcgcctct caacctctta gtcttcgccc cgaggcatgc 120cgacccgccg ccgggggtgc tgttcatacg aggggcttgg acttcgcttg tgatatttac 180atttgggctc cgttggcggg tacgtgcggc gtccttttgt tgtcactcgt tattactttg 240tattgtaatc acaggaatcg c 261122252DNAArtificial SequenceSynthetic 122tttgtcccgg tatttctccc agccaaaccg accacgactc ccgccccgcg ccctccgaca 60cccgctccca ccatcgcctc tcaacctctt agtcttcgcc ccgaggcatg ccgacccgcc 120gccgggggtg ctgttcatac gaggggcttg gacttcgctt gtgatattta catttgggct 180ccgttggcgg gtacgtgcgg cgtccttttg ttgtcactcg ttattacttt gtattgtaat 240cacaggaatc gc 25212384PRTArtificial SequenceSynthetic 123Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro1 5 10 15Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 20 25 30Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 35 40 45Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 50 55 60Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn65 70 75 80His Arg Asn Arg124120PRTArtificial SequenceSynthetic 124Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 120125107PRTArtificial SequenceSynthetic 125Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 10512618PRTArtificial SequenceSynthetic 126Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr1 5 10 15Lys Gly12715PRTArtificial SequenceSynthetic 127Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met His1 5 10 1512811PRTArtificial SequenceSynthetic 128Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe1 5 101297PRTArtificial SequenceSynthetic 129Leu Ala Ser Asn Leu Glu Ser1 51309PRTArtificial SequenceSynthetic 130Gln His Ser Arg Glu Val Pro Trp Thr1 51316PRTArtificial SequenceSynthetic 131Ser Arg Glu Val Pro Trp1 51325PRTArtificial SequenceSynthetic 132Asn Tyr Gly Met Asn1 513310PRTArtificial SequenceSynthetic 133Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn1 5 1013417PRTArtificial SequenceSynthetic 134Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe Lys1 5 10 15Gly1356PRTArtificial SequenceSynthetic 135Asn Thr Tyr Thr Gly Glu1 51369PRTArtificial SequenceSynthetic 136Asp Tyr Gly Asp Tyr Gly Met Asp Tyr1 513714PRTArtificial SequenceSynthetic 137Cys Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly1 5 10138508PRTArtificial SequenceSynthetic 138Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln 50 55 60Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr65 70 75 80Tyr Ala Asp Ala Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser 85 90 95Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr 115 120 125Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asp Ile Val Met Thr145 150 155 160Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile 165 170 175Asn Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met 180 185 190His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 195 200 205Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 210 215 220Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu225 230 235 240Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg Glu Val Pro Trp Thr 245 250 255Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ala Ala Ala Phe Val 260 265 270Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 275 280 285Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 290 295 300Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu305 310 315 320Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 325 330 335Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg 340 345 350Asn Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 355 360 365Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 370 375 380Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe385 390 395 400Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 405 410 415Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 420 425 430Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 435 440 445Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 450 455 460Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys465 470 475 480Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 485 490 495Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505139735DNAArtificial SequenceSynthetic 139gatatagtta tgacccaatc acccgatagt cttgcggtaa gcctggggga gcgagcaaca 60ataaactgtc gggcatcaaa atccgtcagt acaagcgggt attcattcat gcactggtat 120caacagaaac ccggtcagcc acccaagctc ctgatttatc ttgcgtctaa tcttgagtcc 180ggcgtcccag accggttttc cggctccggg agcggcacgg attttactct tactatttct 240agccttcagg ccgaagatgt ggcggtatac tactgccagc attcaaggga agttccttgg 300acgttcggtc agggcacgaa agtggaaatt aaaggcgggg ggggatccgg cgggggaggg 360tctggaggag gtggcagtgg tcaggtccaa ctggtgcagt ccggggcaga ggtaaaaaaa 420cccggcgcgt ctgttaaggt ttcatgcaag gccagtggat atactttcac caattacgga 480atgaactggg tgaggcaggc ccctggtcaa ggcctgaaat ggatgggatg gataaacacg 540tacaccggtg aacctaccta tgccgatgcc tttaagggtc gggttacgat gacgagagac 600acctccatat caacagccta catggagctc agcagattga ggagtgacga tacggcagtc 660tattactgtg caagagacta cggcgattat ggcatggatt actggggcca gggcactaca 720gtaaccgttt ccagc 735140245PRTArtificial SequenceSynthetic 140Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly 100 105 110Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln 115 120 125Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser 130 135 140Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly145 150 155 160Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met Gly 165 170 175Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe Lys 180 185 190Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met 195 200 205Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr225 230 235 240Val Thr Val Ser Ser 245141735DNAArtificial SequenceSynthetic 141caggtccagt tggtgcaaag cggggcggag gtgaaaaaac ccggcgcttc cgtgaaggtg 60tcctgtaagg cgtccggtta tacgttcacg aactacggga tgaattgggt tcgccaagcg 120ccggggcagg gactgaaatg gatggggtgg ataaatacct acaccggcga acctacatac 180gccgacgctt ttaaagggcg agtcactatg acgcgcgata ccagcatatc caccgcatac 240atggagctgt cccgactccg gtcagacgac acggctgtct actattgtgc tcgggactat 300ggcgattatg gcatggacta ctggggtcag ggtacgactg taacagttag tagtggtgga 360ggcggcagtg gcgggggggg aagcggagga gggggttctg gtgacatagt tatgacccaa 420tccccagata gtttggcggt ttctctgggc gagagggcaa cgattaattg tcgcgcatca 480aagagcgttt caacgagcgg atattctttt atgcattggt accagcaaaa acccggacaa 540ccgccgaagc tgctgatcta cttggcttca aatcttgagt ctggggtgcc ggaccgattt 600tctggtagtg gaagcggaac tgactttacg ctcacgatca gttcactgca ggctgaggat 660gtagcggtct attattgcca gcacagtaga gaagtcccct ggaccttcgg tcaaggcacg 720aaagtagaaa ttaaa 735142245PRTArtificial SequenceSynthetic 142Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser 130 135 140Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser145 150 155 160Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met His Trp Tyr Gln Gln 165 170 175Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu 180 185 190Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 210 215 220Tyr Cys Gln His Ser Arg Glu Val Pro Trp Thr Phe Gly Gln Gly Thr225 230 235 240Lys Val Glu Ile Lys

245143118PRTArtificial SequenceSynthetic 143Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 115144111PRTArtificial SequenceSynthetic 144Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1101451524DNAArtificial SequenceSynthetic 145atggcgcttc cggtgacagc actgctcctc cccttggcgc tgttgctcca cgcagcaagg 60ccgcaggtgc agctggtgca gagcggagcc gagctcaaga agcccggagc ctccgtgaag 120gtgagctgca aggccagcgg caacaccctg accaactacg tgatccactg ggtgagacaa 180gcccccggcc aaaggctgga gtggatgggc tacatcctgc cctacaacga cctgaccaag 240tacagccaga agttccaggg cagggtgacc atcaccaggg ataagagcgc ctccaccgcc 300tatatggagc tgagcagcct gaggagcgag gacaccgctg tgtactactg tacaaggtgg 360gactgggacg gcttctttga cccctggggc cagggcacaa cagtgaccgt cagcagcggc 420ggcggaggca gcggcggcgg cggcagcggc ggaggcggaa gcgaaatcgt gatgacccag 480agccccgcca cactgagcgt gagccctggc gagagggcca gcatctcctg cagggctagc 540caaagcctgg tgcacagcaa cggcaacacc cacctgcact ggtaccagca gagacccgga 600caggctccca ggctgctgat ctacagcgtg agcaacaggt tctccgaggt gcctgccagg 660tttagcggca gcggaagcgg caccgacttt accctgacca tcagcagcgt ggagtccgag 720gacttcgccg tgtattactg cagccagacc agccacatcc cttacacctt cggcggcggc 780accaagctgg agatcaaaag tgctgctgcc tttgtcccgg tatttctccc agccaaaccg 840accacgactc ccgccccgcg ccctccgaca cccgctccca ccatcgcctc tcaacctctt 900agtcttcgcc ccgaggcatg ccgacccgcc gccgggggtg ctgttcatac gaggggcttg 960gacttcgctt gtgatattta catttgggct ccgttggcgg gtacgtgcgg cgtccttttg 1020ttgtcactcg ttattacttt gtattgtaat cacaggaatc gcaaacgggg cagaaagaaa 1080ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1140ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgcg agtgaagttt 1200tcccgaagcg cagacgctcc ggcatatcag caaggacaga atcagctgta taacgaactg 1260aatttgggac gccgcgagga gtatgacgtg cttgataaac gccgggggag agacccggaa 1320atggggggta aaccccgaag aaagaatccc caagaaggac tctacaatga actccagaag 1380gataagatgg cggaggccta ctcagaaata ggtatgaagg gcgaacgacg acggggaaaa 1440ggtcacgatg gcctctacca agggttgagt acggcaacca aagatacgta cgatgcactg 1500catatgcagg ccctgcctcc caga 1524146508PRTArtificial SequenceSynthetic 146Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu 20 25 30Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn 35 40 45Thr Leu Thr Asn Tyr Val Ile His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60Arg Leu Glu Trp Met Gly Tyr Ile Leu Pro Tyr Asn Asp Leu Thr Lys65 70 75 80Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Arg Asp Lys Ser 85 90 95Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Thr Arg Trp Asp Trp Asp Gly Phe Phe Asp Pro 115 120 125Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln145 150 155 160Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Ser Ile Ser 165 170 175Cys Arg Ala Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr His Leu 180 185 190His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 195 200 205Ser Val Ser Asn Arg Phe Ser Glu Val Pro Ala Arg Phe Ser Gly Ser 210 215 220Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Glu Ser Glu225 230 235 240Asp Phe Ala Val Tyr Tyr Cys Ser Gln Thr Ser His Ile Pro Tyr Thr 245 250 255Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ala Ala Ala Phe Val 260 265 270Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 275 280 285Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 290 295 300Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu305 310 315 320Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 325 330 335Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg 340 345 350Asn Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 355 360 365Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 370 375 380Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe385 390 395 400Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 405 410 415Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 420 425 430Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 435 440 445Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 450 455 460Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys465 470 475 480Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 485 490 495Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505147735DNAArtificial SequenceSynthetic 147caggtgcagc tggtgcagag cggagccgag ctcaagaagc ccggagcctc cgtgaaggtg 60agctgcaagg ccagcggcaa caccctgacc aactacgtga tccactgggt gagacaagcc 120cccggccaaa ggctggagtg gatgggctac atcctgccct acaacgacct gaccaagtac 180agccagaagt tccagggcag ggtgaccatc accagggata agagcgcctc caccgcctat 240atggagctga gcagcctgag gagcgaggac accgctgtgt actactgtac aaggtgggac 300tgggacggct tctttgaccc ctggggccag ggcacaacag tgaccgtcag cagcggcggc 360ggaggcagcg gcggcggcgg cagcggcgga ggcggaagcg aaatcgtgat gacccagagc 420cccgccacac tgagcgtgag ccctggcgag agggccagca tctcctgcag ggctagccaa 480agcctggtgc acagcaacgg caacacccac ctgcactggt accagcagag acccggacag 540gctcccaggc tgctgatcta cagcgtgagc aacaggttct ccgaggtgcc tgccaggttt 600agcggcagcg gaagcggcac cgactttacc ctgaccatca gcagcgtgga gtccgaggac 660ttcgccgtgt attactgcag ccagaccagc cacatccctt acaccttcgg cggcggcacc 720aagctggaga tcaaa 735148245PRTArtificial SequenceSynthetic 148Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Thr Leu Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Leu Pro Tyr Asn Asp Leu Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Trp Asp Trp Asp Gly Phe Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 130 135 140Ser Val Ser Pro Gly Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Gln145 150 155 160Ser Leu Val His Ser Asn Gly Asn Thr His Leu His Trp Tyr Gln Gln 165 170 175Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ser Val Ser Asn Arg 180 185 190Phe Ser Glu Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Val Glu Ser Glu Asp Phe Ala Val Tyr 210 215 220Tyr Cys Ser Gln Thr Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr225 230 235 240Lys Leu Glu Ile Lys 245149118PRTArtificial SequenceSynthetic 149Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Thr Leu Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Leu Pro Tyr Asn Asp Leu Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Trp Asp Trp Asp Gly Phe Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 115150112PRTArtificial SequenceSynthetic 150Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr His Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ala 35 40 45Pro Arg Leu Leu Ile Tyr Ser Val Ser Asn Arg Phe Ser Glu Val Pro 50 55 60Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70 75 80Ser Ser Val Glu Ser Glu Asp Phe Ala Val Tyr Tyr Cys Ser Gln Thr 85 90 95Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 11015116PRTArtificial SequenceSynthetic 151Arg Ala Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr His Leu His1 5 10 151527PRTArtificial SequenceSynthetic 152Ser Val Ser Asn Arg Phe Ser1 51539PRTArtificial SequenceSynthetic 153Ser Gln Thr Ser His Ile Pro Tyr Thr1 51549PRTArtificial SequenceSynthetic 154Ser Gln Thr Ser His Ile Pro Tyr Thr1 51555PRTArtificial SequenceSynthetic 155Asn Tyr Val Ile His1 51567PRTArtificial SequenceSynthetic 156Gly Asn Thr Leu Thr Asn Tyr1 515717PRTArtificial SequenceSynthetic 157Tyr Ile Leu Pro Tyr Asn Asp Leu Thr Lys Tyr Ser Gln Lys Phe Gln1 5 10 15Gly1586PRTArtificial SequenceSynthetic 158Leu Pro Tyr Asn Asp Leu1 51599PRTArtificial SequenceSynthetic 159Trp Asp Trp Asp Gly Phe Phe Asp Pro1 51609PRTArtificial SequenceSynthetic 160Trp Asp Trp Asp Gly Phe Phe Asp Pro1 5161145DNAArtificial SequenceSynthetic 161ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120gccaactcca tcactagggg ttcct 145162130DNAArtificial SequenceSynthetic 162cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct 130163145DNAArtificial SequenceSynthetic 163aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 120gagcgcgcag agagggagtg gccaa 145164141DNAArtificial SequenceSynthetic 164aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120gagcgcgcag ctgcctgcag g 141165800DNAArtificial SequenceSynthetic 165gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca 800166804DNAArtificial SequenceSynthetic 166tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 60gacaccttct tccccagccc aggtaagggc agctttggtg ccttcgcagg ctgtttcctt 120gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa tgatgtctaa aactcctctg 180attggtggtc tcggccttat ccattgccac caaaaccctc tttttactaa gaaacagtga 240gccttgttct ggcagtccag agaatgacac gggaaaaaag cagatgaaga gaaggtggca 300ggagagggca cgtggcccag cctcagtctc tccaactgag ttcctgcctg cctgcctttg 360ctcagactgt ttgcccctta ctgctcttct aggcctcatt ctaagcccct tctccaagtt 420gcctctcctt atttctccct gtctgccaaa aaatctttcc cagctcacta agtcagtctc 480acgcagtcac tcattaaccc accaatcact gattgtgccg gcacatgaat gcaccaggtg 540ttgaagtgga ggaattaaaa agtcagatga ggggtgtgcc cagaggaagc accattctag 600ttgggggagc ccatctgtca gctgggaaaa gtccaaataa cttcagattg gaatgtgttt 660taactcaggg ttgagaaaac agctaccttc aggacaaaag tcagggaagg gctctctgaa 720gaaatgctac ttgaagatac cagccctacc aagggcaggg agaggaccct atagaggcct 780gggacaggag ctcaatgaga aagg 8041671178DNAArtificial SequenceSynthetic 167ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 960agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140tggttcaaag tttttttctt ccatttcagg tgtcgtga 11781684358DNAArtificial SequenceSynthetic 168gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt

tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatgcttc ttttggttac gtctctgttg ctttgcgaac ttcctcatcc agcgttcttg 2040ctgatccccg atattcagat gactcagacc accagtagct tgtctgcctc actgggagac 2100cgagtaacaa tctcctgcag ggcaagtcaa gacattagca aatacctcaa ttggtaccag 2160cagaagcccg acggaacggt aaaactcctc atctatcata cgtcaaggtt gcattccgga 2220gtaccgtcac gattttcagg ttctgggagc ggaactgact attccttgac tatttcaaac 2280ctcgagcagg aggacattgc gacatatttt tgtcaacaag gtaataccct cccttacact 2340ttcggaggag gaaccaaact cgaaattacc gggtccacca gtggctctgg gaagcctggc 2400agtggagaag gttccactaa aggcgaggtg aagctccagg agagcggccc cggtctcgtt 2460gcccccagtc aaagcctctc tgtaacgtgc acagtgagtg gtgtatcatt gcctgattat 2520ggcgtctcct ggataaggca gcccccgcga aagggtcttg aatggcttgg ggtaatatgg 2580ggctcagaga caacgtatta taactccgct ctcaaaagtc gcttgacgat aataaaagat 2640aactccaaga gtcaagtttt ccttaaaatg aacagtttgc agactgacga taccgctata 2700tattattgtg ctaaacatta ttactacggc ggtagttacg cgatggatta ttgggggcag 2760gggacttctg tcacagtcag tagtgctgct gcctttgtcc cggtatttct cccagccaaa 2820ccgaccacga ctcccgcccc gcgccctccg acacccgctc ccaccatcgc ctctcaacct 2880cttagtcttc gccccgaggc atgccgaccc gccgccgggg gtgctgttca tacgaggggc 2940ttggacttcg cttgtgatat ttacatttgg gctccgttgg cgggtacgtg cggcgtcctt 3000ttgttgtcac tcgttattac tttgtattgt aatcacagga atcgctcaaa gcggagtagg 3060ttgttgcatt ccgattacat gaatatgact cctcgccggc ctgggccgac aagaaaacat 3120taccaaccct atgccccccc acgagacttc gctgcgtaca ggtcccgagt gaagttttcc 3180cgaagcgcag acgctccggc atatcagcaa ggacagaatc agctgtataa cgaactgaat 3240ttgggacgcc gcgaggagta tgacgtgctt gataaacgcc gggggagaga cccggaaatg 3300gggggtaaac cccgaagaaa gaatccccaa gaaggactct acaatgaact ccagaaggat 3360aagatggcgg aggcctactc agaaataggt atgaagggcg aacgacgacg gggaaaaggt 3420cacgatggcc tctaccaagg gttgagtacg gcaaccaaag atacgtacga tgcactgcat 3480atgcaggccc tgcctcccag ataataataa aatcgctatc catcgaagat ggatgtgtgt 3540tggttttttg tgtgtggagc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca 3600gcattattcc agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg 3660caggctgttt ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt 3720ctaaaactcc tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta 3780ctaagaaaca gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg 3840aagagaaggt ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg 3900cctgcctgcc tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc 3960cccttctcca agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc 4020actaagtcag tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat 4080gaatgcacca ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg 4140aagcaccatt ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag 4200attggaatgt gttttaactc agggttgaga aaacagctac cttcaggaca aaagtcaggg 4260aagggctctc tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga 4320ccctatagag gcctgggaca ggagctcaat gagaaagg 43581694364DNAArtificial SequenceSynthetic 169gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040aggccgcagg tccagttggt gcaaagcggg gcggaggtga aaaaacccgg cgcttccgtg 2100aaggtgtcct gtaaggcgtc cggttatacg ttcacgaact acgggatgaa ttgggttcgc 2160caagcgccgg ggcagggact gaaatggatg gggtggataa atacctacac cggcgaacct 2220acatacgccg acgcttttaa agggcgagtc actatgacgc gcgataccag catatccacc 2280gcatacatgg agctgtcccg actccggtca gacgacacgg ctgtctacta ttgtgctcgg 2340gactatggcg attatggcat ggactactgg ggtcagggta cgactgtaac agttagtagt 2400ggtggaggcg gcagtggcgg ggggggaagc ggaggagggg gttctggtga catagttatg 2460acccaatccc cagatagttt ggcggtttct ctgggcgaga gggcaacgat taattgtcgc 2520gcatcaaaga gcgtttcaac gagcggatat tcttttatgc attggtacca gcaaaaaccc 2580ggacaaccgc cgaagctgct gatctacttg gcttcaaatc ttgagtctgg ggtgccggac 2640cgattttctg gtagtggaag cggaactgac tttacgctca cgatcagttc actgcaggct 2700gaggatgtag cggtctatta ttgccagcac agtagagaag tcccctggac cttcggtcaa 2760ggcacgaaag tagaaattaa aagtgctgct gcctttgtcc cggtatttct cccagccaaa 2820ccgaccacga ctcccgcccc gcgccctccg acacccgctc ccaccatcgc ctctcaacct 2880cttagtcttc gccccgaggc atgccgaccc gccgccgggg gtgctgttca tacgaggggc 2940ttggacttcg cttgtgatat ttacatttgg gctccgttgg cgggtacgtg cggcgtcctt 3000ttgttgtcac tcgttattac tttgtattgt aatcacagga atcgcaaacg gggcagaaag 3060aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 3120gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gcgagtgaag 3180ttttcccgaa gcgcagacgc tccggcatat cagcaaggac agaatcagct gtataacgaa 3240ctgaatttgg gacgccgcga ggagtatgac gtgcttgata aacgccgggg gagagacccg 3300gaaatggggg gtaaaccccg aagaaagaat ccccaagaag gactctacaa tgaactccag 3360aaggataaga tggcggaggc ctactcagaa ataggtatga agggcgaacg acgacgggga 3420aaaggtcacg atggcctcta ccaagggttg agtacggcaa ccaaagatac gtacgatgca 3480ctgcatatgc aggccctgcc tcccagataa taataaaatc gctatccatc gaagatggat 3540gtgtgttggt tttttgtgtg tggagcaaca aatctgactt tgcatgtgca aacgccttca 3600acaacagcat tattccagaa gacaccttct tccccagccc aggtaagggc agctttggtg 3660ccttcgcagg ctgtttcctt gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa 3720tgatgtctaa aactcctctg attggtggtc tcggccttat ccattgccac caaaaccctc 3780tttttactaa gaaacagtga gccttgttct ggcagtccag agaatgacac gggaaaaaag 3840cagatgaaga gaaggtggca ggagagggca cgtggcccag cctcagtctc tccaactgag 3900ttcctgcctg cctgcctttg ctcagactgt ttgcccctta ctgctcttct aggcctcatt 3960ctaagcccct tctccaagtt gcctctcctt atttctccct gtctgccaaa aaatctttcc 4020cagctcacta agtcagtctc acgcagtcac tcattaaccc accaatcact gattgtgccg 4080gcacatgaat gcaccaggtg ttgaagtgga ggaattaaaa agtcagatga ggggtgtgcc 4140cagaggaagc accattctag ttgggggagc ccatctgtca gctgggaaaa gtccaaataa 4200cttcagattg gaatgtgttt taactcaggg ttgagaaaac agctaccttc aggacaaaag 4260tcagggaagg gctctctgaa gaaatgctac ttgaagatac cagccctacc aagggcaggg 4320agaggaccct atagaggcct gggacaggag ctcaatgaga aagg 43641704364DNAArtificial SequenceSynthetic 170gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040aggccgcagg tgcagctggt gcagagcgga gccgagctca agaagcccgg agcctccgtg 2100aaggtgagct gcaaggccag cggcaacacc ctgaccaact acgtgatcca ctgggtgaga 2160caagcccccg gccaaaggct ggagtggatg ggctacatcc tgccctacaa cgacctgacc 2220aagtacagcc agaagttcca gggcagggtg accatcacca gggataagag cgcctccacc 2280gcctatatgg agctgagcag cctgaggagc gaggacaccg ctgtgtacta ctgtacaagg 2340tgggactggg acggcttctt tgacccctgg ggccagggca caacagtgac cgtcagcagc 2400ggcggcggag gcagcggcgg cggcggcagc ggcggaggcg gaagcgaaat cgtgatgacc 2460cagagccccg ccacactgag cgtgagccct ggcgagaggg ccagcatctc ctgcagggct 2520agccaaagcc tggtgcacag caacggcaac acccacctgc actggtacca gcagagaccc 2580ggacaggctc ccaggctgct gatctacagc gtgagcaaca ggttctccga ggtgcctgcc 2640aggtttagcg gcagcggaag cggcaccgac tttaccctga ccatcagcag cgtggagtcc 2700gaggacttcg ccgtgtatta ctgcagccag accagccaca tcccttacac cttcggcggc 2760ggcaccaagc tggagatcaa aagtgctgct gcctttgtcc cggtatttct cccagccaaa 2820ccgaccacga ctcccgcccc gcgccctccg acacccgctc ccaccatcgc ctctcaacct 2880cttagtcttc gccccgaggc atgccgaccc gccgccgggg gtgctgttca tacgaggggc 2940ttggacttcg cttgtgatat ttacatttgg gctccgttgg cgggtacgtg cggcgtcctt 3000ttgttgtcac tcgttattac tttgtattgt aatcacagga atcgcaaacg gggcagaaag 3060aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 3120gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gcgagtgaag 3180ttttcccgaa gcgcagacgc tccggcatat cagcaaggac agaatcagct gtataacgaa 3240ctgaatttgg gacgccgcga ggagtatgac gtgcttgata aacgccgggg gagagacccg 3300gaaatggggg gtaaaccccg aagaaagaat ccccaagaag gactctacaa tgaactccag 3360aaggataaga tggcggaggc ctactcagaa ataggtatga agggcgaacg acgacgggga 3420aaaggtcacg atggcctcta ccaagggttg agtacggcaa ccaaagatac gtacgatgca 3480ctgcatatgc aggccctgcc tcccagataa taataaaatc gctatccatc gaagatggat 3540gtgtgttggt tttttgtgtg tggagcaaca aatctgactt tgcatgtgca aacgccttca 3600acaacagcat tattccagaa gacaccttct tccccagccc aggtaagggc agctttggtg 3660ccttcgcagg ctgtttcctt gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa 3720tgatgtctaa aactcctctg attggtggtc tcggccttat ccattgccac caaaaccctc 3780tttttactaa gaaacagtga gccttgttct ggcagtccag agaatgacac gggaaaaaag 3840cagatgaaga gaaggtggca ggagagggca cgtggcccag cctcagtctc tccaactgag 3900ttcctgcctg cctgcctttg ctcagactgt ttgcccctta ctgctcttct aggcctcatt 3960ctaagcccct tctccaagtt gcctctcctt atttctccct gtctgccaaa aaatctttcc 4020cagctcacta agtcagtctc acgcagtcac tcattaaccc accaatcact gattgtgccg 4080gcacatgaat gcaccaggtg ttgaagtgga ggaattaaaa agtcagatga ggggtgtgcc 4140cagaggaagc accattctag ttgggggagc ccatctgtca gctgggaaaa gtccaaataa 4200cttcagattg gaatgtgttt taactcaggg ttgagaaaac agctaccttc aggacaaaag 4260tcagggaagg gctctctgaa gaaatgctac ttgaagatac cagccctacc aagggcaggg 4320agaggaccct atagaggcct gggacaggag ctcaatgaga aagg 436417123DNAArtificial SequenceSynthetic 171ggtcatcgat gggagcaacg tgg 2317223DNAArtificial SequenceSynthetic 172caccaccccg cgggactaga ggg 2317323DNAArtificial SequenceSynthetic 173ggtctggcgc tcccgctcgg tgg 2317423DNAArtificial SequenceSynthetic 174ttcacaccat cacgacgcgt ggg 2317523DNAArtificial SequenceSynthetic 175acaccatcac gacgcgtggg tgg 2317623DNAArtificial SequenceSynthetic 176ctacgagtct gacgggatcg tgg 2317723DNAArtificial SequenceSynthetic 177ttgccaccca cgcgtcgtga tgg 2317823DNAArtificial SequenceSynthetic 178gttcacacca tcacgacgcg tgg 2317923DNAArtificial SequenceSynthetic 179cacgatcccg tcagactcgt agg 2318023DNAArtificial SequenceSynthetic 180acgacgcgtg ggtggcaagc ggg 2318121DNAArtificial SequenceSynthetic 181gatgggagca aacgtggcca t 2118219DNAArtificial SequenceSynthetic 182gatgggagca cgtggccat 1918317DNAArtificial SequenceSynthetic 183gatgggaacg tggccat 1718412DNAArtificial SequenceSynthetic 184gatgggagcc at 1218510DNAArtificial SequenceSynthetic 185gatgggccat 1018610DNAArtificial SequenceSynthetic 186acgtggccat 1018721DNAArtificial SequenceSynthetic 187ccgcgggact tagagggagc t 2118811DNAArtificial SequenceSynthetic 188ccgcgggagc t 1118919DNAArtificial SequenceSynthetic 189ccgcgggata gagggagct 1919010DNAArtificial SequenceSynthetic 190ccgcgggact 1019118DNAArtificial SequenceSynthetic 191ccgcgggtag agggagct 1819212DNAArtificial

SequenceSynthetic 192ccgcggggag ct 1219319DNAArtificial SequenceSynthetic 193ccgcgggaca gagggagct 1919419DNAArtificial SequenceSynthetic 194ccgcgggact gagggagct 1919511DNAArtificial SequenceSynthetic 195ccgagggagc t 1119615DNAArtificial SequenceSynthetic 196ccgcgggagg gagct 1519714DNAArtificial SequenceSynthetic 197ccgtagaggg agct 1419821DNAArtificial SequenceSynthetic 198cgctcccgct tcggtggctg t 2119919DNAArtificial SequenceSynthetic 199cgctcccgcc ggtggctgt 1920019DNAArtificial SequenceSynthetic 200cgctcccgtc ggtggctgt 1920118DNAArtificial SequenceSynthetic 201cgctcccgcg gtggctgt 1820219DNAArtificial SequenceSynthetic 202cgctcccgct ggtggctgt 1920318DNAArtificial SequenceSynthetic 203cgctccctcg gtggctgt 1820422DNAArtificial SequenceSynthetic 204cgctcccgct ttcggtggct gt 2220516DNAArtificial SequenceSynthetic 205cgctcccggt ggctgt 1620618DNAArtificial SequenceSynthetic 206catcacgacg tgggtggc 1820715DNAArtificial SequenceSynthetic 207catcacgtgg gtggc 1520821DNAArtificial SequenceSynthetic 208catcacgacg ccgtgggtgg c 2120914DNAArtificial SequenceSynthetic 209catcacgacg tggc 1421021DNAArtificial SequenceSynthetic 210catcacgacg gcgtgggtgg c 2121110DNAArtificial SequenceSynthetic 211cgtgggtggc 1021217DNAArtificial SequenceSynthetic 212catcacgacg tggtggc 1721321DNAArtificial SequenceSynthetic 213catcacgacg tcgtgggtgg c 2121416DNAArtificial SequenceSynthetic 214catcacgacg ggtggc 1621515DNAArtificial SequenceSynthetic 215catcacgacg gtggc 1521621DNAArtificial SequenceSynthetic 216cacgacgcgt tgggtggcaa g 2121718DNAArtificial SequenceSynthetic 217cacgacgcgg gtggcaag 1821811DNAArtificial SequenceSynthetic 218cacgacgcaa g 1121916DNAArtificial SequenceSynthetic 219cacgacgcgt ggcaag 1622019DNAArtificial SequenceSynthetic 220cacgacgcgg ggtggcaag 1922121DNAArtificial SequenceSynthetic 221tctgacggga atcgtggttt c 2122213DNAArtificial SequenceSynthetic 222tctgacgggt ttc 1322314DNAArtificial SequenceSynthetic 223tctgacgtgg tttc 1422421DNAArtificial SequenceSynthetic 224tctgacggga ttcgtggttt c 2122519DNAArtificial SequenceSynthetic 225tctgacggga cgtggtttc 1922614DNAArtificial SequenceSynthetic 226tctgtcgtgg tttc 1422712DNAArtificial SequenceSynthetic 227tctgacggtt tc 1222819DNAArtificial SequenceSynthetic 228tctgacgggt cgtggtttc 1922921DNAArtificial SequenceSynthetic 229tctgacggga gtcgtggttt c 2123021DNAArtificial SequenceSynthetic 230tctgacggga ctcgtggttt c 2123110DNAArtificial SequenceSynthetic 231tcgtggtttc 1023218DNAArtificial SequenceSynthetic 232tctgacggtc gtggtttc 1823318DNAArtificial SequenceSynthetic 233tctgacggga gtggtttc 1823421DNAArtificial SequenceSynthetic 234ccacgcgtcg gtgatggtgt g 2123521DNAArtificial SequenceSynthetic 235ccacgcgtcg ttgatggtgt g 2123611DNAArtificial SequenceSynthetic 236ccacgcgtgt g 1123721DNAArtificial SequenceSynthetic 237ccacgcgtcg atgatggtgt g 2123818DNAArtificial SequenceSynthetic 238ccacgcgtcg atggtgtg 1823919DNAArtificial SequenceSynthetic 239ccacgcgtct gatggtgtg 1924021DNAArtificial SequenceSynthetic 240ccacgcgtcg ctgatggtgt g 2124110DNAArtificial SequenceSynthetic 241ccacgcgtcg 1024215DNAArtificial SequenceSynthetic 242ccacgcgtcg gtgtg 1524315DNAArtificial SequenceSynthetic 243ccacgcgtgg gtgtg 1524417DNAArtificial SequenceSynthetic 244ccacgcgtga tggtgtg 1724514DNAArtificial SequenceSynthetic 245ccacgcgtcg tgtg 1424613DNAArtificial SequenceSynthetic 246ccacgcgtcg tga 1324713DNAArtificial SequenceSynthetic 247ccacgcgtgg gtg 1324821DNAArtificial SequenceSynthetic 248ccatcacgac cgcgtgggtg g 2124915DNAArtificial SequenceSynthetic 249ccatcacgtg ggtgg 1525017DNAArtificial SequenceSynthetic 250ccatcacgcg tgggtgg 1725121DNAArtificial SequenceSynthetic 251ccatcacgac agcgtgggtg g 2125221DNAArtificial SequenceSynthetic 252ccgtcagact tcgtaggcca g 2125311DNAArtificial SequenceSynthetic 253ccgtaggcca g 1125410DNAArtificial SequenceSynthetic 254ccgtcagact 1025512DNAArtificial SequenceSynthetic 255ccgtcagacc ag 1225618DNAArtificial SequenceSynthetic 256ccgtcagacg taggccag 1825712DNAArtificial SequenceSynthetic 257ccgtcaggcc ag 1225819DNAArtificial SequenceSynthetic 258ccgtcagacc gtaggccag 1925919DNAArtificial SequenceSynthetic 259ccgtcagact gtaggccag 1926021DNAArtificial SequenceSynthetic 260gtgggtggca aagcgggtgg t 2126119DNAArtificial SequenceSynthetic 261gtgggtggca gcgggtggt 1926221DNAArtificial SequenceSynthetic 262gtgggtggca tagcgggtgg t 2126318DNAArtificial SequenceSynthetic 263gtgggtggag cgggtggt 18264101RNAArtificial SequenceSynthetic 264ccgacuucug aacgugcggu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcmuuu u 101265103RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(100)..(103)modified with 2'-O-methyl phosphorothioate 265ccgacuucug aacgugcggu gggguuuuag agcuagaaau agcaaguuaa aauaaggcua 60guccguuauc aacuugaaaa aguggcaccg agucggugcu uuu 10326620RNAArtificial SequenceSynthetic 266ccgacuucug aacgugcggu 2026721RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 267cccgacuucu gaacgugcgg u 2126820DNAArtificial SequenceSynthetic 268ccgcgggact agagggagct 2026920DNAArtificial SequenceSynthetic 269ccgacttctg aacgtgcggt 20270101RNAArtificial SequenceSynthetic 270ugcuggcgau acgcguccac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcmuuu u 101271101RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(98)..(101)modified with 2'-O-methyl phosphorothioate 271ugcuggcgau acgcguccac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcmuuu u 10127220RNAArtificial SequenceSynthetic 272ugcuggcgau acgcguccac 2027320RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 273ugcuggcgau acgcguccac 2027420DNAArtificial SequenceSynthetic 274cgctcccgct cggtggctgt 2027520DNAArtificial SequenceSynthetic 275tgctggcgat acgcgtccac 20276100RNAArtificial SequenceSynthetic 276ucggucuaug acgagcagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100277100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 277ucggucuaug acgagcagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10027820RNAArtificial SequenceSynthetic 278ucggucuaug acgagcagcg 2027920RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 279ucggucuaug acgagcagcg 2028020DNAArtificial SequenceSynthetic 280catcacgacg cgtgggtggc 2028120DNAArtificial SequenceSynthetic 281tcggtctatg acgagcagcg 20282101RNAArtificial SequenceSynthetic 282augggcaguc cuauuacagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcmuuu u 101283100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 283augggcaguc cuauuacagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10028420RNAArtificial SequenceSynthetic 284augggcaguc cuauuacagc 2028520RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 285augggcaguc cuauuacagc 2028620DNAArtificial SequenceSynthetic 286cacgacgcgt gggtggcaag 2028720DNAArtificial SequenceSynthetic 287atgggcagtc ctattacagc 20288100RNAArtificial SequenceSynthetic 288auuguucacu uguuagcccc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100289103RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(100)..(103)modified with 2'-O-methyl phosphorothioate 289auuguucacu uguuagcccc aggguuuuag agcuagaaau agcaaguuaa aauaaggcua 60guccguuauc aacuugaaaa aguggcaccg agucggugcu uuu 10329020RNAArtificial SequenceSynthetic 290auuguucacu uguuagcccc 2029120RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 291auuguucacu uguuagcccc 2029220DNAArtificial SequenceSynthetic 292tctgacggga tcgtggtttc 2029320DNAArtificial SequenceSynthetic 293attgttcact tgttagcccc 20294100RNAArtificial SequenceSynthetic 294gcugaagaac ugccucuaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100295100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 295gcugaagaac ugccucuaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10029620RNAArtificial SequenceSynthetic 296gcugaagaac ugccucuaua 2029720RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 297gcugaagaac ugccucuaua 2029820DNAArtificial SequenceSynthetic 298ccacgcgtcg tgatggtgtg 2029920DNAArtificial SequenceSynthetic 299gctgaagaac tgcctctata 20300100RNAArtificial SequenceSynthetic 300gcaggauuuc ugguugucac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100301100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 301gcaggauuuc ugguugucac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10030220RNAArtificial SequenceSynthetic 302gcaggauuuc ugguugucac 2030320RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 303gcaggauuuc ugguugucac 2030420DNAArtificial SequenceSynthetic 304ccatcacgac gcgtgggtgg 2030520DNAArtificial SequenceSynthetic 305gcaggatttc tggttgtcac 20306100RNAArtificial SequenceSynthetic 306cuccaucugu gagaagccac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100307100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 307cuccaucugu gagaagccac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10030820RNAArtificial SequenceSynthetic 308cuccaucugu gagaagccac 2030920RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 309cuccaucugu gagaagccac 2031020DNAArtificial SequenceSynthetic 310ccgtcagact cgtaggccag 2031120DNAArtificial SequenceSynthetic 311ctccatctgt gagaagccac 20312100RNAArtificial SequenceSynthetic 312ccccuaccau gacuuuauuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100313100RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioatemisc_feature(97)..(100)modified with 2'-O-methyl phosphorothioate 313ccccuaccau gacuuuauuc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 10031423RNAArtificial SequenceSynthetic 314ccccuaccau gacuuuauuc ugg 2331520RNAArtificial SequenceSyntheticmisc_feature(1)..(4)modified with 2'-O-methyl phosphorothioate 315ccccuaccau gacuuuauuc

2031620DNAArtificial SequenceSynthetic 316gtgggtggca agcgggtggt 2031720DNAArtificial SequenceSynthetic 317cccctaccat gactttattc 2031820DNAArtificial SequenceSynthetic 318ggtcatcgat gggagcaacg 2031920DNAArtificial SequenceSynthetic 319caccaccccg cgggactaga 2032020DNAArtificial SequenceSynthetic 320ggtctggcgc tcccgctcgg 2032120DNAArtificial SequenceSynthetic 321ttcacaccat cacgacgcgt 2032220DNAArtificial SequenceSynthetic 322acaccatcac gacgcgtggg 2032320DNAArtificial SequenceSynthetic 323ctacgagtct gacgggatcg 2032420DNAArtificial SequenceSynthetic 324ttgccaccca cgcgtcgtga 2032520DNAArtificial SequenceSynthetic 325gttcacacca tcacgacgcg 2032620DNAArtificial SequenceSynthetic 326cacgatcccg tcagactcgt 2032720DNAArtificial SequenceSynthetic 327acgacgcgtg ggtggcaagc 203285PRTArtificial SequenceSynthetic 328Ser Tyr Tyr Ile His1 532917PRTArtificial SequenceSynthetic 329Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Gln1 5 10 15Gly3309PRTArtificial SequenceSynthetic 330Glu Val Arg Leu Arg Tyr Phe Asp Val1 533117PRTArtificial SequenceSynthetic 331Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu1 5 10 15Ala3327PRTArtificial SequenceSynthetic 332Trp Ala Ser Thr Arg Glu Ser1 53338PRTArtificial SequenceSynthetic 333His Gln Tyr Leu Ser Ser Arg Thr1 5334118PRTArtificial SequenceSynthetic 334Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 35 40 45Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 50 55 60Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser 115335112PRTArtificial SequenceSynthetic 335Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110336246PRTArtificial SequenceSynthetic 336Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 130 135 140Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr145 150 155 160Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 165 170 175Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 180 185 190Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 195 200 205Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr225 230 235 240Thr Val Thr Val Ser Ser 245337738DNAArtificial SequenceSynthetic 337gaaatcgtcc tcacacaatc cccggggagc ctcgcagtca gtcctgggga acgagtcact 60atgagctgca aatccagtca gagtgttttt ttctcaagta gccagaagaa ctacctcgca 120tggtaccaac aaataccggg gcaatctccc cgcttgctta tatactgggc aagtacccgc 180gaatccggcg taccggatcg attcacggga tctgggtcag gtactgattt cactttgact 240atcagctctg ttcagcctga agatttggca atttactact gtcaccaata cttgagtagc 300cgaactttcg gccagggcac gaagctcgaa atcaagggcg gagggggagg ttctggtggg 360ggcggttctg gcggtggagg aagccaagta cagttgcaac agccaggggc ggaggtcgta 420aaacctgggg cgtctgtcaa gatgagctgt aaagcaagtg gatacacctt cacctcctac 480tatatacatt ggattaagca aactccgggt caggggctgg aatgggttgg cgttatatac 540cccgggaacg atgatatatc atacaaccaa aaatttcaag gcaaggcgac tctgactgcc 600gataagagta gcacaacagc ttacatgcag ctttcttccc tgaccagcga agattcagca 660gtttactact gcgctcggga agtgcgcctg cgatactttg atgtctgggg tcaaggaact 720acagttactg tatcaagc 738338507PRTArtificial SequenceSynthetic 338Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu 20 25 30Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln 35 40 45Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr65 70 75 80Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile 100 105 110Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr 115 120 125Lys Leu Glu Ile Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val145 150 155 160Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 165 170 175Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 180 185 190Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 195 200 205Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser225 230 235 240Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 245 250 255Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Ala Ala Ala Phe 260 265 270Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 275 280 285Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 290 295 300Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly305 310 315 320Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 325 330 335Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 340 345 350Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 355 360 365Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 370 375 380Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser385 390 395 400Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 405 410 415Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 420 425 430Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 435 440 445Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 450 455 460Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly465 470 475 480His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 485 490 495Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505339509PRTArtificial SequenceSynthetic 339Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu 20 25 30Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln 35 40 45Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr65 70 75 80Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile 100 105 110Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr 115 120 125Lys Leu Glu Ile Lys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val145 150 155 160Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 165 170 175Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 180 185 190Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 195 200 205Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser225 230 235 240Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 245 250 255Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Ala Ala Ala Phe 260 265 270Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 275 280 285Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 290 295 300Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly305 310 315 320Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 325 330 335Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 340 345 350Arg Asn Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 355 360 365Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 370 375 380Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys385 390 395 400Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 405 410 415Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 420 425 430Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 435 440 445Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 450 455 460Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly465 470 475 480Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 485 490 495Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 5053405PRTArtificial SequenceSynthetic 340Ser Tyr Gly Met His1 534117PRTArtificial SequenceSynthetic 341Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys1 5 10 15Gly34217PRTArtificial SequenceSynthetic 342Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly Thr Asp1 5 10 15Val34311PRTArtificial SequenceSynthetic 343Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala1 5 103447PRTArtificial SequenceSynthetic 344Asp Ala Ser Asn Arg Ala Thr1 534510PRTArtificial SequenceSynthetic 345Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr1 5 10346126PRTArtificial SequenceSynthetic 346Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly 100 105 110Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125347108PRTArtificial SequenceSynthetic 347Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105348249PRTArtificial SequenceSynthetic 348Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly 100 105 110Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 130 135 140Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala145 150 155 160Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

Leu Leu Ile Tyr Asp Ala 180 185 190Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe 210 215 220Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr Phe225 230 235 240Gly Pro Gly Thr Lys Val Asp Ile Lys 245349510PRTArtificial SequenceSynthetic 349Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val 20 25 30Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr65 70 75 80Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe 115 120 125Asn Ser Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr 130 135 140Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 165 170 175Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 180 185 190Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 195 200 205Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe 210 215 220Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu225 230 235 240Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp 245 250 255Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala 260 265 270Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro 275 280 285Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 290 295 300Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His305 310 315 320Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 340 345 350Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 355 360 365Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 370 375 380Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val385 390 395 400Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 405 410 415Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 420 425 430Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 435 440 445Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 450 455 460Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg465 470 475 480Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 485 490 495Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 510350512PRTArtificial SequenceSynthetic 350Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val 20 25 30Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys 50 55 60Gly Leu Glu Trp Val Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr65 70 75 80Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 85 90 95Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe 115 120 125Asn Ser Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr 130 135 140Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 165 170 175Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 180 185 190Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 195 200 205Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe 210 215 220Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu225 230 235 240Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp 245 250 255Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala 260 265 270Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro 275 280 285Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 290 295 300Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His305 310 315 320Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 340 345 350Cys Asn His Arg Asn Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 355 360 365Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 370 375 380Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu385 390 395 400Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 405 410 415Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 420 425 430Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 435 440 445Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 450 455 460Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg465 470 475 480Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 485 490 495Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 5103514361DNAArtificial SequenceSynthetic 351gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040aggccggaaa tcgtcctcac acaatccccg gggagcctcg cagtcagtcc tggggaacga 2100gtcactatga gctgcaaatc cagtcagagt gtttttttct caagtagcca gaagaactac 2160ctcgcatggt accaacaaat accggggcaa tctccccgct tgcttatata ctgggcaagt 2220acccgcgaat ccggcgtacc ggatcgattc acgggatctg ggtcaggtac tgatttcact 2280ttgactatca gctctgttca gcctgaagat ttggcaattt actactgtca ccaatacttg 2340agtagccgaa ctttcggcca gggcacgaag ctcgaaatca agggcggagg gggaggttct 2400ggtgggggcg gttctggcgg tggaggaagc caagtacagt tgcaacagcc aggggcggag 2460gtcgtaaaac ctggggcgtc tgtcaagatg agctgtaaag caagtggata caccttcacc 2520tcctactata tacattggat taagcaaact ccgggtcagg ggctggaatg ggttggcgtt 2580atataccccg ggaacgatga tatatcatac aaccaaaaat ttcaaggcaa ggcgactctg 2640actgccgata agagtagcac aacagcttac atgcagcttt cttccctgac cagcgaagat 2700tcagcagttt actactgcgc tcgggaagtg cgcctgcgat actttgatgt ctggggtcaa 2760ggaactacag ttactgtatc aagcagtgct gctgcctttg tcccggtatt tctcccagcc 2820aaaccgacca cgactcccgc cccgcgccct ccgacacccg ctcccaccat cgcctctcaa 2880cctcttagtc ttcgccccga ggcatgccga cccgccgccg ggggtgctgt tcatacgagg 2940ggcttggact tcgcttgtga tatttacatt tgggctccgt tggcgggtac gtgcggcgtc 3000cttttgttgt cactcgttat tactttgtat tgtaatcaca ggaatcgctc aaagcggagt 3060aggttgttgc attccgatta catgaatatg actcctcgcc ggcctgggcc gacaagaaaa 3120cattaccaac cctatgcccc cccacgagac ttcgctgcgt acaggtcccg agtgaagttt 3180tcccgaagcg cagacgctcc ggcatatcag caaggacaga atcagctgta taacgaactg 3240aatttgggac gccgcgagga gtatgacgtg cttgataaac gccgggggag agacccggaa 3300atggggggta aaccccgaag aaagaatccc caagaaggac tctacaatga actccagaag 3360gataagatgg cggaggccta ctcagaaata ggtatgaagg gcgaacgacg acggggaaaa 3420ggtcacgatg gcctctacca agggttgagt acggcaacca aagatacgta cgatgcactg 3480catatgcagg ccctgcctcc cagataataa taaaatcgct atccatcgaa gatggatgtg 3540tgttggtttt ttgtgtgtgg agcaacaaat ctgactttgc atgtgcaaac gccttcaaca 3600acagcattat tccagaagac accttcttcc ccagcccagg taagggcagc tttggtgcct 3660tcgcaggctg tttccttgct tcaggaatgg ccaggttctg cccagagctc tggtcaatga 3720tgtctaaaac tcctctgatt ggtggtctcg gccttatcca ttgccaccaa aaccctcttt 3780ttactaagaa acagtgagcc ttgttctggc agtccagaga atgacacggg aaaaaagcag 3840atgaagagaa ggtggcagga gagggcacgt ggcccagcct cagtctctcc aactgagttc 3900ctgcctgcct gcctttgctc agactgtttg ccccttactg ctcttctagg cctcattcta 3960agccccttct ccaagttgcc tctccttatt tctccctgtc tgccaaaaaa tctttcccag 4020ctcactaagt cagtctcacg cagtcactca ttaacccacc aatcactgat tgtgccggca 4080catgaatgca ccaggtgttg aagtggagga attaaaaagt cagatgaggg gtgtgcccag 4140aggaagcacc attctagttg ggggagccca tctgtcagct gggaaaagtc caaataactt 4200cagattggaa tgtgttttaa ctcagggttg agaaaacagc taccttcagg acaaaagtca 4260gggaagggct ctctgaagaa atgctacttg aagataccag ccctaccaag ggcagggaga 4320ggaccctata gaggcctggg acaggagctc aatgagaaag g 43613524367DNAArtificial SequenceSynthetic 352gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040aggccggaaa tcgtcctcac acaatccccg gggagcctcg cagtcagtcc tggggaacga 2100gtcactatga gctgcaaatc cagtcagagt gtttttttct caagtagcca gaagaactac 2160ctcgcatggt accaacaaat accggggcaa tctccccgct tgcttatata ctgggcaagt 2220acccgcgaat ccggcgtacc ggatcgattc acgggatctg ggtcaggtac tgatttcact 2280ttgactatca gctctgttca gcctgaagat ttggcaattt actactgtca ccaatacttg 2340agtagccgaa ctttcggcca gggcacgaag ctcgaaatca agggcggagg gggaggttct 2400ggtgggggcg gttctggcgg tggaggaagc caagtacagt tgcaacagcc aggggcggag 2460gtcgtaaaac ctggggcgtc tgtcaagatg agctgtaaag caagtggata caccttcacc 2520tcctactata tacattggat taagcaaact ccgggtcagg ggctggaatg ggttggcgtt 2580atataccccg ggaacgatga tatatcatac aaccaaaaat ttcaaggcaa ggcgactctg 2640actgccgata agagtagcac aacagcttac atgcagcttt cttccctgac cagcgaagat 2700tcagcagttt actactgcgc tcgggaagtg cgcctgcgat actttgatgt ctggggtcaa 2760ggaactacag ttactgtatc aagcagtgct gctgcctttg tcccggtatt tctcccagcc 2820aaaccgacca cgactcccgc cccgcgccct ccgacacccg ctcccaccat cgcctctcaa 2880cctcttagtc ttcgccccga ggcatgccga cccgccgccg ggggtgctgt tcatacgagg 2940ggcttggact tcgcttgtga tatttacatt tgggctccgt tggcgggtac gtgcggcgtc 3000cttttgttgt cactcgttat tactttgtat tgtaatcaca ggaatcgcaa acggggcaga 3060aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 3120gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgcgagtg 3180aagttttccc gaagcgcaga cgctccggca tatcagcaag gacagaatca gctgtataac 3240gaactgaatt tgggacgccg cgaggagtat gacgtgcttg ataaacgccg ggggagagac 3300ccggaaatgg ggggtaaacc ccgaagaaag aatccccaag aaggactcta caatgaactc 3360cagaaggata agatggcgga ggcctactca gaaataggta tgaagggcga acgacgacgg 3420ggaaaaggtc acgatggcct ctaccaaggg ttgagtacgg caaccaaaga tacgtacgat 3480gcactgcata tgcaggccct gcctcccaga taataataaa atcgctatcc atcgaagatg 3540gatgtgtgtt ggttttttgt gtgtggagca acaaatctga ctttgcatgt gcaaacgcct 3600tcaacaacag cattattcca gaagacacct tcttccccag cccaggtaag ggcagctttg 3660gtgccttcgc aggctgtttc cttgcttcag gaatggccag gttctgccca gagctctggt 3720caatgatgtc taaaactcct ctgattggtg gtctcggcct tatccattgc caccaaaacc 3780ctctttttac taagaaacag tgagccttgt tctggcagtc cagagaatga cacgggaaaa 3840aagcagatga agagaaggtg gcaggagagg gcacgtggcc cagcctcagt ctctccaact

3900gagttcctgc ctgcctgcct ttgctcagac tgtttgcccc ttactgctct tctaggcctc 3960attctaagcc ccttctccaa gttgcctctc cttatttctc cctgtctgcc aaaaaatctt 4020tcccagctca ctaagtcagt ctcacgcagt cactcattaa cccaccaatc actgattgtg 4080ccggcacatg aatgcaccag gtgttgaagt ggaggaatta aaaagtcaga tgaggggtgt 4140gcccagagga agcaccattc tagttggggg agcccatctg tcagctggga aaagtccaaa 4200taacttcaga ttggaatgtg ttttaactca gggttgagaa aacagctacc ttcaggacaa 4260aagtcaggga agggctctct gaagaaatgc tacttgaaga taccagccct accaagggca 4320gggagaggac cctatagagg cctgggacag gagctcaatg agaaagg 4367353484PRTArtificial SequenceSynthetic 353Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser145 150 155 160Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser225 230 235 240Val Thr Val Ser Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala 245 250 255Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 260 265 270Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 275 280 285Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 290 295 300Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser305 310 315 320Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser 325 330 335Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 340 345 350Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 355 360 365Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg385 390 395 400Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 420 425 430Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 435 440 445Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 450 455 460Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala465 470 475 480Leu Pro Pro Arg354487PRTArtificial SequenceSynthetic 354Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Ala Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Tyr Gly Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser 130 135 140Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser145 150 155 160Lys Ser Val Ser Thr Ser Gly Tyr Ser Phe Met His Trp Tyr Gln Gln 165 170 175Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu 180 185 190Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 210 215 220Tyr Cys Gln His Ser Arg Glu Val Pro Trp Thr Phe Gly Gln Gly Thr225 230 235 240Lys Val Glu Ile Lys Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro 245 250 255Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu305 310 315 320Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Lys Arg Gly 325 330 335Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn385 390 395 400Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His465 470 475 480Met Gln Ala Leu Pro Pro Arg 485355487PRTArtificial SequenceSynthetic 355Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Thr Leu Thr Asn Tyr 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Leu Pro Tyr Asn Asp Leu Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Trp Asp Trp Asp Gly Phe Phe Asp Pro Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu 130 135 140Ser Val Ser Pro Gly Glu Arg Ala Ser Ile Ser Cys Arg Ala Ser Gln145 150 155 160Ser Leu Val His Ser Asn Gly Asn Thr His Leu His Trp Tyr Gln Gln 165 170 175Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ser Val Ser Asn Arg 180 185 190Phe Ser Glu Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205Phe Thr Leu Thr Ile Ser Ser Val Glu Ser Glu Asp Phe Ala Val Tyr 210 215 220Tyr Cys Ser Gln Thr Ser His Ile Pro Tyr Thr Phe Gly Gly Gly Thr225 230 235 240Lys Leu Glu Ile Lys Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro 245 250 255Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280 285Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu305 310 315 320Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Lys Arg Gly 325 330 335Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn385 390 395 400Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His465 470 475 480Met Gln Ala Leu Pro Pro Arg 485356486PRTArtificial SequenceSynthetic 356Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 130 135 140Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr145 150 155 160Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 165 170 175Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 180 185 190Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 195 200 205Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr225 230 235 240Thr Val Thr Val Ser Ser Ser Ala Ala Ala Phe Val Pro Val Phe Leu 245 250 255Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 275 280 285Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu305 310 315 320Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Ser Lys 325 330 335Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 340 345 350Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 355 360 365Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu385 390 395 400Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met465 470 475 480Gln Ala Leu Pro Pro Arg 485357488PRTArtificial SequenceSynthetic 357Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45Ser Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95Tyr Leu Ser Ser Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 130 135 140Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr145 150 155 160Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val 165 170 175Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe 180 185 190Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 195 200 205Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 210 215 220Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Gln Gly Thr225 230 235 240Thr Val Thr Val Ser Ser Ser Ala Ala Ala Phe Val Pro Val Phe Leu 245 250 255Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 275 280 285Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu305 310 315 320Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Lys Arg 325 330 335Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu385 390 395 400Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425

430Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu465 470 475 480His Met Gln Ala Leu Pro Pro Arg 485358489PRTArtificial SequenceSynthetic 358Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly 100 105 110Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 130 135 140Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala145 150 155 160Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala 180 185 190Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe 210 215 220Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr Phe225 230 235 240Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala Ala Ala Phe Val Pro 245 250 255Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315 320Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn 325 330 335Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 340 345 350Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 355 360 365Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser 370 375 380Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu385 390 395 400Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala465 470 475 480Leu His Met Gln Ala Leu Pro Pro Arg 485359491PRTArtificial SequenceSynthetic 359Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly 100 105 110Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly 115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val 130 135 140Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala145 150 155 160Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala Trp 165 170 175Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala 180 185 190Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser 195 200 205Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe 210 215 220Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr Phe225 230 235 240Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala Ala Ala Phe Val Pro 245 250 255Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly305 310 315 320Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn 325 330 335Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr385 390 395 400Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr465 470 475 480Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 49036020DNAArtificial SequenceSynthetic 360ctgaacgtgc ggtgggatcg 2036110DNAArtificial SequenceSynthetic 361ctgaacgtgc 1036215DNAArtificial SequenceSynthetic 362ctgaacgtgg gatcg 1536321DNAArtificial SequenceSynthetic 363ctgaacgtgc cggtgggatc g 2136419DNAArtificial SequenceSynthetic 364ctgaacgtgg gtgggatcg 1936510DNAArtificial SequenceSynthetic 365ggtgggatcg 1036618DNAArtificial SequenceSynthetic 366ctgaacgtgg tgggatcg 1836716DNAArtificial SequenceSynthetic 367ctgaacggtg ggatcg 1636812DNAArtificial SequenceSynthetic 368ctggtgggat cg 1236921DNAArtificial SequenceSynthetic 369ctgaacgtgc aggtgggatc g 2137018DNAArtificial SequenceSynthetic 370ctgaacgtgc gtggatcg 1837120DNAArtificial SequenceSynthetic 371gatacgcgtc cacaggacga 2037219DNAArtificial SequenceSynthetic 372gatacgcgtc acaggacga 1937321DNAArtificial SequenceSynthetic 373gatacgcgtc ccacaggacg a 2137418DNAArtificial SequenceSynthetic 374gatacgcgtc caggacga 1837516DNAArtificial SequenceSynthetic 375gatacgcaca ggacga 1637614DNAArtificial SequenceSynthetic 376gatacacagg acga 1437713DNAArtificial SequenceSynthetic 377gatacgcgtc cga 1337812DNAArtificial SequenceSynthetic 378gatacgcgtc ga 1237917DNAArtificial SequenceSynthetic 379gatacgcgtc aggacga 1738012DNAArtificial SequenceSynthetic 380gatacaggac ga 1238117DNAArtificial SequenceSynthetic 381gatacgccac aggacga 1738210DNAArtificial SequenceSynthetic 382gatacgcgtc 1038321DNAArtificial SequenceSynthetic 383gatacgcgtc acacaggacg a 2138419DNAArtificial SequenceSynthetic 384gatacgctgc acaggacga 1938514DNAArtificial SequenceSynthetic 385gatacgcagg acga 1438613DNAArtificial SequenceSynthetic 386acgcacagga cga 1338720DNAArtificial SequenceSynthetic 387atgacgagca gcggggtctg 2038821DNAArtificial SequenceSynthetic 388atgacgagca agcggggtct g 2138917DNAArtificial SequenceSynthetic 389atgacgagcg gggtctg 1739018DNAArtificial SequenceSynthetic 390atgacgaagc ggggtctg 1839114DNAArtificial SequenceSynthetic 391atgagcgggg tctg 1439222DNAArtificial SequenceSynthetic 392atgacgagca aagcggggtc tg 2239313DNAArtificial SequenceSynthetic 393atgacggggt ctg 1339420DNAArtificial SequenceSynthetic 394catgacttta ttctggaaga 2039513DNAArtificial SequenceSynthetic 395catgactgga aga 1339616DNAArtificial SequenceSynthetic 396catgacttct ggaaga 1639717DNAArtificial SequenceSynthetic 397catgactttc tggaaga 1739821DNAArtificial SequenceSynthetic 398catgacttta tttctggaag a 2139921DNAArtificial SequenceSynthetic 399catgacttta attctggaag a 2140018DNAArtificial SequenceSynthetic 400catgactttt ctggaaga 1840111DNAArtificial SequenceSynthetic 401catctggaag a 1140219DNAArtificial SequenceSynthetic 402catgactttt tctggaaga 1940319DNAArtificial SequenceSynthetic 403catgacttta tctggaaga 1940413DNAArtificial SequenceSynthetic 404catgacttta aga 1340510DNAArtificial SequenceSynthetic 405ttctggaaga 1040618DNAArtificial SequenceSynthetic 406catgacttta ctggaaga 1840720DNAArtificial SequenceSynthetic 407gtcctattac agctggggca 2040818DNAArtificial SequenceSynthetic 408gtcctattag ctggggca 1840919DNAArtificial SequenceSynthetic 409gtcctattaa gctggggca 1941017DNAArtificial SequenceSynthetic 410gtcctatagc tggggca 1741116DNAArtificial SequenceSynthetic 411gtcctaagct ggggca 1641215DNAArtificial SequenceSynthetic 412gtcctagctg gggca 1541317DNAArtificial SequenceSynthetic 413gtcctattac tggggca 1741414DNAArtificial SequenceSynthetic 414gtccagctgg ggca 1441521DNAArtificial SequenceSynthetic 415gtcctattac cagctggggc a 2141617DNAArtificial SequenceSynthetic 416gtcctattgc tggggca 1741719DNAArtificial SequenceSynthetic 417gtcctattac gctggggca 1941811DNAArtificial SequenceSynthetic 418gtcctggggc a 1141920DNAArtificial SequenceSynthetic 419acttgttagc cccagggcca 2042018DNAArtificial SequenceSynthetic 420acttgttagc cagggcca 1842119DNAArtificial SequenceSynthetic 421acttgttagc ccagggcca 1942217DNAArtificial SequenceSynthetic 422acttgttagc agggcca 1742312DNAArtificial SequenceSynthetic 423acttgttagc ca 1242414DNAArtificial SequenceSynthetic 424acttgttagg gcca 1442521DNAArtificial SequenceSynthetic 425acttgttagc ccccagggcc a 2142617DNAArtificial SequenceSynthetic 426acttgttccc agggcca 1742710DNAArtificial SequenceSynthetic 427cccagggcca 1042818DNAArtificial SequenceSynthetic 428acttgttacc cagggcca 1842915DNAArtificial SequenceSynthetic 429acttgcccag ggcca 1543016DNAArtificial SequenceSynthetic 430acttgtccca gggcca 1643110DNAArtificial SequenceSynthetic 431accagggcca 1043213DNAArtificial SequenceSynthetic 432acttgcaggg cca 1343313DNAArtificial SequenceSynthetic 433acttgttagc cca 1343420DNAArtificial SequenceSynthetic 434aactgcctct atatggtgtg 2043521DNAArtificial SequenceSynthetic 435aactgcctct tatatggtgt g 2143618DNAArtificial SequenceSynthetic 436aactgcctat atggtgtg 1843718DNAArtificial SequenceSynthetic 437aactgcctct atggtgtg 1843819DNAArtificial SequenceSynthetic 438aactgcctca tatggtgtg 1943916DNAArtificial SequenceSynthetic 439aactgtatat ggtgtg 1644017DNAArtificial SequenceSynthetic 440aactgctata tggtgtg 1744114DNAArtificial SequenceSynthetic 441aactatatgg tgtg 1444219DNAArtificial SequenceSynthetic 442aactgcctta tatggtgtg 1944310DNAArtificial SequenceSynthetic 443aactgcctct 1044411DNAArtificial SequenceSynthetic 444tatatggtgt g 1144511DNAArtificial SequenceSynthetic 445aactgcctct a 1144619DNAArtificial SequenceSynthetic 446aactgcctct tatggtgtg

1944710DNAArtificial SequenceSynthetic 447aactggtgtg 1044820DNAArtificial SequenceSynthetic 448ttctggttgt cacaggtgga 2044921DNAArtificial SequenceSynthetic 449ttctggttgt tcacaggtgg a 2145010DNAArtificial SequenceSynthetic 450ttctggtgga 1045110DNAArtificial SequenceSynthetic 451ttcaggtgga 1045218DNAArtificial SequenceSynthetic 452ttctggttca caggtgga 1845322DNAArtificial SequenceSynthetic 453ttctggttgt ttcacaggtg ga 2245411DNAArtificial SequenceSynthetic 454ttctggttgg a 1145521DNAArtificial SequenceSynthetic 455ttctggttgt ccacaggtgg a 2145613DNAArtificial SequenceSynthetic 456ttccacaggt gga 1345719DNAArtificial SequenceSynthetic 457ttctggtttc acaggtgga 1945819DNAArtificial SequenceSynthetic 458ttctggttgc acaggtgga 1945921DNAArtificial SequenceSynthetic 459ttctggttgt acacaggtgg a 2146010DNAArtificial SequenceSynthetic 460ttctggttga 1046119DNAArtificial SequenceSynthetic 461ttctggttgt acaggtgga 1946220DNAArtificial SequenceSynthetic 462tgtgagaagc cacaggaagt 2046310DNAArtificial SequenceSynthetic 463tgtgagaagt 1046419DNAArtificial SequenceSynthetic 464tgtgagaagc acaggaagt 1946521DNAArtificial SequenceSynthetic 465tgtgagaagc ccacaggaag t 2146611DNAArtificial SequenceSynthetic 466tgtgaggaag t 1146718DNAArtificial SequenceSynthetic 467tgtgagaagc caggaagt 1846814DNAArtificial SequenceSynthetic 468tgtgagaagg aagt 1446910DNAArtificial SequenceSynthetic 469cacaggaagt 1047021DNAArtificial SequenceSynthetic 470tgtgagaagc acacaggaag t 2147117DNAArtificial SequenceSynthetic 471tgtgagaagc aggaagt 1747214DNAArtificial SequenceSynthetic 472tgtgcacagg aagt 1447311DNAArtificial SequenceSynthetic 473ccacaggaag t 1147417DNAArtificial SequenceSynthetic 474tgtgagacac aggaagt 1747515DNAArtificial SequenceSynthetic 475tgtgagacag gaagt 1547618DNAArtificial SequenceSynthetic 476tgtgagaaca caggaagt 1847710DNAArtificial SequenceSynthetic 477tgtgagaagc 1047822DNAArtificial SequenceSynthetic 478tgtgagaagc cacacaggaa gt 22

Claims

1. A population of genetically engineered T cells, comprising: (i) a disrupted Regnase-1 (Reg1) gene; and/or (ii) a disrupted Transforming Growth Factor Beta Receptor II (TGFBRII) gene.

2. The population of genetically engineered T cells of claim 1, which comprises both (i) and (ii).

3. The population of genetically engineered T cells of claim 1, wherein the population of genetically engineered T cells, as compared to non-engineered T cell counterparts, has one or more of the following features: (a) improved cell growth activity; (b) enhanced persistence; (c) reduced T cell exhaustion; (d) resistant to inhibitory effects induced by TGF-.beta.; (e) enhanced cell killing capacity; and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby.

4. The population of genetically engineered T cells of claim 1, wherein the T cells are further engineered to express a chimeric antigen receptor (CAR).

5. The population of genetically engineered T cells of claim 1, wherein the disrupted Reg1 gene is genetically edited in exon 2 and/or exon 4.

6. The population of genetically engineered T cells of claim 1, wherein the disrupted TGFBRII gene is genetically edited in exon 1, exon 2, exon 3, exon 4, or exon 5, optionally wherein the disrupted TGFBRII gene is genetically edited in exon 4 or exon 5.

7. The population of genetically engineered T cells of claim 1, wherein the disrupted Reg1 gene, the disrupted TGFBRII gene, or both are 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 comprises a guide RNA (gRNA) targeting a site in the Reg1 gene that comprises a nucleotide sequence listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: SEQ ID NO: 320, 322, 323, and 327.

9. The population of genetically engineered T cells of claim 8, wherein the gRNA targeting the Reg1 gene comprises a nucleotide sequence listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: 24, 32, 36, or 52.

10. The population of genetically engineered T cells of claim 9, wherein the disrupted Reg1 gene comprises a nucleotide sequence selected from those listed in Table 31, 33, 34, or 38.

11. The population of genetically engineered T cells of claim 7, wherein the CRISPR/Cas-mediated gene editing system comprises a guide RNA (gRNA) targeting a site in the TGFBRII gene that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 269, 275, 281, 287, 293, 299, 305, 311, and 317.

12. The population of genetically engineered T cells of claim 11, wherein the gRNA targeting the TGFBRII gene comprises a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 266, 272, 278, 284, 290, 296, 302, 308, and 314.

13. The population of genetically engineered T cells of claim 7, wherein the gRNA further comprises a scaffold sequence.

14. The population of genetically engineered T cells of claim 13, wherein: the gRNA targeting the Reg1 gene comprises the nucleotide sequence of any of SEQ ID NO: 22, 30, 34, or 50; and/or the gRNA targeting the TGFBRII gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 270, 300, 306, or 312.

15. The population of genetically engineered T cells of claim 1, which further comprises: (iii) a disrupted T cell receptor alpha chain constant region (TRAC) gene, (iv) a disrupted beta-2-microglobulin (.beta.2M) gene, (v) a disrupted CD70 gene, or (vi) a combination of any of (iii)-(v).

16. The population of genetically engineered T cells of claim 15, wherein the T cells comprise a disrupted T cell receptor alpha chain constant region (TRAC) gene.

17. The population of genetically engineered T cells of claim 15, wherein the T cells comprise a disrupted beta-2-microglobulin (.beta.2M) gene.

18. The population of genetically engineered T cells of claim 15, wherein the T cells comprise a disrupted CD70 gene.

19. The population of genetically engineered T cells of claim 15, wherein the disrupted TRAC gene, the disrupted .beta.2M gene, and/or the disrupted CD70 gene is genetically edited by one or more CRISPR/Cas-mediated gene editing system

20. The population of genetically engineered T cells of claim 4, wherein the T cells comprise a nucleic acid encoding the CAR, and wherein the nucleic acid is inserted in the genome of the T cells.

21. The population of genetically engineered T cells of claim 20, wherein the nucleic acid encoding the CAR is inserted in the disrupted Reg1 gene, the disrupted TGFBRII gene, the disrupted TRAC gene, the disrupted .beta.2M, or the disrupted CD70 gene.

22. The population of genetically engineered T cells of claim 21, wherein the nucleic acid encoding the CAR is inserted in the disrupted TRAC gene, and optionally wherein the nucleic acid encoding the CAR replaces the deleted fragment comprising SEQ ID NO: 69 in the TRAC gene.

23. The population of genetically engineered T cells of claim 15, wherein the disrupted Reg1 gene comprises a nucleotide sequence listed in Table 31, 33, 34, or 38, the disrupted TRAC gene comprises a nucleotide sequence listed in Table 24; the disrupted .beta.2M comprises a nucleotide sequence listed in Table 25 and/or the disrupted CD70 gene comprises a nucleotide sequence listed in Table 26.

24. The population of genetically engineered T cells of claim 4, 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 CD3.zeta..

25. The population of genetically engineered T cells of claim 24, wherein the tumor antigen is CD19, BCMA, CD70, CD33, or PTK7.

26. The population of genetically engineered T cells of claim 24, wherein the CAR binds CD19 (anti-CD19 CAR) and wherein the extracellular antigen binding domain in the anti-CD19 CAR is a single chain variable fragment (scFv) that binds CD19 (anti-CD19 scFv).

27. The population of genetically engineered T cells of claim 24, wherein the anti-CD19 scFv comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 125; optionally wherein the V.sub.H comprises the amino acid sequence of SEQ ID NO: 124 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 125.

28. The population of genetically engineered T cells of claim 27, wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 120.

29. The population of genetically engineered T cells of claim 28, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:353.

30. The population of genetically engineered T cells of claim 24, wherein the CAR binds CD70 (anti-CD70 CAR) and wherein extracellular antigen binding domain in the anti-CD70 CAR is a single chain variable fragment (scFv) that binds CD70 (anti-CD70 scFv).

31. The population of genetically engineered T cells of claim 30, wherein the anti-CD70 scFv comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 144; optionally wherein the V.sub.H comprises the amino acid sequence of SEQ ID NO: 143 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 144.

32. The population of genetically engineered T cells of claim 31, wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 140 or 142.

33. The population of genetically engineered T cells of claim 32, wherein the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO:354.

34. The population of genetically engineered T cells of claim 24, wherein the CAR binds BCMA (anti-BCMA CAR) and wherein the extracellular antigen binding domain in the anti-BCMA CAR is a single chain variable fragment (scFv) that binds BCMA (anti-BCMA CAR).

35. The population of genetically engineered T cells of claim 34, wherein the anti-BCMA scFv comprises (i) a heavy chain variable region (V.sub.H) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 149; and (ii) a light chain variable region (V.sub.L) that comprises the same light chain CDRs as those in SEQ ID NO: 150; optionally wherein the V.sub.H comprises the amino acid sequence of SEQ ID NO: 149 and the V.sub.L comprises the amino acid sequence of SEQ ID NO: 150.

36. The population of genetically engineered T cells of claim 35, wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 148.

37. The population of genetically engineered T cells of claim 36, wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 146 or SEQ ID NO:355.

38. 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.

39. The population of genetically engineered T cells of claim 1, wherein the genetically engineered T cells show cytokine-dependent growth.

40. 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; (b) genetically editing the Reg1 gene and/or the TGFBRII gene; and (c) producing the population of genetically engineered T cells having disrupted Reg1 gene and/or the TGFBRII gene.

41. The method of claim 40, wherein step (b) comprises genetically editing both the Reg1 gene and the TGFBRII gene.

42. The method of claim 40, wherein step (b) is performed by one or more CRISPR/Cas-mediated gene editing systems.

43. The method of claim 40, wherein step (b) is performed by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the Reg1 gene.

44. The method of claim 43, wherein the gRNA targeting the Reg1 gene is specific to an exon of the Reg1 gene selected from the group consisting of exon 2 and exon 4.

45. The method of claim 44, wherein the gRNA targeting the Reg1 gene comprises a spacer having a nucleotide sequence of SEQ ID NO: 24, 32, 36, or 52.

46. The method of claim 40, wherein step (b) is performed by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the TGFBRII gene.

47. The method of claim 46, wherein the gRNA targeting the TGFBRII gene is specific to an exon of the TGFBRII gene selected from the group consisting of exon 1, exon 2, exon 3, exon 4, and exon 5, preferably wherein the gRNA targeting the TGFBRII gene is specific to exon 4 or exon 5.

48. The method of claim 47, wherein the gRNA targeting the TGFBRII gene comprises a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 272, 300, 308, and 314.

49. The method of claim 40, wherein the gRNA targeting the Reg1 gene and the gRNA targeting the TGFBRII gene further comprises a scaffold sequence.

50. The method of claim 49, wherein the gRNA targeting the Reg1 gene comprises a nucleotide sequence of SEQ ID NO: 22, 30, 34, or 50; and/or wherein the gRNA targeting the TGFBRII gene comprises a nucleotide sequence of SEQ ID NOs: 270, 300, 306, or 312.

51. The method of claim 40, wherein the plurality of T cells in step (a) comprises one or more of the following genetic modifications: (i) engineered to express a chimeric antigen receptor (CAR); (ii) has a disrupted T cell receptor alpha chain constant region (TRAC) gene; (iii) has a disrupted .beta.2M gene; and (iv) has a disrupted CD70 gene.

52. The method of claim 40, wherein the method further comprises: (i) delivering to the T cells a nucleic acid encoding a chimeric antigen receptor (CAR); (ii) genetically editing a TRAC gene to disrupt its expression; (iii) genetically editing a .beta.2M gene to disrupt its expression; (iv) genetically editing a CD70 gene to disrupt its expression; or (v) a combination thereof.

53. The method of claim 52, wherein one or more of (i)-(iv) are performed by one or more CRISPR/Cas-mediated gene editing system comprising one or more RNA-guided nucleases and one or more gRNAs targeting the TRAC gene, the .beta.2M gene, and/or the CD70 gene.

54. The method of claim 53, wherein the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 59.

55. The method of claim 53, wherein the gRNA targeting the .beta.2M gene comprises the nucleotide sequence of SEQ ID NO: 63.

56. The method of claim 53, wherein the gRNA targeting the CD70 gene comprises the nucleotide sequence of SEQ ID NO: 55.

57. The method of claim 56, wherein the method comprises delivering to the T cells one or more ribonucleoprotein particles (RNP), comprising the RNA-guided nuclease, one or more of the gRNAs, and the nucleic acid encoding the CAR.

58. The method of claim 50, wherein the RNA-guided nuclease is a Cas9 nuclease, which optionally is a S. pyogenes Cas9 nuclease.

59. The method of claim 50, wherein the nucleic acid encoding the CAR is in an AAV vector.

60. The method of claim 50, wherein the nucleic acid encoding the CAR comprises a left homology arm and a right homology arm flanking the nucleotide sequence encoding the CAR; and wherein the left homology arm and the right homology arm are homologous to a genomic locus in the T cells, allowing for insertion of the nucleic acid into the genomic locus.

61. The method of claim 60, wherein the genomic locus is in the Reg1 gene, the TGFBRII gene, the TRAC gene, the .beta.2M gene, or the CD70 gene; optionally wherein the genomic locus is the TRAC gene.

62. The method of claim 50, wherein the method comprising disrupting the TRAC gene by a CRISPR/Cas-mediated gene editing system comprising a gRNA comprising nucleotide sequence of SEQ ID NO: 47 and the nucleic acid encoding the CAR is inserted at the site targeted by the gRNA.

63. The method of claim 40, wherein the method comprising delivering to the T cells a nucleic acid encoding a CAR, which is specific to CD70, and genetically editing the CD70 gene to disrupt its expression.

64. The method of claim 40, wherein the T cells of step (a) are derived from primary T cells of one or more human donors.

65. A population of genetically engineered T cells, which is prepared by a method of claim 40.

66. A method for eliminating undesired cells in a subject, the method comprising administering to a subject in need thereof a population of genetically engineered T cells set forth in claim 4.

67. The method of claim 66, wherein the undesired cells are cancer cells, which optionally are hematopoietic cancer cells or solid tumor cells.

68. The method of claim 66, wherein the undesired cells are CD19.sup.+, BCMA.sup.+, CD70.sup.+, CD33.sup.+, or PTK7.sup.+.

69. A guide RNA (gRNA) targeting a Reg1 gene, comprising a nucleotide sequence specific to a fragment in exon 1, exon2, exon3, or exon4, optionally in exon 2 or exon 4 of the Reg1 gene.

70. The gRNA of claim 69, wherein the gRNA comprises a spacer listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: 24, 32, 36, and 52.

71. The gRNA of claim 69, wherein the gRNA further comprises a scaffold sequence.

72. The gRNA of claim 69, wherein the gRNA comprises one or more modified nucleotides.

73. The gRNA of claim 72, wherein the gRNA comprises one or more 2'-O-methyl phosphorothioate residues at the 5' and/or 3' terminus of the gRNA.

74. The gRNA of claim 73, which comprises the nucleotide sequence listed in Table 22, which optionally is any of SEQ ID NOs: 22, 23, 30, 31, 34, 35, 50, or 51.

75. A guide RNA (gRNA) targeting a TGFBRII gene, comprising a nucleotide sequence specific to a fragment in exon 1, exon 2, exon 3, exon 4, or exon5 of the TGFBRII gene, preferably wherein the gRNA comprises a nucleotide sequence specific to exon 4 or exon 5 of the TGFBRII gene.

76. The gRNA of claim 75, wherein the gRNA comprises a spacer listed in Table 32, which optionally has the nucleotide sequence selected from the group consisting of SEQ ID NOs: 272, 302, 308, and 314.

77. The gRNA of claim 76, wherein the gRNA further comprises a scaffold sequence.

78. The gRNA of claim 75, wherein the gRNA comprises one or more modified nucleotides.

79. The gRNA of claim 78, wherein the gRNA comprises one or more 2'-O-methyl phosphorothioate residues at the 5' and/or 3' terminus of the gRNA.

80. The gRNA of claim 79, which comprises the nucleotide sequence listed in Table 39, optionally wherein the gRNA comprises the nucleotide sequence of any one of SEQ ID NO: SEQ ID NOs: 270, 271, 300, 301, 306, 307, 312, and 313.