HK40055048A - Anti-ptk7 immune cell cancer therapy - Google Patents

Description

anti-PTK 7 immune cell cancer therapy

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 62/756,638 filed on 7/11/2018 and U.S. provisional application No. 62/910,586 filed on 4/10/2019, which are hereby incorporated by reference in their entirety.

Electronically-submitted material is incorporated by reference

The sequence listing as part of this disclosure will be filed concurrently with the specification as a text file. The name of the text file containing the sequence listing is "CT 111_ seqliking. txt", which was created on day 11/7 in 2019 and has a size of 119,776 bytes. The subject matter of the sequence listing is incorporated herein by reference in its entirety.

Background

Chimeric Antigen Receptor (CAR) T cell therapy uses genetically modified T cells to more specifically and efficiently target and kill cancer cells. After the T cells are collected from the blood, the cells are engineered to contain CARs on their surface. The CAR can be introduced into T cells using CRISPR/Cas9 gene editing technology. When these allogeneic CAR T cells are injected into a patient, the recipient enables the T cells to kill cancer cells.

Disclosure of Invention

A variety of tumor-associated antigen targets have entered clinical trials, primarily using logical selection that expression in cancer tissues should be selective compared to normal tissues to avoid toxicity (Morgan, R.blood 2013; 122(2): 3392-3394). However, PTK7 did not meet this criterion due to its apparent overexpression in normal tissues, including lung, smooth muscle, stomach, kidney and bladder. Thus, PTK7 has not been considered a live CAR T cell target. Nevertheless, the data provided herein unexpectedly demonstrate that animals are actually resistant to therapy by anti-PTK 7CAR T cells, and that these anti-PTK 7CAR T cells effectively and selectively kill cells expressing PTK 7.

Some aspects of the disclosure provide an engineered T cell comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds PTK 7. In some embodiments, the engineered T cell further comprises a disrupted T cell receptor alpha chain constant region (TRAC) gene. For example, the TRAC gene may be disrupted by insertion of a nucleic acid encoding a CAR. In some embodiments, the engineered T cell further comprises a disrupted β -2-microglobulin (β 2M) gene.

In some embodiments, the extracellular domain of the CAR comprises an anti-PTK 7 antibody. In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv). In some embodiments, the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, or 82. In some embodiments, the anti-PTK 7 scFv comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs 55, 69, 76, or 83 and/or a VL comprising the amino acid sequence of any one of SEQ ID NOs 56, 70, 77, or 84. In some embodiments, the anti-PTK 7 scFv comprises a VH comprising the CDR amino acid sequences of SEQ ID NO 57, SEQ ID NO 58, and/or SEQ ID NO 59; and/or the anti-PTK 7 scFv comprises a VL sequence comprising the CDR amino acid sequences of SEQ ID NO:60, SEQ ID NO:61 and/or SEQ ID NO: 62. In some embodiments, the anti-PTK 7 scFv comprises a VH comprising the CDR amino acid sequences of SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO: 87; and/or the anti-PTK 7 scFv comprises a VL sequence comprising the CDR amino acid sequences of SEQ ID NO:88, SEQ ID NO:89 and/or SEQ ID NO: 90.

In some embodiments, the CAR comprises a CD3 ζ cytoplasmic signaling domain. In some embodiments, the CAR comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

In some embodiments, the TRAC gene comprises a nucleotide sequence encoding the Left Homology Arm (LHA) and/or the Right Homology Arm (RHA) within any of SEQ ID NOs 63, 64, 71, 78 or 91, or the nucleotide sequence of SEQ ID NOs 92 or 100, and/or wherein the CAR is encoded by the nucleotide sequence of any of SEQ ID NOs 49, 51, 65, 72, 79 or 112. In some embodiments, the disrupted β 2M gene comprises at least one nucleotide sequence selected from any one of SEQ ID NOs 9-14.

In some aspects, also provided herein are engineered T cells comprising: (i) a disrupted TRAC gene; (ii) a disrupted β 2M gene; and (iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment.

In some aspects, also provided herein are cell populations comprising engineered T cells, wherein the engineered T cells comprise: (i) a disrupted TRAC gene; (ii) a disrupted β 2M gene; and (iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment. In other aspects, provided herein are cell populations comprising engineered T cells, wherein the engineered T cells comprise: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112 and encodes a CAR of SEQ ID NO 50, 52, 66, 73, or 80; and (ii) a disrupted β 2M gene.

In some aspects, also provided herein is a population of engineered T cells (e.g., comprising a nucleic acid encoding an anti-PTK 7 CAR), wherein at least 25% or at least 50% of the engineered T cells of the population express the CAR. For example, at least 70% of the engineered T cells of the population express the CAR.

In some embodiments, at least 25% of the engineered T cells of the population express the CAR after at least 7 days or at least 14 days of in vitro proliferation.

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels of a T Cell Receptor (TCR) protein. For example, at least 90% of the engineered T cells of the population may not express detectable levels of TCR protein.

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels of β 2M protein. For example, at least 70% of the engineered T cells of the population may not express detectable levels of β 2M protein.

In some embodiments, when co-cultured in vitro with a population of cancer cells expressing PTK7, the engineered T cells of the population induce cell lysis of at least 50% of the cancer cells of the population. For example, the engineered T cells of the population may induce cell lysis of at least 70%, at least 80%, or at least 90% of the cancer cells of the population. In some embodiments, when co-cultured in vitro with a population of cancer cells expressing PTK7, the engineered T cells of the population induce cell lysis of at least 10%, at least 25%, or at least 50% of the cancer cells of the population. In some embodiments, the engineered T cells of the population secrete IFN γ when co-cultured in vitro with the population of cancer cells. In some embodiments, the ratio of engineered T cells to cancer cells is 1:1 to 2: 1. The cancer cells can be, for example, sarcoma cells or breast cancer cells. Other cancer cells can be targeted. In some embodiments, the cancer cells can be breast cancer cells, ovarian cancer cells, small cell lung cancer cells, and/or colon cancer cells

In some embodiments, the proliferative capacity of the engineered T cells of the population is within 10% of the proliferative capacity of the control cells. In still other embodiments, the population of T cells does not induce toxicity in a subject when administered in vivo to the subject.

Other aspects of the disclosure provide a method comprising administering a population of engineered T cells described herein. In some embodiments, the percentage of body weight of the subject after 5-10 days of administration is within 10% of the initial body weight of the subject, wherein the initial body weight of the subject is the body weight of the subject at the time of administration. In some embodiments, the subject is a human subject. In some embodiments, the subject has cancer. The cancer may express, for example, PTK 7. In various embodiments, the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma (liver hepatocholangiocarcinoma), osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct cancer.

Other aspects of the disclosure provide a method of producing an engineered T cell, the method comprising (a) delivering to the T cell: an RNA-guided nuclease (RNA-guided nuclease), a gRNA targeting the TRAC gene, and a vector comprising a donor template comprising a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds PTK7, wherein the nucleic acid encoding the CAR is flanked by the left and right homology arms of the TRAC gene, and (b) generating an engineered T cell. In some embodiments, the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID No. 18 or 19, or the nucleotide sequence targeting SEQ ID No. 40. In a related embodiment, there is provided a method wherein the nucleic acid encoding the CAR is flanked by the left and right homology arms of the TRAC gene.

In some embodiments, the method further comprises delivering a gRNA targeting the β 2M gene to the T cell. In some embodiments, the gRNA targeting the β 2M gene comprises the nucleotide sequence of SEQ ID No. 20 or 21, or the nucleotide sequence targeting SEQ ID No. 41.

In some embodiments, the RNA-guided nuclease is Cas9 nuclease, optionally streptococcus pyogenes (s. pyogenes) Cas9 nuclease. In some embodiments, the extracellular domain of the CAR is an anti-PTK 7 antibody, optionally an anti-PTK 7 single chain variable fragment (scFv).

In some embodiments, the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78, or 91.

In some embodiments, the CAR comprises a nucleotide sequence encoding any one of SEQ ID NOs 50, 52, 66, 73, or 80.

In some embodiments, methods of production are provided wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Region (CDR) and the same light chain variable domain (VL) CDR as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84. In one embodiment, methods are provided wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody. In yet another embodiment, the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75 or 82.

In one embodiment, the aforementioned method of generating is provided, wherein the CAR comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain. In a related embodiment, the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

In some embodiments, the foregoing methods of generating are provided, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78, or 91. In still other embodiments, the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs 49, 51, 65, 72, 79, or 112.

Drawings

FIG. 1 shows that PTK7 expresses normal human tissue.

Fig. 2A and 2B show PTK7 expression in human diseased and normal tissues.

Figure 3 shows the prevalence of PTK7 patients in solid tumors by Immunohistochemistry (IHC).

FIG. 4 shows the binding affinity of PTK7/CTX181 Ab in human and murine cell lines.

Fig. 5A and 5B show PTK7 expression in frozen normal tissue panels (FDA standard) from mice (fig. 5A) and humans (fig. 5B).

FIG. 6 shows PTK7 expression in frozen murine embryonic development arrays.

FIG. 7 includes a graph showing TRAC^-/β2M^-anti-PTK 7CAR⁺Efficient multigene editing in T cells. Four different anti-PTK 7CAR are shown⁺Editing phenotype of constructs (PTK7-4, PTK7-7, PTK7-13, and PTK7-17) as measured by FACS (left panel) and anti-PTK 7CAR as measured by immunohistochemistry (left panel)⁺Expression (right panel).

Figure 8 includes a graph showing that PTK7CAR T cells with a CD28 co-stimulatory domain (PTK7-4) are more potent than PTK7CAR T cells with a 4-1BB co-stimulatory domain (PTK7-4 b).

FIGS. 9A-9C include graphs showing TRAC^-/β2M^-anti-PTK 7CAR⁺Cell killing of T cells against adherent sarcoma cell lines A-204 (FIG. 9A) and Saos-2 (FIG. 9B) and the breast cancer cell line MCF7 (FIG. 9C). Cell ratios of 2:1 and 1:1 (CAR T cells: target cancer cells) were used.

FIGS. 10A-10C show PTK7CAR T cell specificity in PTK7-KO Saos2 cells (FIG. 10A) and A498 cells overexpressing PTK7 (FIG. 10B). FIG. 10C shows PTK7 cell surface expression in Saos2 cells, PTK7-KO Saos2 cells, A498 cells, and A498 cells overexpressing PTK 7.

FIGS. 11A-11C show the expression of: in vitro potency of PTK7CAR T cells with a tendency to express patterns in breast (fig. 11A), pancreatic (fig. 11B) and lung (NSCLC) (fig. 11C) cancers.

FIG. 12 includes graphs showing TRAC after gene editing compared to controls^-/β2M^-anti-PTK 7CAR⁺Cell proliferation of T cells.

FIGS. 13A-13B include graphs showing the persistence of multiple gene editing in T cells. The editing phenotype as measured by FACS remained consistent from day 7 (fig. 13A) to day 14 (fig. 13B) post-editing. Figures 13C-13D show the editing phenotype of PTK7-4CAR-T cells measured by FACS at day 7 post-editing, presented as FACS plots (figure 13C) and graphs (figure 13D).

FIGS. 14A-14F include graphs showing TRAC when in contact with MCF7 (FIGS. 14A-14C) and Saos-2 (FIGS. 14D-14F) cells^-/β2M^-anti-PTK 7CAR⁺T cell ratio TRAC^-/β2M^-anti-CD 19CAR⁺T cells are more efficient in cell killing and secrete higher levels of IFN γ.

Figure 15 shows that anti-PTK 7CAR T cells were equally effective in vitro in human and murine cell lines.

FIG. 16 includes graphs showing TRAC^-/β2M^-anti-PTK 7CAR⁺T cell treated mice showed minimal weight loss up to 10 days after treatment/injection.

FIG. 17 shows the cell surface expression level of PTK7 in human cancer cell lines.

Figures 18A-18C show the efficacy of anti-PTK 7CAR T cells in various in vivo xenograft models.

Figures 19A-19C show subsequent blind in vivo studies to evaluate PTK7CAR T cell efficacy in ovarian (figure 19A), colon and SCLC (figure 19B) and breast (figure 19C) cancer types.

Figure 20 shows the effect of PTK7CAR T cell treatment on body weight in an Hs-766T pancreatic tumor xenograft mouse model.

Detailed Description

PTK7 cancer antigen

In some embodiments, T cells of the present disclosure are engineered with a Chimeric Antigen Receptor (CAR) designed to target PTK 7. Protein tyrosine kinase 7(PTK7), also known as colon cancer kinase 4(CCK4), is a receptor protein tyrosine kinase involved in atypical Wnt signaling and comprising an extracellular domain. PTK7 lacks detectable catalytic tyrosine kinase activity; however, it does include signal transduction activity and is presumed to play a role in cell adhesion. PTK7 is further believed to be a marker of tumor progression in cancer because it is expressed in cancer cell lines (e.g., colon and breast cancer cell lines).

Thus, in some embodiments, the T cells of the disclosure are engineered to express a CAR comprising an anti-PTK 7 antibody (e.g., anti-PTK 7 scFv). In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 scFv encoded by the sequence of any one of SEQ ID NOs 53, 67, 74, or 81. In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 scFv encoded by the sequence of any one of SEQ ID NOs 113. In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 scFv comprising the sequence of any one of SEQ ID NOs 54, 68, 75, or 82. In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 scFv comprising a VH comprising the amino acid sequence of any one of SEQ ID NOs 55, 69, 76, or 83. In some embodiments, the anti-PTK 7 antibody is an anti-PTK 7 scFv comprising a VL comprising the amino acid sequence of any one of SEQ ID NOs 56, 70, 77, or 84. In some embodiments, the CAR comprising the anti-PTK 7 antibody is encoded by the sequence of any one of SEQ ID NOs 49, 51, 65, 72, or 79. In some embodiments, a CAR comprising an anti-PTK 7 antibody is encoded by a sequence comprising a nucleic acid that is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112. In some embodiments, a CAR comprising an anti-PTK 7 antibody is encoded by the sequence of any one of SEQ ID NOs 112. In some embodiments, a CAR comprising an anti-PTK 7 antibody comprises the sequence of any one of SEQ ID NOs 50, 52, 66, 73, or 80. In some embodiments, the CAR comprising an anti-PTK 7 antibody comprises an anti-PTK 7 antibody as described in US 9,102,738 or US 9,409,995.

Multiple gene editing

In some embodiments, an engineered T cell of the disclosure comprises more than one gene edit, e.g., in more than one gene. For example, the engineered T cells may comprise a disrupted CD70 gene, a disrupted T cell receptor alpha chain constant region (TRAC) gene, a disrupted beta-2-microglobulin (beta 2M) gene, a disrupted programmed cell death-1 (PD-1 or PDCD1) gene, or any combination of two or more of the foregoing disrupted genes. In some embodiments, the engineered T cells comprise a disrupted TRAC gene, a disrupted β 2M gene, and a disrupted CD70 gene. In some embodiments, the engineered T cell comprises a disrupted TRAC gene, a disrupted β 2M gene, and a disrupted PD-1 gene. In some embodiments, the engineered T cell comprises a disrupted TRAC gene, a disrupted β 2M gene, a disrupted CD70 gene, and a disrupted PD-1 gene.

It is understood that gene disruption includes gene modification by gene editing (e.g., insertion or deletion of one or more nucleotides using CRISPR/Cas gene editing). In some embodiments, the disrupted gene is a gene that does not encode a functional protein. In some embodiments, a cell comprising a disrupted gene does not express (e.g., does not express on the cell surface) a detectable level of a protein encoded by the gene (e.g., by an antibody, e.g., by flow cytometry). A cell that does not express detectable levels of a protein may be referred to as a knockout cell. For example, if β 2M protein cannot be detected on the cell surface using an antibody that specifically binds to β 2M protein, a cell with β 2M gene editing can be considered a β 2M knockout cell.

In some embodiments, provided herein are cell populations in which a percentage of cells have been edited (e.g., the β 2M gene is edited), resulting in a percentage of cells not expressing a particular gene and/or protein. In some embodiments, at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 85%) of the cells of the gene-edited cell population are β 2M knockout cells. In some embodiments, at least 50% of the cells (e.g., T cells) of the population do not express detectable levels of β 2M protein. In some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells of the gene-edited population of cells can be β 2M knockout cells.

Methods of generating genomic deletions in cells using CRISPR-Cas gene editing techniques (e.g., to knock out genes in cells) are known (Bauer DE et al vis. exp. [ visualization experiments ] 2015; 95; e 52118).

TRAC Gene editing

In some embodiments, the engineered T cell comprises a disrupted TRAC gene. This disruption results in loss of TCR function and renders the engineered T cells non-alloreactive and suitable for allogeneic transplantation, thereby minimizing the risk of graft-versus-host disease. In some embodiments, expression of endogenous TRAC genes is eliminated to prevent graft versus host responses. In some embodiments, disruption of TRAC gene expression is produced by knocking-in a Chimeric Antigen Receptor (CAR) into the TRAC gene (e.g., using an adeno-associated virus (AAV) vector and a donor template). In some embodiments, disruption of TRAC gene expression is produced by a gRNA targeting the TRAC genomic region. In some embodiments, the genomic deletion of the TRAC gene is generated by knocking-in a Chimeric Antigen Receptor (CAR) into the TRAC gene (e.g., using an AAV vector and donor template). In some embodiments, disruption of TRAC gene expression is generated using grnas that target the TRAC genomic region and knock-in a Chimeric Antigen Receptor (CAR) into the TRAC gene.

Non-limiting examples of modified and unmodified TRAC gRNA sequences that may be used to generate genomic disruption in the TRAC gene as provided herein are listed in Table 4 (e.g., SEQ ID NOS: 18 and 19). See also international application number PCT/US 2018/032334 filed on 11/5/2018, which is incorporated herein by reference. Other gRNA sequences may be designed using the TRAC gene sequence located on chromosome 14 (GRCh 38: chromosome 14: 22,547,506-22,552, 154; Ensembl; ENSG 00000277734). In some embodiments, grnas targeting the TRAC genomic region create an insertion (indel) in the TRAC gene, disrupting expression of mRNA or protein.

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels of T Cell Receptor (TCR) 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 population may not express detectable levels of TCR 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 population of engineered T cells do not express detectable levels of TCR surface protein.

In some embodiments, the gRNA targeting the TRAC genomic region produces an insertion in a TRAC gene comprising at least one nucleotide sequence selected from the following sequences in table 1:

table 1.

In some embodiments, the engineered T cells comprise a deletion in the TRAC gene relative to unmodified T cells. In some embodiments, the engineered T cells comprise a deletion of 15-30 base pairs in the TRAC gene relative to unmodified T cells. In some embodiments, the engineered T cell comprises a deletion of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in the gene in the TRAC relative to an unmodified T cell. In some embodiments, the engineered T cells comprise a deletion of more than 30 base pairs in the TRAC gene relative to unmodified T cells. In some embodiments, the engineered T cells comprise a deletion of 20 base pairs in the TRAC gene relative to unmodified T cells. In some embodiments, the engineered T cell comprises a deletion of SEQ ID NO:104(AGAGCAACAGTGCTGTGGCC) in the TRAC gene relative to an unmodified T cell. In some embodiments, the engineered T cell comprises a deletion in the TRAC gene comprising SEQ ID NO:104(AGAGCAACAGTGCTGTGGCC) relative to an unmodified T cell. In some embodiments, the engineered T cells comprise a deletion of SEQ ID NO 40 in the TRAC gene relative to unmodified T cells. In some embodiments, the engineered T cells comprise a deletion in the TRAC gene comprising SEQ ID NO:40 relative to unmodified T cells.

Beta 2M Gene editing

In some embodiments, the engineered T cell comprises a disrupted β 2M gene. β 2M is a common (invariant) component of the MHC I complex. Disruption of its expression by gene editing will prevent the host from responding to therapeutic allogeneic T cells, resulting in increased persistence of allogeneic T cells. In some embodiments, expression of the endogenous β 2M gene is eliminated to prevent graft versus host response.

Non-limiting examples of modified and unmodified β 2M gRNA sequences that can be used to generate genomic disruption in the β 2M gene as provided herein are listed in table 4 (e.g., SEQ ID NOs: 20 and 21). See also international application number PCT/US 2018/032334 filed on 11/5/2018, which is incorporated herein by reference. Other gRNA sequences can be designed using the sequence of the β 2M gene located on chromosome 15 (GRCh38 coordinates: chromosome 15: 44,711,477-44,718, 877; Ensembl: ENSG 00000166710).

In some embodiments, grnas targeting the β 2M genomic region create an insertion in the β 2M gene that disrupts mRNA or protein expression.

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels of β 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 engineered T cells of the population may not express detectable levels of β 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 population of engineered T cells do not express detectable levels of β 2M surface protein.

In some embodiments, the edited β 2M gene comprises at least one nucleotide sequence selected from the sequences in table 2 below.

Table 2.

PD-1 gene editing

PD-1 is an immune checkpoint molecule that is upregulated in activated T cells and is used to suppress or terminate T cell responses. Disruption of PD-1 by gene editing may result in a more durable and/or efficient therapeutic T cell response and/or reduced immunosuppression in the subject. In some embodiments, the engineered T cell comprises a disrupted PD-1 gene. In some embodiments, expression of the endogenous PD-1 gene is eliminated to enhance the anti-tumor efficacy of the CAR T cells of the present disclosure.

Non-limiting examples of modified and unmodified PD-1gRNA sequences that can be used to generate genomic deletions in the PD-1 gene as provided herein are listed in Table 4 (e.g., SEQ ID NOS: 22 and 23). See also international application number PCT/US 2018/032334 filed on 11/5/2018, which is incorporated herein by reference. Additional gRNA sequences can be designed using the PD-1 gene sequence located on chromosome 2 (GRCh38 coordinates: chromosome 2: 241,849,881-241,858, 908; Ensembl: ENSG 00000188389).

In some embodiments, grnas targeting a PD-1 genomic region create an insertion in the PD-1 gene that disrupts expression of PD-1mRNA or protein.

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels of PD-1 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 engineered T cells of the population may not express detectable levels of PD-1 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 population of engineered T cells do not express detectable levels of PD-1 surface protein.

CD70 gene editing

Cluster of differentiation 70(CD70) is a member of the tumor necrosis factor superfamily, and its expression is restricted to activated T and B lymphocytes as well as mature dendritic cells. CD70 has also been detected on hematological tumors and cancers. CD70 is involved in the survival of tumor cells and regulatory T cells by interacting with its ligand, CD 27. Disruption of CD70 by gene editing may increase cell expansion and decrease cell exhaustion. In some embodiments, the engineered T cell comprises a disrupted CD70 gene. In some embodiments, expression of the endogenous CD70 gene is eliminated to enhance the anti-tumor efficacy of the CAR T cells of the present disclosure. In some embodiments, grnas targeting the CD70 genomic region create an insertion in or around the CD70 gene, disrupting expression of CD70 mRNA and/or protein.

Non-limiting examples of modified and unmodified CD70 gRNA sequences that can be used to generate genomic disruption in the CD70 gene as provided herein are listed in table 4 (e.g., SEQ ID NOs: 24-27). Other gRNA sequences may be designed using the CD70 gene sequence located on chromosome 19 (GRCh38 coordinates: chromosome 19: 6,583,183-6,604,103; Ensembl: ENSG 00000125726).

In some embodiments, at least 50% of the engineered T cells of the population do not express detectable levels 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%, or at least 95% of the engineered T cells of the population may not express detectable levels 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%, or 90% -100% of the population of engineered T cells do not express detectable levels of CD70 surface protein.

Cell phenotype

In some embodiments, one or more gene edits within the population of cells results in a phenotype associated with an alteration in cell proliferation capacity, cell depletion, cell viability, cell lysis capacity (e.g., increased cytokine production and/or release), or any combination thereof.

In some embodiments, the engineered T cells of the disclosure exhibit at least 20% greater cell proliferative capacity relative to control T cells. For example, an engineered T cell may exhibit at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 90% greater cell proliferative capacity relative to a control cell. In some embodiments, an engineered T cell of the disclosure exhibits a greater cell proliferative capacity of 20% -100%, 20% -90%, 20% -80%, 20% -70%, 20% -60%, 20% -50%, 30% -100%, 30% -90%, 30% -80%, 30% -70%, 30% -60%, 30% -50%, 40% -100%, 40% -90%, 40% -80%, 40% -70%, 40% -60%, 40% -50%, 50% -100%, 50% -90%, 50% -80%, 50% -70%, or 50% -60% relative to a control T cell.

In some embodiments, the engineered T cells of the disclosure exhibit at least a 20% increase in cell viability relative to control cells. For example, the cell viability of an engineered T cell of the present disclosure may exhibit an increase of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 90% relative to a control cell. In some embodiments, the cell viability of an engineered T cell of the present disclosure exhibits an increase of 20% -100%, 20% -90%, 20% -80%, 20% -70%, 20% -60%, 20% -50%, 30% -100%, 30% -90%, 30% -80%, 30% -70%, 30% -60%, 30% -50%, 40% -100%, 40% -90%, 40% -80%, 40% -70%, 40% -60%, 40% -50%, 50% -100%, 50% -90%, 50% -80%, 50% -70%, or 50% -60% relative to a control cell.

In some embodiments, engineered T cells of the disclosure exhibit at least a 20% increase in cytolytic capacity relative to control cells (killing at least 20% more of the target cells). For example, the cell lytic capacity of an engineered T cell of the disclosure may exhibit an increase of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 90% relative to a control cell. In some embodiments, the engineered T cells of the disclosure exhibit an increase in cell lytic capacity relative to a control cell of 20% -100%, 20% -90%, 20% -80%, 20% -70%, 20% -60%, 20% -50%, 30% -100%, 30% -90%, 30% -80%, 30% -70%, 30% -60%, 30% -50%, 40% -100%, 40% -90%, 40% -80%, 40% -70%, 40% -60%, 40% -50%, 50% -100%, 50% -90%, 50% -80%, 50% -70%, or 50% -60%. For example, the engineered T cells secrete cytokines (e.g., IL-2 and/or IFN- γ) at a level that is at least 2-fold (e.g., at least 3-fold, at least 4-fold, or at least 5-fold) greater than the level of cytokines secreted by control T cells.

In some embodiments, the control T cell is an engineered T cell (e.g., a genetically edited T cell). In some embodiments, the control T cell is an engineered T cell comprising a disrupted TRAC gene, a nucleic acid encoding a CAR (e.g., an anti-PTK 7 CAR) inserted into the TRAC gene, and/or a disrupted β 2M gene. In some embodiments, the control T cell is an unedited T cell.

Gene editing method

Gene editing (including genome editing) is a type of genetic engineering in which one or more nucleotides/one or more nucleic acids are inserted, deleted and/or substituted in a DNA sequence, for example, in the genome of a target cell. Targeted gene editing enables insertions, deletions, and/or substitutions at preselected sites in the genome of a target cell (e.g., in a target gene or target DNA sequence). When the sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of one or more nucleotides/one or more nucleic acids, the endogenous gene comprising the affected sequence may be knocked out or knocked down as a result of the sequence modification. Thus, targeted editing can be used to disrupt endogenous gene expression. "Targeted integration" refers to a process that involves the insertion of one or more exogenous sequences, with or without deletion of endogenous sequences at the insertion site. Targeted integration can result from targeted gene editing when a donor template comprising the exogenous sequence is present.

Targeted editing can be achieved by nuclease-independent or nuclease-dependent methods. In nuclease-independent targeted editing methods, homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide introduced into the endogenous sequence by the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce a deletion, insertion or substitution of nucleotides in the endogenous sequence.

Alternatively, nuclease-dependent methods can achieve a higher frequency of targeted editing by specifically introducing double-strand breaks (DSBs) with specific rare-cutting nucleases (e.g., endonucleases). This nuclease-dependent targeted editing also utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ), which occurs in response to DSBs. NHEJ repair of DNA typically results in random insertion or deletion (insertions) of small numbers of endogenous nucleotides. Repair can also be performed by Homology Directed Repair (HDR) as compared to NHEJ mediated repair. When a donor template is present that contains exogenous genetic material flanked by a pair of homology arms, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

Useful endonucleases capable of introducing specificity and targeting DSBs include, but are not limited to, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas9 nucleases (CRISPR/Cas 9; clustered regularly interspaced short palindromic repeats related 9). In addition, the DICE (Dual integrase cassette exchange) system using phiC31 and Bxb1 integrase can also be used for targeted integration.

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 in the zinc finger binding domain, the structure of which is stabilized by coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. Designed zinc finger domains are domains that do not exist in nature, whose design/composition comes primarily from reasonable criteria, e.g., substitution rules and the application of computer algorithms for processing information in databases storing existing ZFP design information and binding data. See, e.g., U.S. patent 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. The zinc finger domain of choice is a domain not found in nature, which results primarily from empirical processes such as phage display, interaction traps, or hybridization selection. ZFNs are described in more detail in U.S. patent No. 7,888,121 and U.S. patent No. 7,972,854. The most widely known example of ZFNs is the fusion of FokI nucleases with zinc finger DNA binding domains.

TALENs are targeted nucleases that comprise 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 a TAL effector protein that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus flavobacterium (Xanthomonas) during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription on these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on the effector variable number of incomplete 34 amino acid repeats, which comprise a polymorphism at a selected repeat position called Repeat Variable Diresidue (RVD). TALENs are described in more detail in U.S. patent application No. 2011/0145940. The most recognized example of a TALEN in the art is a fusion polypeptide of a FokI nuclease and a TAL effector DNA binding domain.

Other examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and W β/SPBc/TP901-1, whether used alone or in combination.

Other non-limiting examples of targeted nucleases include naturally occurring nucleases and recombinant nucleases, such as CRISPR/Cas9, restriction endonucleases, meganuclease homing endonucleases and the like.

CRISPR-Cas9 gene editing

The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been reused as an RNA-guided DNA targeting platform for gene editing. It relies on the DNA nuclease Cas9 and two non-coding RNAs (criprpr RNA (crrna) and transactivating RNA (tracrrna)) to target cleavage of DNA.

crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex by watson-crick base pairing, typically with a20 nucleotide (nt) sequence in the target DNA. Altering the 5' 20nt sequence in the crRNA can target the CRISPR-Cas9 complex to a specific locus. If the target sequence is followed by a specific short DNA sequence (sequence NGG) as a Protospacer Adjacent Motif (PAM), the CRISPR-Cas9 complex binds only to DNA sequences comprising a match to the first 20nt sequence of the crRNA, i.e. a single guide rna (sgrna).

The TracrRNA hybridizes to the 3' end of the crRNA to form an RNA duplex structure that binds to Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

Once the CRISPR-Cas9 complex binds to the DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cut one of the DNA strands upstream of the PAM site, leaving a Double Strand Break (DSB) where both strands of DNA terminate in base pairs (blunt ends).

After the CRISPR-Cas9 complex binds to DNA at a particular target site and forms a site-specific DSB, the next key step is to repair the DSB. Cells use two major DNA repair pathways to repair DSBs: non-homologous end joining (NHEJ) and Homologous Directed Repair (HDR).

NHEJ is a robust repair mechanism that exhibits high activity in most cell types, including non-dividing cells. NHEJ is error prone and often results in deletions or additions of between one and several hundred nucleotides at the site of the DSB, although such modifications are typically <20 nt. The resulting insertions and deletions (insertions) can disrupt the coding or non-coding regions of the gene. Alternatively, HDR uses long stretches of homologous donor DNA provided endogenously or exogenously to repair DSBs with high fidelity. HDR is only effective 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 a spontaneous repair.

In some embodiments, the Cas9 (CRISPR-associated protein 9) endonuclease is from streptococcus pyogenes, although other Cas9 homologs can be used. It is understood that as provided herein, a wild-type Cas9 can be used or a modified version of Cas9 can be used (e.g., an evolved version of Cas9, or a Cas9 ortholog or variant). In some embodiments, Cas9 may be substituted with another RNA-guided endonuclease such as Cpf1 (of class II CRISPR/Cas system).

Guide RNA

The present disclosure provides genome-targeted nucleic acids that can direct the activity of a polypeptide of interest (e.g., a site-directed polypeptide) to a particular target sequence within a target nucleic acid. The nucleic acid that targets the genome can be RNA. The genome-targeted RNA is referred to herein as a "guide RNA" or "gRNA". The guide RNA comprises at least one spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat. In type II systems, the gRNA also contains a second RNA called a tracrRNA sequence. In type II guide rnas (grnas), CRISPR repeats and tracrRNA sequences hybridize to each other to form duplexes. In type V guide rna (grna), crRNA forms a duplex. In both systems, the duplex binds to the site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. In some embodiments, the genome-targeted nucleic acid provides target specificity to the complex due to its association with the site-directed polypeptide. Thus, the genome-targeted nucleic acid directs the activity of the site-directed polypeptide.

As understood by one of ordinary skill in the art, each guide RNA is designed to include a spacer sequence that is complementary to its genomic target sequence. See Jinek et al, Science [ Science ],337, 816-.

In some embodiments, the genome-targeted nucleic acid is a bimolecular guide RNA. In some embodiments, the genome-targeted nucleic acid is a single-molecule guide RNA.

The bimolecular guide RNA comprises two-stranded RNA. The first strand comprises in the 5 'to 3' direction an optional spacer extension sequence, a spacer sequence and a minimal CRISPR repeat. The second strand comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat), a 3' tracrRNA sequence, and optionally a tracrRNA extension sequence.

The single-molecule guide rna (sgrna) in a type II system comprises in the 5' to 3' direction an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension sequence may comprise elements that contribute additional functions (e.g., stability) to the guide RNA. A single-molecule guide linker connects the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins.

Single molecule guide RNAs (referred to as "sgrnas" or "grnas") in type V systems comprise a minimal CRISPR repeat and a spacer sequence in the 5 'to 3' direction.

The sgRNA can comprise a spacer sequence of 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable length spacer sequence of 17-30 nucleotides at the 5' end of the sgRNA sequence (see table 3).

The sgRNA can comprise no uracil at the 3' end of the sgRNA sequence. The sgRNA can comprise one or more uracils at the 3' end of the sgRNA sequence. For example, the sgRNA can include 1 uracil (U) at the 3' end of the sgRNA sequence. The sgRNA can comprise 2 uracils (UUs) at the 3' end of the sgRNA sequence. The sgRNA can comprise 3 uracils (UUUs) at the 3' end of the sgRNA sequence. The sgRNA can comprise 4 uracils (uuuuuu) at the 3' end of the sgRNA sequence. The sgRNA can comprise 5 uracils (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 6 uracils (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 7 uracils (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 8 uracils (UUUUUUUU) at the 3' end of the sgRNA sequence.

The sgrnas can be unmodified or modified. For example, a modified sgRNA can comprise one or more 2' -O-methyl phosphorothioate nucleotides.

Table 3.

By way of example, guide RNAs or other smaller RNAs for use in the CRISPR/Cas/Cpf1 system can be readily synthesized by chemical methods, as shown below and described in the art. With the continued development of chemical synthesis procedures, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to be more challenging as the length of the polynucleotide increases significantly beyond about a hundred nucleotides. One method for producing RNA of greater length is to produce two or more molecules linked together. Longer RNAs (such as those encoding Cas9 or Cpf1 endonuclease) are easier to enzymatically produce. As described in the art, various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNA, for example, modifications that enhance stability, reduce the likelihood or extent of an innate immune response, and/or enhance other attributes.

Spacer sequences

The gRNA comprises a spacer sequence. A spacer sequence is a sequence (e.g., a20 nucleotide sequence) that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target nucleic acid of interest. In some embodiments, the spacer sequence is 15 to 30 nucleotides. In some embodiments, the spacer sequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence is 20 nucleotides.

The "target sequence" is adjacent to the PAM sequence and is a sequence modified by an RNA-guided nuclease (e.g., Cas 9). A "target nucleic acid" is a double-stranded molecule: one strand comprises the target sequence and is referred to as the "PAM strand", and the other complementary strand is referred to as the "non-PAM strand". One skilled in the art recognizes that the gRNA spacer sequence hybridizes to the reverse complement of the target 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:104), the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3' (SEQ ID NO: 105). Spacers of grnas interact in a sequence-specific manner with a target nucleic acid of interest via hybridization (i.e., base pairing). Thus, the nucleotide sequence of the spacer varies depending on the target sequence of the target nucleic acid of interest.

In the CRISPR/Cas system herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid that is 5' to the PAM of the Cas9 enzyme used in the system. The spacer may be a perfect match to the target sequence or may have a mismatch. Each Cas9 enzyme has a specific PAM sequence, allowing the enzyme to recognize the target DNA. For example, streptococcus pyogenes recognizes a PAM in a target nucleic acid comprising the sequence 5' -NRG-3', where R comprises a or G, where N is any nucleotide and N is immediately 3' to the target nucleic acid sequence targeted by the spacer sequence.

In some embodiments, the target nucleic acid sequence comprises 20 nucleotides. In some embodiments, the target nucleic acid comprises less than 20 nucleotides. In some embodiments, the target nucleic acid comprises more than 20 nucleotides. In some embodiments, the target nucleic acid comprises at least: 5. 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid comprises at most: 5. 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence comprises 20 bases immediately 5' to the first nucleotide of the PAM. For example, in the list comprising 5' -NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid comprises a sequence corresponding to a plurality of N, wherein N is any nucleotide, and the underlined NRG sequence is streptococcus pyogenes PAM.

Table 4 and PCT/US 2018/032334 filed 2018, 5, 11, provide non-limiting examples of grnas that may be used as described herein.

TABLE 4 gRNA sequences/target sequences

2' -O-methyl phosphorothioate residue

Chimeric Antigen Receptor (CAR) T cells

Chimeric antigen receptors refer to artificial immune cell receptors that are engineered to recognize and bind antigens expressed by tumor cells. Typically, the CAR is designed for T cells and is a chimera of the signaling domain of the T Cell Receptor (TCR) complex with an antigen recognition domain (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) (Enblad et al, Human Gene Therapy [ Human Gene Therapy ]. 2015; 26(8): 498-) 505). T cells expressing CARs are referred to as CAR T cells. CARs have the ability to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner. non-MHC restricted antigen recognition confers CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs do not advantageously dimerize with endogenous T Cell Receptor (TCR) alpha and beta chains.

There are four generations of CARs, each containing different components. The first generation CARs linked an antibody-derived scFv to the CD3 ζ (ζ or z) intracellular signaling domain of the T cell receptor via a hinge domain and a transmembrane domain. Second generation CARs incorporate additional domains (e.g., CD28, 4-1BB (41BB) or ICOS) to provide costimulatory signals. The third generation CARs contain two costimulatory domains fused to the TCR CD3 zeta chain. The third generation costimulatory domain can include, for example, a combination of CD3 ζ, CD27, CD28, 4-1BB, ICOS, or OX 40. In some embodiments, the CAR contains an extracellular domain, a transmembrane domain, and an intracellular domain (e.g., CD3 ζ) having one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3Z and/or a costimulatory molecule (Maude et al, Blood [ Blood ] 2015; 125(26): 4017. sup., [ Kakarla and Gottschalk. sup. [ Cancer J. [ 2014; 20(2): 151. sup. ] 155).

CARs often differ in functional characteristics. The CD3 zeta signaling domain of the T cell receptor (when involved) activates and induces T cell proliferation but may lead to anergy (lack of response in the body's defense mechanisms leading to direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered non-responsive when they are non-responsive to a particular antigen. The addition of a costimulatory domain in second generation CARs improved the replicative capacity and persistence of the modified T cells. Similar anti-tumor effects were observed in vitro with either CD28 or 4-1BB CARs, but in-vivo studies indicate that 4-1BB CARs can produce excellent proliferation and/or persistence. Clinical trials have shown that both of these second generation CARs are able to induce massive T cell proliferation in vivo, but CARs containing the 4-1BB co-stimulatory domain can appear for longer periods of time. Third generation CARs combine multiple signaling domains (co-stimulation) to enhance potency.

In some embodiments, the chimeric antigen receptor is a first generation CAR. In other embodiments, the chimeric antigen receptor is a second generation CAR. In still other embodiments, the chimeric antigen receptor is a third generation CAR.

In some embodiments, the CAR comprises an extracellular (extracellular) domain comprising an antigen binding domain (e.g., an antibody, such as an scFv), a transmembrane domain, and a cytoplasmic (intracellular) domain.

Extracellular domain

The extracellular domain is a region of the CAR that is exposed to extracellular fluid, and in some embodiments, the CAR comprises an antigen binding domain, and optionally a signal peptide, a spacer domain, and/or a hinge domain. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising the VL and VH of an immunoglobulin linked to a short linker peptide. In some embodiments, the linker includes hydrophilic residues, i.e., multiple stretches of glycine and serine for flexibility, and multiple stretches of glutamic acid and lysine for increased solubility. Single chain variable fragments (scFv) are not actually fragments of antibodies, but rather fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, linked to a short chain linker peptide of ten to about 25 amino acids. The linker is typically glycine rich for flexibility and serine or threonine for solubility, and may link the N-terminus of VH with the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the specificity of the original immunoglobulin is retained by this protein. Non-limiting examples of VH and VL protein sequences that can be used to generate anti-PTK 7 scFv can include the amino acid sequences of SEQ ID NO:55, 69, 76 or 83(VH) and SEQ ID NO:56, 70, 77 or 83 (VL). In some embodiments, the scFv of the present disclosure is humanized. In other embodiments, the scFv is fully human. In still other embodiments, the scFv is a chimera (e.g., a chimera of mouse and human sequences). In some embodiments, the scFv is an anti-PTK 7 scFv (specifically binding PTK 7). Non-limiting examples of anti-PTK 7 scFv proteins that can be used as provided herein can include the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, 82. Other scFv proteins may be used.

The signal peptide can enhance the antigen specificity of CAR binding. The signal peptide may be derived from an antibody (such as but not limited to CD8), and an epitope tag (such as but not limited to GST or FLAG). Examples of signal peptides include MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:106) and MALPVTALLLPLALLLHAARP (SEQ ID NO: 93). Other signal peptides may be used.

In some embodiments, the spacer domain or hinge domain is located between the extracellular domain (comprising the antigen binding domain) and the transmembrane domain of the CAR or between the cytoplasmic domain and the transmembrane domain of the CAR. A spacer domain is any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain and/or a cytoplasmic domain in a polypeptide chain. A hinge domain is any oligopeptide or polypeptide that serves to provide flexibility to the CAR or domain thereof or to prevent steric hindrance of the CAR or domain thereof. In some embodiments, the spacer domain or hinge domain can 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 spacer domains may be included in other regions of the CAR. In some embodiments, the hinge domain is a CD8 hinge domain. Other hinge domains may be used.

Transmembrane domain

The transmembrane domain is a hydrophobic alpha helix that spans the membrane. The transmembrane domain provides stability to the CAR. In some embodiments, the transmembrane domain of a CAR as provided herein is a CD8 transmembrane domain. In other embodiments, the transmembrane domain is a CD28 transmembrane domain. In still other embodiments, the transmembrane domain is a chimera of CD8 and CD28 transmembrane domains. Other transmembrane domains may be used as provided herein. In some embodiments, the transmembrane domain is a CD8a transmembrane domain: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR (SEQ ID NO: 107). Other transmembrane domains may be used.

Intracellular domains

The intracellular domain is the functional end of the receptor. Upon antigen recognition, the receptors cluster and a signal is transmitted to the cell. The most commonly used intracellular domain component is CD 3-zeta, which contains three (3) immunoreceptor tyrosine-based activation motifs (ITAMs). This will transmit an activation signal to the T cell upon antigen binding. In many cases, CD 3-zeta may not provide a fully effective activation signal, and thus, co-stimulatory signaling is used. For example, CD28 and/or 4-1BB may be used with CD 3-zeta (CD3 zeta) to deliver a proliferation/survival signal. Thus, in some embodiments, the co-stimulatory molecule of a CAR as provided herein is a CD28 co-stimulatory molecule. In other embodiments, the co-stimulatory molecule is a 4-1BB co-stimulatory molecule. In some embodiments, the CAR comprises CD3 ζ and CD 28. In other embodiments, the CAR comprises CD 3-zeta and 4-1 BB. In still other embodiments, the CAR comprises CD3 ζ, CD28, and 4-1 BB. Table 5 provides examples of signaling molecules that may be used as described herein.

TABLE 5

Antibodies

An antibody (used interchangeably in various forms) is an immunoglobulin molecule capable of specifically binding to a target, e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes not only intact (i.e., full-length) monoclonal antibodies, but also antigen-binding fragments (e.g., Fab ', F (ab')2, Fv), single chain variable fragments (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, single domain antibodies (e.g., camel or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising a desired specific antigen recognition site (including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies).

A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL) that are normally associated with antigen binding. These regions/residues responsible for antigen binding can be identified from the amino acid sequence of the VH/VL sequence of a reference antibody (e.g., the anti-PTK 7 antibody described herein) using methods known in the art. The VH and VL regions may be further subdivided into hypervariable regions (termed "complementarity determining regions" ("CDRs")) interspersed with more conserved regions (termed "framework regions" ("FRs")). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The extent of framework regions and CDRs can be precisely identified using methods known in the art, e.g., by Kabat definition, Chothia definition, AbM definition, and/or contact definition (all of which are well known in the art). As used herein, a CDR may refer to a CDR defined by any method known in the art. Two antibodies having the same CDR mean that the two antibodies have the same amino acid sequence of the CDR determined by the same method. See, e.g., Kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest [ protein Sequences of Immunological Interest ], fifth edition, U.S. department of health and public service, NIH Pub. No. 91-3242, Chothia et al, (1989) Nature [ Nature ]342: 877; chothia, C, et al, (1987) J.mol.biol. [ J.M. 196: 901-917; al-lazikani et Al, (1997) J.Molec.biol. [ J.M. 273: 927-948; and Almagro, J.mol.Recognit. [ journal of molecular recognition ]17: 132-. See also hgmp.mrc.ac.uk and bio in.org.uk/abs.

In some embodiments, the antibody is a scFv, such as an anti-PTK 7 scFv. Antibodies include any class of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Immunoglobulins can be classified into different classes according to the antibody amino acid sequence of the constant domain of the heavy chain of the immunoglobulin. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The antibody used herein may be murine, rat, human or any other source of antibody (including chimeric or humanized antibodies). In some examples, the antibody comprises a modified constant region, such as an immunologically inert constant region, e.g., does not trigger complement-mediated lysis or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, an antibody of the disclosure is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof (which fragments comprise minimal sequence derived from non-human immunoglobulins). In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a recipient Complementarity Determining Region (CDR) are replaced by CDR residues from a non-human species (e.g., mouse, rat, rabbit) (donor antibody) having the desired specificity, affinity, and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDRs or framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody also optionally includes at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) altered relative to the original antibody, also referred to as one or more CDRs "derived" from the original antibody. Humanized antibodies may also be involved in affinity maturation.

In some embodiments, the antibodies of the present disclosure are chimeric antibodies, which may include heavy and light constant regions from human antibodies. A chimeric antibody is an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one mammalian species (e.g., non-human mammals (e.g., mice, rabbits, and rats)), while the constant portions are homologous to sequences in antibodies derived from another mammal (e.g., human). In some embodiments, amino acid modifications may be made in the variable and/or constant regions.

In some embodiments, an antibody of the disclosure specifically binds a target antigen, such as human PTK 7. Antibodies that "specifically bind" (used interchangeably herein) to a target or epitope are terms well known in the art, and methods of determining such specific binding are also well known in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, more permanently, and/or with greater affinity with a particular target antigen than with other target antigens. An antibody "specifically binds" a target antigen if it has greater affinity, avidity, binds more easily and/or more permanently to the target antigen than it binds to other substances. For example, an antibody that specifically (or preferably) binds to an epitope of PTK7 is an antibody that binds with greater affinity, avidity, more readily and/or more permanently to this PTK7 epitope than to other PTK7 or non-PTK 7 epitopes. It is also understood by reading this definition that, for example, an antibody that specifically binds a first target antigen may or may not specifically bind or preferentially bind a second target antigen. As such, "specific binding" or "preferential binding" does not necessarily require (although may include) exclusive binding. Typically, but not necessarily, reference to binding refers to preferential binding.

In some embodiments, the equilibrium dissociation constant (K) between the antibody and PTK7_D) From 100pM to 1. mu.M. In some embodiments, the K between the antibody and PTK7_DFrom 1nM to 100 nM.

Also within the scope of the present disclosure are functional variants of any of the exemplary antibodies disclosed herein. A functional variant can comprise one or more amino acid residue variations in VH and/or VL or in one or more VH CDRs and/or one or more VL CDRs, relative to a reference antibody, while maintaining substantially similar binding and biological activity (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, anti-tumor activity, or a combination thereof) as the reference antibody.

In some examples, an antibody disclosed herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 that collectively comprise NO more than 10 amino acid variations (e.g., NO more than 9,8, 7, 6,5, 4, 3,2, or1 amino acid variation) as compared to a VH CDR1, VH CDR2, and VH CDR3 of a reference antibody, e.g., antibody a (VH: SEQ ID NO: 55; VL: SEQ ID NO: 56). By "total" is meant that the total number of amino acid variations in all three VH CDRs is within a defined range. Alternatively or additionally, the antibody can comprise a VL CDR1, VL CDR2, and VL CDR3 that collectively comprise no more than 10 amino acid variations (e.g., no more than 9,8, 7, 6,5, 4, 3,2, or1 amino acid variation) as compared to the VL CDR1, VL CDR2, and VL CDR3 of the reference antibody.

In some examples, an antibody disclosed herein can comprise a VH CDR1, VH CDR2, and VH CDR3, at least one of which comprises NO more than 5 amino acid variations (e.g., NO more than 4, 3,2, or1 amino acid variation), as a corresponding VH CDR of a reference antibody (e.g., antibody a (VH: SEQ ID NO: 55; VL: SEQ ID NO: 56)). In particular examples, the antibody comprises a VH CDR3 comprising NO more than 5 amino acid variations (e.g., NO more than 4, 3,2, or1 amino acid variations) as the VH CDR3 of a reference antibody, such as antibody A (VH: SEQ ID NO: 55; VL: SEQ ID NO: 56). Alternatively or additionally, the antibody may comprise as the corresponding VL CDRs of the reference antibody at least one of VL CDR1, VL CDR2, and VL CDR3 comprising no more than 5 amino acid variations (e.g., no more than 4, 3,2, or1 amino acid variations). In particular examples, the antibody comprises a VL CDR3 comprising no more than 5 amino acid variations (e.g., no more than 4, 3,2, or1 amino acid variation) as the VL CDR3 of the reference antibody.

In some cases, the amino acid residue variation may be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, for example methods for altering polypeptide sequences can be found in the following references which compile such methods: for example, Molecular Cloning A Laboratory Manual [ Molecular Cloning: a laboratory manual, edited by j.sambrook et al, second edition, cold spring harbor laboratory press, cold spring harbor, new york, 1989, or Current Protocols in Molecular Biology, edited by f.m.ausubel et al, John Wiley & Sons, Inc., new york. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) a → G, S; (b) r → K, H; (c) n → Q, H; (d) d → E, N; (e) c → S, A; (f) q → N; (g) e → D, Q; (h) g → A; (i) h → N, Q; (j) i → L, V; (k) l → I, V; (l) K → R, H; (M) M → L, I, Y; (n) F → Y, M, L; (o) P → A; (p) S → T; (q) T → S; (r) W → Y, F; (s) Y → W, F; and (t) V → I, L.

In some embodiments, an antibody disclosed herein can comprise at least 80% (e.g., 85%, 90%, 95%, or 98%) total VH CDRs that are identical to the VH CDRs of a reference antibody (e.g., antibody a (VH: SEQ ID NO: 55; VL: SEQ ID NO: 56)). Alternatively or additionally, an antibody can comprise VL CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical in total to the VL CDRs of a reference antibody. In some embodiments, the antibody can comprise a VH that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the VH of a reference antibody (e.g., antibody A (VH: SEQ ID NO: 55; VL: SEQ ID NO:56)) and/or a VL that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the VL of a reference antibody.

Donor template

A nucleic acid encoding a CAR can be delivered to a T cell that comprises a donor template (also referred to as a donor polynucleotide) referred to herein. The donor template may comprise a non-homologous sequence, e.g. a nucleic acid encoding a CAR, flanked by two homologous regions to allow efficient HDR at the genomic location of interest. In some embodiments, the homologous region can comprise the nucleotide sequence of SEQ ID NO 92 or 100. In some embodiments, the non-homologous sequence is flanked by the nucleotide sequence of SEQ ID NO 92 and the nucleotide sequence of SEQ ID NO 100. Alternatively, the donor template may not have a region of homology to the target site in the DNA and may be integrated by NHEJ-dependent end-linking following cleavage at the target site.

The donor template may be single-and/or double-stranded DNA or RNA, and may be introduced into the cell in linear or circular form. If introduced in a linear form, the ends of the donor sequence can be protected by methods known to those skilled in the art (e.g., to prevent exonucleolytic degradation). For example, one or more dideoxynucleotide residues are added to the 3' end 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 [ Proc. Natl.Acad. Sci. USA ]84: 4959-; nehls et al (1996) Science 272: 886-. Other methods of protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of one or more terminal amino groups and the use of modified internucleotide linkages, such as phosphorothioate, phosphoramidate, and O-methyl ribose or deoxyribose residues.

The donor template may be introduced into the cell as part of a vector molecule having additional sequences, such as an origin of replication, a promoter, and a gene encoding antibiotic resistance. In addition, the donor template can be introduced as naked nucleic acid (as nucleic acid complexed with an agent, such as a liposome or poloxamer), or can be delivered by a virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

In some embodiments, the donor template is inserted such that its expression is driven by an endogenous promoter at the integration site, i.e., a promoter that drives expression of the endogenous gene into which the donor is inserted. However, in some embodiments, the donor template comprises an exogenous promoter and/or enhancer, such as a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In some embodiments, the exogenous promoter is the EF1a promoter comprising the sequence of SEQ ID No. 101. Other promoters may be used.

In addition, the exogenous sequence may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and/or polyadenylation signals.

In some embodiments, the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78, or 91.

Delivery methods and constructs

The nuclease and/or donor template can be delivered using a vector system including, but not limited to, plasmid vectors, DNA miniloops, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors, and combinations thereof.

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor templates into cells (e.g., T cells). Non-viral vector delivery systems include DNA plasmids, DNA miniloops, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses, which have either an episomal or an integrative genome upon delivery to a cell.

Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids, nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virosomes, and agents that enhance uptake of DNA. Sonication using, for example, the Sonitron 2000 system (Rich-Mar corporation) may also be used to deliver nucleic acids.

Adeno-associated virus delivery

The donor nucleic acid encoding the CAR construct can be delivered to the cell using an adeno-associated virus (AAV). AAV is a parvovirus that site-specifically integrates into the host genome and can therefore deliver a transgene, such as a CAR. Inverted Terminal Repeats (ITRs) are present, flank the AAV genome and/or the transgene of interest, and serve as replication origins. Rep and cap proteins are also present in the AAV genome, which when transcribed form a capsid that encapsulates the AAV genome for delivery into a target cell. Surface receptors on these capsids can confer an AAV serotype that determines which target organ the capsid primarily binds to, and thus which cells the AAV will most efficiently infect. Twelve human AAV serotypes are currently known. In some embodiments, the AAV is AAV serotype 6(AAV 6).

Adeno-associated virus is one of the most commonly used viruses for gene therapy for a number of reasons. First, AAV does not elicit an immune response after administration to mammals, including humans. Second, AAV is efficiently delivered to target cells, especially when considering the selection of an appropriate AAV serotype. Finally, AAV has the ability to infect both dividing and non-dividing cells because the genome can persist without integration in the host cell. This property makes them ideal candidates for gene therapy.

Homologous Directed Repair (HDR)

The donor nucleic acid encoding the CAR is inserted into the target locus by Homology Directed Repair (HDR). The CRISPR Cas9 enzyme cleaves both DNA strands at the target locus. HDR then occurs to repair Double Strand Breaks (DSBs) and insert donor DNA. For this to occur correctly, the flanking residues of the donor sequence are designed to be complementary to the sequence surrounding the DSB site in the target gene (hereinafter referred to as the "homology arm"). These homology arms serve as templates for DSB repair and make HDR a substantially error-free mechanism. The rate of Homologous Directed Repair (HDR) is a function of the distance between the mutation and the cleavage site, and therefore it is important to select target sites that overlap or are nearby. The template may include additional sequences flanking the homologous regions or may contain sequences different from the genomic sequence, allowing sequence editing.

The target gene may be associated with an immune response in the subject, wherein a permanent deletion of at least a portion of the target gene modulates the immune response. For example, to generate CAR T cells, the target gene may be the TCR alpha constant region (TRAC). Disruption of TRACs results in loss of function of endogenous TCRs.

In some embodiments, the target gene is in a safe harbor locus.

Engineered T cells

Engineered (genetically edited) CAR T cells of the present disclosure can be autologous ("autologous") or non-autologous ("non-autologous", e.g., allogeneic, syngeneic, or allogeneic). By "autologous" is meant cells from the same subject. "allogeneic" refers to cells of the same species as the subject, but genetically distinct from the cells in the subject. In some embodiments, the T cell is obtained from a mammalian subject. In some embodiments, the T cells are obtained from a human subject.

T cells can be obtained from a variety of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, a number of techniques known to those skilled in the art may be used, such as precipitation (e.g., FICOLL)^TMIsolated), T cells are obtained from a unit of blood collected from the subject.

In some embodiments, an isolated population of T cells is used. In some embodiments, after isolation of Peripheral Blood Mononuclear Cells (PBMCs), cytotoxic T cells and helper T lymphocytes may be classified into naive, memory and effector T cell subpopulations, either before or after activation, expansion and/or genetic modification.

Specific subpopulations 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 protein and/or MCH-II protein may be further isolated by positive or negative selection techniques. In some embodiments, the specific subpopulation of T cells that express 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, the population of engineered T cells does not express, or does not substantially express, one or more of the following markers: CD70, CD57, CD244, CD160, PD-1, CTLA4, hmm 3 and LAG 3. In some embodiments, a subpopulation of T cells may be isolated by positive or negative selection before and/or after genetic engineering.

In some embodiments, the isolated population of T cells expresses one or more markers including, but not limited to, CD3+, CD4+, CD8+, or a combination thereof. In some embodiments, T cells are isolated from a subject and first activated and stimulated for proliferation in vitro prior to undergoing gene editing.

To obtain a sufficient therapeutic dose of the T cell composition, the T cells are typically subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using, for example, the methods described in the following U.S. patents: us patent 6,352,694; 6,534,055, respectively; 6,905,680, respectively; 6,692,964, respectively; 5,858,358, respectively; 6,887,466, respectively; 6,905,681, respectively; 7,144,575, respectively; 7,067,318, respectively; 7,172,869, respectively; 7,232,566, respectively; 7,175,843, respectively; 5,883,223, respectively; 6,905,874, respectively; 6,797,514, respectively; and 6,867,041. In some embodiments, prior to introducing the genome-editing composition into the T cell, the T cell is 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.

In some embodiments, prior to introducing the genome-editing composition into the T cell, the T cell is 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.

In some embodiments, the T cell is activated at the same time as the genome editing composition is introduced into the T cell.

Also provided are populations of engineered T cells described herein. In some embodiments, at least 25% to 100% of the engineered T cells of the population express the CAR. In some embodiments, at least 25% or at least 50% of the engineered T cells of the population express the CAR. In some embodiments, at least 70% of the engineered T cells of the population express the CAR. In some embodiments, at least 25% of the engineered T cells of the population express the CAR after at least 7 days or at least 14 days of in vitro proliferation.

Therapeutic methods and compositions

In some embodiments, provided herein are methods of treating cancer (e.g., a solid tumor). Non-limiting examples of solid tumors that can be treated as provided herein include: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, and/or melanoma. In some embodiments, the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma. In some embodiments, the methods comprise delivering a CAR T cell of the disclosure (e.g., an anti-Ptk 7CAR T cell) to a subject having a cancer (e.g., a solid tumor), including pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, and/or melanoma. In some embodiments, the methods comprise delivering CAR T cells of the disclosure (e.g., anti-PTK 7CAR T cells) to a subject having a leukemia or lymphoma (e.g., a T cell, B cell, NK cell, dendritic cell leukemia or lymphoma). Non-limiting examples of leukemias include Acute Lymphoblastic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), and Chronic Myelogenous Leukemia (CML).

The administering step can include placing (e.g., transplanting) cells (e.g., engineered T cells) into the subject by a method or route in which the introduced cells are at least partially localized at a desired site (e.g., a tumor), thereby producing a desired effect or effects. The engineered T cells may be administered by any suitable route that results in delivery to a desired location in a subject where at least a portion of the implanted cells or cell components remain viable. After administration to a subject, the viability phase of the cells may be as short as several hours (e.g., twenty-four hours), days, up to years, or even the life span of the subject (i.e., long-term implantation). For example, in some aspects described herein, an effective amount of an engineered T cell is administered via a systemic route of administration (e.g., intraperitoneal or intravenous route).

The 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.

The donor is an individual that is not the subject being treated. The donor is a non-patient individual. In some embodiments, the donor is an individual who does not have or is not suspected of having the cancer to be treated. In some embodiments, multiple donors, e.g., two or more donors, can be used.

In some embodiments, the population of engineered T cells administered according to the methods described herein comprises allogeneic T cells obtained from one or more donors. "allogeneic" refers to cells, cell populations, or biological samples comprising cells obtained from one or more different donors of the same species, wherein the genes at one or more loci are different from the recipient. For example, the population of engineered T cells administered to a subject may be derived from one or more unrelated donors, or from one or more different siblings. In some embodiments, a homogenous cell population 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.

In some embodiments, the population of engineered T cells administered according to the methods described herein does not induce toxicity in the subject, e.g., the engineered T cells do not induce toxicity of non-cancer cells. In some embodiments, the population of engineered T cells administered does not trigger complement-mediated lysis, or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC).

An effective amount refers to the amount of the 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 refers to the amount of the composition sufficient to provide the desired effect (e.g., treating a subject suffering from a medical condition). An effective amount also includes an amount sufficient to prevent or delay the development of a disease symptom, alter the progression of a disease symptom (e.g., without limitation, slow the progression of a disease symptom), or reverse a disease symptom. It will be appreciated that, for any given situation, one of ordinary skill in the art can determine an appropriate effective amount using routine experimentation.

For use in various aspects described herein, an effective amount of cells (e.g., engineered T cells) includes at least 10²Individual cell, at least 5X 10²A cell, at least 10³Individual cell, at least 5X 10³A cell, at least 10⁴Individual cell, at least 5X 10⁴A cell, at least 10⁵Individual cell, at least 2X 10⁵A cell, at least3X 10⁵Individual cell, at least 4X 10⁵Individual cell, at least 5X 10⁵Individual cell, at least 6X 10⁵Individual cell, at least 7X 10⁵Individual cell, at least 8X 10⁵Individual cell, at least 9X 10⁵Individual cell, at least 1X 10⁶Individual cell, at least 2X 10⁶Individual cell, at least 3X 10⁶Individual cell, at least 4X 10⁶Individual cell, at least 5X 10⁶Individual cell, at least 6X 10⁶Individual cell, at least 7X 10⁶Individual cell, at least 8X 10⁶Individual cell, at least 9X 10⁶Individual cells or multiples thereof. The cells are derived from one or more donors or obtained from an autologous source. In some examples described herein, the cells are expanded in culture prior to administration to a subject in need thereof.

Modes of administration include injection, infusion, instillation, or ingestion. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subepithelial, subarachnoid, intraspinal and intrasternal injections and infusions. In some embodiments, the route is intravenous.

In some embodiments, engineered T cells are administered systemically, which refers to a population of cells that are administered in a manner other than directly to a target site, tissue, or organ, but rather are passed into the circulatory system of a subject, undergoing metabolism and other similar processes.

The efficacy of a treatment comprising a composition for treating a medical condition can be determined by a skilled clinician. A treatment is considered to be an "effective treatment" if any or all signs or symptoms of a disease (e.g., cancer), as one example, altering the level of a functional target in a beneficial manner (e.g., by at least 10%) or other clinically acceptable symptoms or markers, are improved or alleviated. Efficacy can also be measured by failure of a subject to worsen (e.g., cessation or at least slowing of disease progression) as assessed by hospitalization or need for medical intervention. Methods of measuring these indices are known to those skilled in the art and/or described herein. Treatment includes any treatment of a disease in a subject, including: (1) inhibiting disease, e.g., arresting or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of development of symptoms.

The disclosure is illustrated by the following examples:

example 1 an engineered T cell comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds PTK 7.

Example 2. the engineered T cell of example 1, further comprising a disrupted T cell receptor alpha chain constant region (TRAC) gene.

Example 3. the engineered T cell of example 2, wherein the nucleic acid encoding the CAR is inserted into the disrupted TRAC gene.

Example 4. the engineered T cell of any one of examples 1-3, further comprising a disrupted β -2-microglobulin (β 2M) gene.

Embodiment 5 the engineered T cell of any one of embodiments 1-4, wherein the extracellular domain of the CAR comprises an anti-PTK 7 antibody.

Example 6. the engineered T cell of example 5, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

Example 7 the engineered T cell of example 6, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Regions (CDRs) and the same light chain variable domain (VL) CDRs as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84.

Example 8 the engineered T cell of example 7, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

Example 9. the engineered T cell of example 7, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, or 82.

The engineered T cell of any one of embodiments 1-9, wherein the CAR comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

Example 11 the engineered T cell of example 10, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

Example 12 the engineered T cell of any one of examples 3-11, wherein the TRAC gene comprises a nucleotide sequence within any one of SEQ ID NOs 63, 64, 71, 78 or 91 encoding LHA and/or RHA or a nucleotide sequence of SEQ ID NOs 92 or 100, and/or wherein the CAR is encoded by a nucleotide sequence of any one of SEQ ID NOs 49, 51, 65, 72, 79 or 112.

Example 13 the engineered T cell of any one of examples 4-12, wherein the disrupted β 2M gene comprises at least one nucleotide sequence selected from any one of SEQ ID NOs 9-14.

Example 14a population of engineered T cells as described in any one of examples 1-13, wherein at least 25% or at least 50% of the engineered T cells of the population express the CAR.

The population of embodiment 14, wherein at least 70% of the engineered T cells of the population express the CAR.

The population of embodiment 14 or 15, wherein at least 25% of the engineered T cells of the population express the CAR after at least 7 days or at least 14 days of in vitro proliferation.

The population of any one of embodiments 14-16, wherein at least 50% of the engineered T cells of the population do not express detectable levels of a T Cell Receptor (TCR) protein.

The population of embodiment 17, wherein at least 90% of the engineered T cells of the population do not express detectable levels of TCR protein.

The population of any one of embodiments 14-18, wherein at least 50% of the engineered T cells of the population do not express detectable levels of β 2M protein.

Example 20. the population of example 19, wherein at least 70% of the engineered T cells of the population do not express detectable levels of β 2M protein.

The population of any one of embodiments 14-20, wherein the engineered T cells of the population induce cell lysis of at least 10%, at least 25%, or at least 50% of the cancer cells of the population when co-cultured in vitro with a population of cancer cells expressing PTK 7.

Example 22. the population of example 21, wherein the engineered T cells of the population induce cell lysis of at least 70%, at least 80%, or at least 90% of the cancer cell population that expresses PTK7 when co-cultured in vitro.

Example 23. the population of examples 21 or 22, wherein the engineered T cells of the population secrete IFN γ when co-cultured in vitro with a population of cancer cells.

The population of any one of embodiments 21-23, wherein the ratio of engineered T cells to cancer cells is 1:1 to 2: 1.

The population of any one of embodiments 21-24, wherein the cancer cells comprise sarcoma cells.

Example 26. the population of any one of examples 21-24, wherein the cancer cells comprise breast cancer cells, ovarian cancer cells, small cell lung cancer cells, and/or colon cancer cells.

Example 28 the population of any one of examples 14-27, which when administered in vivo to a subject, does not induce toxicity in the subject.

The method of embodiment 29, comprising administering to a subject the population of engineered T cells of any one of embodiments 14-28.

Example 30 the method of example 29, wherein the subject is a human subject.

The method of embodiment 30, wherein the subject has cancer.

Embodiment 32 the method of embodiment 31, wherein the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma.

Embodiment 33 the method of embodiment 31 or 32, wherein the cancer comprises cancer cells that express PTK 7.

Example 34 a method for producing an engineered T cell, the method comprising (a) delivering to a T cell: an RNA-guided nuclease, a gRNA targeting the TRAC gene, and a vector comprising a donor template comprising a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds PTK 7; and (b) generating engineered T cells having the disrupted TRAC gene and expressing the CAR.

Example 35 the method of example 34, wherein the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID NO:18 or 19, or the nucleotide sequence targeting SEQ ID NO: 40.

Example 36. the method of example 34 or 35, wherein the nucleic acid encoding the CAR is flanked by the left and right homology arms of the TRAC gene.

The method of any one of embodiments 34-36, further comprising delivering a gRNA targeting the β 2M gene to a T cell.

Example 38. the method of example 37, wherein the gRNA targeting the β 2M gene comprises the nucleotide sequence of SEQ ID NO:20 or 21, or the nucleotide sequence targeting SEQ ID NO: 41.

Example 39. the method of any one of examples 34-38, wherein the RNA-guided nuclease is Cas9 nuclease, optionally streptococcus pyogenes Cas9 nuclease.

The method of any of embodiments 34-39, wherein the extracellular domain of the CAR is an anti-PTK 7 antibody.

Example 41. the method of example 40, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

Example 42. the method of example 41, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Regions (CDRs) and the same light chain variable domain (VL) CDRs as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84.

Example 43 the method of example 42, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

The method of embodiment 42, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75 or 82.

The method of any of embodiments 34-44, wherein the CAR comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

Embodiment 46. the method of embodiment 45, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

Embodiment 47 the method of any one of embodiments 34-46, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78, or 91.

The method of any one of embodiments 34-47, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs 49, 51, 65, 72, 79, or 112.

The disclosure is further illustrated by the following examples:

example a1. an engineered T cell comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds PTK 7.

Example a2. the engineered T cell of example a1, further comprising a disrupted T cell receptor alpha chain constant region (TRAC) gene.

Example A3. the engineered T cell of example a1 or a2, further comprising a disrupted β -2-microglobulin (β 2M) gene.

The engineered T cell of any one of embodiments a1-A3, wherein the extracellular domain of the CAR comprises an anti-PTK 7 antibody.

Example a5. the engineered T cell of example a4, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

Example a6. the engineered T cell of example a5, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Regions (CDRs) and the same light chain variable domain (VL) CDRs as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84.

Example A7. an engineered T cell as in example a6, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

Example A8. an engineered T cell as in example a6, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75 or 82.

Embodiment A9. the engineered T cell of any one of embodiments a1-A8, wherein the CAR further comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

The engineered T cell of embodiment a9, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

Example a11 the engineered T cell of any one of examples a1-a10, wherein the CAR is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs 49, 51, 65, 72, 79, or 112 or a nucleotide sequence comprising a nucleic acid sequence at least 90% identical to SEQ ID NOs 49, 51, 65, 72, 79, or 112.

The engineered T cell of any one of embodiments a1-a11, wherein the nucleic acid encoding the CAR is inserted into the disrupted TRAC gene.

Example a13 the engineered T cell of any one of examples a2-a12, wherein the disrupted TRAC gene comprises a nucleotide sequence encoding LHA and/or RHA within any one of SEQ ID NOs 63, 64, 71, 78 or 91, a nucleotide sequence of SEQ ID NOs 92 or 100, and/or a nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78 or 91.

Example a14. the engineered T cell of any one of examples a1-a13, wherein the disrupted β 2M gene comprises at least one nucleotide sequence selected from any one of SEQ ID NOs 9-14.

Example a15. an engineered T cell comprising: (i) a disrupted TRAC gene; (ii) a disrupted β 2M gene; and (iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment.

Example a16. the engineered T cell of example a15, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta cytoplasmic signaling domain.

The engineered T cell of embodiment a17. the engineered T cell of embodiment a15 or a16, wherein the disrupted TRAC gene comprises a nucleic acid encoding the CAR.

Example a18. an engineered T cell comprising: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 co-stimulatory domain and a CD3 zeta cytoplasmic signaling domain; and (ii) a disrupted β 2M gene.

Example a19. an engineered T cell comprising: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs 50, 52, 66, 73, or 80; and (ii) a disrupted β 2M gene.

Example a20. an engineered T cell comprising: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112 and encodes a CAR comprising the amino acid sequence of SEQ ID NO 50, 52, 66, 73, or 80; and (ii) a disrupted β 2M gene.

The engineered T cell of any one of embodiments a1-a20, wherein the T cell is a human T cell.

A population of cells comprising the engineered T cell of any one of embodiments a1-a21, wherein at least 25% or at least 50% of the engineered T cells of the population express the CAR.

The population of embodiment a23. the population of embodiment a22, wherein at least 70% of the engineered T cells of the population express the CAR.

The population of embodiment a24. the population of embodiment a22, wherein at least 25% of the engineered T cells of the population express the CAR after at least 7 days or at least 14 days of in vitro proliferation.

The population of any one of embodiments a22-a24, wherein at least 50% of the engineered T cells of the population do not express detectable levels of a T Cell Receptor (TCR) protein.

The population of embodiment a26. the population of embodiment a25, wherein at least 90% of the engineered T cells of the population do not express detectable levels of TCR protein.

The population of any one of embodiments a22-a26, wherein at least 50% of the engineered T cells of the population do not express detectable levels of β 2M protein.

Example a28. the population of example a27, wherein at least 70% of the engineered T cells of the population do not express detectable levels of β 2M protein.

The population of any one of embodiments a22-a28, wherein the engineered T cells of the population induce cell lysis of at least 10%, at least 25%, or at least 50% of the cancer cells of the population when co-cultured in vitro with a population of cancer cells expressing PTK 7.

Example a30. the population of example a29, wherein the engineered T cells of the population induce cell lysis of at least 70%, at least 80%, or at least 90% of a population of cancer cells expressing PTK7 when co-cultured in vitro.

Example a31. the population of examples a29 or a30, wherein the engineered T cells of the population secrete IFN γ when co-cultured in vitro with a population of cancer cells.

Embodiment a32 the population of any one of embodiments a29-a31, wherein the ratio of engineered T cells to cancer cells is 1:1 to 2: 1.

The population of any one of embodiments a29-a32, wherein the cancer cells comprise sarcoma cells.

The population of any one of embodiments a29-3a2, wherein the cancer cells comprise breast cancer cells, ovarian cancer cells, small cell lung cancer cells, and/or colon cancer cells.

Example a35 the population of any one of examples a22-a34, which when administered in vivo to a subject, does not induce toxicity in the subject.

Example a36. a cell population comprising engineered T cells, wherein the engineered T cells comprise: (i) a disrupted TRAC gene; (ii) a disrupted β 2M gene; and (iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment.

Example a37. the population of cells of example a36, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta cytoplasmic signaling domain.

The population of cells of embodiment a38. the population of cells of embodiment a36 or a37, wherein the disrupted TRAC gene comprises a nucleic acid encoding the CAR.

Example a39. a cell population comprising engineered T cells, wherein the engineered T cells comprise: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 co-stimulatory domain and a CD3 zeta cytoplasmic signaling domain; and (ii) a disrupted β 2M gene.

Example a40. a cell population comprising engineered T cells, wherein the engineered T cells comprise: (i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112 and encodes a CAR of SEQ ID NO 50, 52, 66, 73, or 80; and (ii) a disrupted β 2M gene.

Example a41. a method comprising administering to a subject a population of engineered T cells as described in any one of examples a22-a 40.

Example a42. the method of example a41, wherein the subject is a human subject.

The method of embodiment a43. the method of embodiment a42, wherein the subject has cancer.

Embodiment a44. the method of embodiment a43, wherein the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma.

Example a45. the method of example a43 or a44, wherein the cancer comprises cancer cells that express PTK 7.

Example a46. a method of generating an engineered T cell, the method comprising (a) (i) an RNA-guided nuclease, (ii) a gRNA targeting the TRAC gene, and (iii) a vector comprising a donor template comprising a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds PTK 7; and (b) generating engineered T cells having the disrupted TRAC gene and expressing the CAR.

Example a47. the method of example a46, wherein the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID No. 18 or 19, or the nucleotide sequence targeting SEQ ID No. 40.

Example a48. the method of examples a46 or a47, further comprising delivering a gRNA targeting the β 2M gene to the T cell.

Example a49. the method of example a48, wherein the gRNA targeting the β 2M gene comprises the nucleotide sequence of SEQ ID No. 20 or 21, or the nucleotide sequence of SEQ ID No. 41.

The method of any one of embodiments a46-a49, wherein the extracellular domain of the CAR comprises an anti-PTK 7 antibody.

Example a51. the method of example a50, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

Example a52. the method of example a51, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Regions (CDRs) and the same light chain variable domain (VL) CDRs as a reference antibody, wherein the reference antibody comprises (i) a VH shown in SEQ ID NO:55 and a VL shown in SEQ ID NO:56, (ii) a VH shown in SEQ ID NO:69 and a VL shown in SEQ ID NO:70, (iii) a VH shown in SEQ ID NO:76 and a VL shown in SEQ ID NO:77, or (iv) a VH shown in SEQ ID NO:83 and a VL shown in SEQ ID NO: 84.

Example a53. the method of example a52, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

Example a54. the method of example a52, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, or 82.

The method of any one of embodiments a46-a54, wherein the CAR further comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

The method of embodiment a56. the method of embodiment a55, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

Example a57 the method of any one of examples a46-a56, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs 49, 51, 65, 72, 79, or 112 or a nucleotide sequence comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NOs 49, 51, 65, 72, 79, or 112.

The method of any one of embodiments a46-a57, wherein the nucleic acid encoding the CAR is flanked by the left and right homology arms of the TRAC gene.

Embodiment A59. the method of any one of embodiments A46-A58, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78, or 91.

Example a60. the method of any one of examples a46-a59, wherein the RNA-guided nuclease is Cas9 nuclease, optionally streptococcus pyogenes Cas9 nuclease.

Example a61. an engineered T cell produced by the method of any one of examples a46-a 60.

Example a62. a cell population comprising engineered T cells as described in example a61.

Example a63. a method of treating cancer in a subject, the method comprising administering to the subject the population of cells of any one of examples a22-a40 or a62.

Embodiment a64. the method of embodiment a63, wherein the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma.

Example a65. the method of example a63 or a64, wherein the cancer comprises cancer cells that express PTK 7.

Examples of the invention

Example 1 expression of PTK7 in Normal human tissues

Expression of PTK7 on frozen normal human tissue panels (FDA standard, Bouchi Inc. (Biochain)) was evaluated using a custom made recombinant monoclonal biotinylated antibody (CTX181 mAb, Creative Biolabs, 1mg/ml, SEQ ID NO:108 and SEQ ID NO:109) specific for PTK7CAR construct or biotinylated mouse isotype control (Rofors, Novus, NBP 2-21948). Slides were fixed with-20 ℃ acetone for 10 min at ambient temperature, followed by manual immunohistochemical staining at ambient temperature. Blocking was performed with peroxidase 1(Peroxidased 1) avidin and biotin in ordered steps, as well as background Sniper (biochar Medical, PX968M, AB972L, BS966M) to minimize non-specific staining. Slides were stained with biotinylated CTX181 primary monoclonal antibody (1:600) for 30 minutes and subsequently incubated with 4plus streptavidin-HRP labeled (bio healthcare medical) reagent for 15 minutes. Visualization of the target antigen was performed with DAB (brown) substrate chromogen (Dako, K3468). Nuclei were counterstained with Mayer' S Hematoxylin (dacco, S3309). Fig. 1 shows that PTK7 expression is not prevalent in normal human tissues.

Sequence of CTX181 mAb:

>181_HC(SEQ ID NO:108)

>181_LC(SEQ ID NO:109)

example 2 expression of PTK7 in diseased human tissues

Formalin-fixed, paraffin-embedded (FFPE) diseased and normal patient tumor microarrays were evaluated for PTK7 expression using the mouse monoclonal anti-human PTK7 antibody clone 4F9(EMD Millipore, MABN721) (fig. 2A and 2B). Table 6 lists FFPE Tissue Microarrays (TMAs) from bemanstan, usa (US Biomax, Inc.). FFPE sections were baked at 60 ℃ for 30 minutes, deparaffinized and rehydrated. Antigen retrieval was performed in 1X Reveal desoker solution at 95 ℃ for 40 minutes using a desoker chamber (bio healthcare medical company). The remaining steps were carried out at ambient temperature. Blocking was performed with peroxidase 1 (bio healthcare medical, PX968M) and background Sniper (bio healthcare medical, BS966M) in ordered steps to minimize non-specific staining. Slides were stained with primary monoclonal anti-PTK 7 antibody or mouse IgG isotype control (Novus Biologicals, LLC) for 60 minutes, washed and then stained with secondary EnVision goat anti-mouse horseradish peroxidase antibody (dacco, K400111-2) for 30 minutes. Visualization of the target antigen was performed with DAB (brown) substrate chromogen (Dako, K3468). Nuclei were counterstained with Mayer' S Hematoxylin (dacco, S3309). Slides were scanned with a panoramic MIDI II scanner (3DHistech, thiele fisher Scientific) and scored using a semiquantitative scoring system that evaluates staining intensity (1+ -3+, where 1+ represents low antigen expression) and percentage of stained sections (1% -100%). The results were tabulated to establish patient prevalence summary data (fig. 3). PTK7 was shown to be expressed in a wide range of solid tumor cancers.

TABLE 6 FFPE TMA

Tissue of	FFPE TMA (American Bemanse company name/number)
		Glioblastoma	GL806f
Hepatocellular carcinoma	LV631
		Hepatobiliary cell carcinoma	LV642
Osteosarcoma	OS804c
		Stomach cancer	ST483e
Ovarian cancer	OVC962
		Squamous cell carcinoma of esophagus	HEso-Squ127Lym-01
Advanced pancreatic cancer	PA1921a
		Adenocarcinoma of lung	BCS04017b
Squamous cell carcinoma of lung	HLug-Squ090Lym-01
		Small cell lung cancer	BS04116
Intrahepatic bile duct cancer	HIBD-Ade100PG-01

Example 3 human/mouse Cross-reactivity against PTK7 CTX181

To evaluate the species cross-reactivity of the custom CTX181 antibodies, their binding affinities were evaluated in murine 3T3L1 fibroblasts and murine M158 breast cancer cell line, and compared to their binding affinities in human Saos2 (osteosarcoma), a498 (renal carcinoma), and HCC70 (breast cancer). For each cell line, 2X 10 pairs at each concentration⁶Dose titration binding assays (200nM to 0.0032nM final CTX181 antibody) were run for each cell. Cells were stained with CTX181 antibody on ice for 30 min, then washed and stained with secondary APC fluorophore conjugated human IgG-Fc antibody for 15 min at room temperature. Staining of cells was assessed on a Novocyte flow cytometer (Acea Biosciences). Figure 4 shows the equivalent binding affinity of CTX181 antibody in mouse and human cell lines.

Example 4 expression of PTK7 in Normal human and mouse tissues

PTK7 expression on frozen normal mouse tissue panels (FDA standard, bocheng; fig. 5A and 5B) and frozen murine embryonic development arrays (hiyagen; fig. 6) was evaluated using CTX181 specific to PTK7CAR constructs or biotinylated mouse isotype control (lofos, NBP 2-21948). Slides were fixed with-20 ℃ acetone for 10 min at ambient temperature, followed by manual immunohistochemical staining at ambient temperature. Blocking was performed with peroxidase 1 avidin and biotin and background Sniper (biochar Medical), PX968M, AB972L, BS966M) in ordered steps to minimize non-specific staining. Slides were stained with biotinylated CTX181 primary monoclonal antibody (human 1: 300; murine 1:600) for 30 minutes, and then incubated with 4plus streptavidin-HRP-labeled (Biohealthcare medical company) reagents for 15 minutes with each other. Visualization of the target antigen was performed with DAB (brown) substrate chromogen (Dako, K3468). Nuclei were counterstained with Mayer' S Hematoxylin (dacco, S3309).

Example 5 TRAC^-/β2M^-anti-PTK 7CAR⁺Generation of T cells

This example describes the absence of the T Cell Receptor (TCR) gene (the gene edited in the TCR alpha constant (TRAC) region), beta 2-microglobulin (beta 2)M) expression and expression of genes targeting protein tyrosine kinase 7(PTK7) and PTK7⁺Production of allogeneic human T cells for Chimeric Antigen Receptors (CARs) of cancer. In TRAC^-/β2M^-Four unique anti-PTK 7 CARs (PTK7-4, PTK7-7, PTK7-13 and PTK7-17) comprising a CD28 co-stimulatory domain were expressed separately in T cells for experiments and evaluation. The 41BB co-stimulatory domain was also used in place of CD28 to generate PTK7-4(PTK7-4 b). Table 7 lists the PTK7CAR structures. Table 11 lists the PTK7CAR component sequences. The donor components are listed in table 12.

Table 7.

Electroporation activation of primary human T cells with Cas9 gRNA-RNP complexes and adeno-Associated Adenovirus Vectors (AAV) to generate TRACs^-/β2M^-anti-PTK 7CAR⁺T cells. Recombinant AAV serotype 6(AAV6) comprising one of the nucleotide sequences encoding an anti-PTK 7CAR (SEQ ID NO:49, 51, 65, 72, 79 or 112) was delivered to activated allogeneic human T cells with Cas9: sgRNA RNP (1 μ M Cas9, 5 μ M gRNA). The following sgrnas were used: TRAC (SEQ ID NO:28) and β 2M (SEQ ID NO: 30). Unmodified versions (or other modified versions) of the gRNAs (e.g., SEQ ID NO:18 or SEQ ID NO:20) may also be used. See also table 4.

Approximately one (1) week after electroporation, cells were treated for flow cytometry to assess TRAC, β 2M, and anti-PTK 7CAR expression levels on the cell surfaces of the edited cell populations (fig. 7). The antibodies used are listed in table 8. For all anti-PTK 7CAR T cells and TRACs^-/β2M^-A control cell, wherein the control cell is selected from the group consisting of,>90% of the living cells lack TCR expression and>60% lack of expression of β 2M. Cells treated with the construct encoding PTK7-4CAR had the highest percentage of expressing anti-PTK 7CAR⁺The living cells of (a)>70%). The orientation of VH and VL sequences in scFV appears to affect the expression of CAR. PTK7-4CAR and PTK7-7 CAR differ in the orientation of VH and VL sequences, however, PTK7-7 is only in living cell populations<Expressed in 50%. PTK7CAR T fine with CD28 costimulatory domainCells (PTK7-4) were more potent than those of the 4-1BB co-stimulatory domain (PTK7-4b) (FIG. 8).

Table 8.

Cell killing assay. TRAC was then assessed using a cell killing assay^-/β2M^-anti-PTK 7CAR⁺T cells induced the ability to lyse in adherent sarcoma cell lines expressing PTK7 (A-204 and Saos-2) and a breast cancer cell line expressing PTK7 (MCF 7). Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37 ℃. On the following day, T cells were added to the wells containing target cells at a ratio of 2:1 or 1:1T cells to target cells. Will TRAC^-/β2M^-T cells were used as negative controls. After approximately 20 hours, T cells were removed from the culture by aspiration and 100 μ L of 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 from each well was then quantified using a microplate reader. anti-PKT 7CAR T cells, particularly those expressing PTK7-4, PTK7-7, and PTK7-13 constructs, showed high cytotoxicity against a-204 (fig. 9A) and Saos (fig. 9B). Furthermore, anti-PKT 7CAR T cells expressing PTK7-4CAR showed the highest cytotoxic activity against the MCF7 cell line (fig. 9C), which is known to have lower expression of PTK7 mRNA than the tested sarcoma cell line.

The cell specificity of PTK7-4CAR T cells was exemplified using PTK7 knock-out (KO) Saos2 cells (fig. 10A) and a498 cells overexpressing PTK7 (fig. 10B). PTK7KO Saos-2 cells were generated by electroporation of ribonucleoprotein particle (RNP) complexes (1. mu.M Cas9 and 5. mu.M PTK7 gRNA (SEQ ID NO: 111; see Table 9)) according to established methods. The loss of cell surface expression of PTK7 from the cells was analyzed by flow cytometry using CTX181 mAb (fig. 10C), and subsequently expanded. Flow cytometry analysis showed 88.6% reduction in protein expression, indicating efficient gene editing. The in vitro efficacy of PTK7KO Saos-2 cells was evaluated in a cytotoxicity assay as compared to Saos-2 WT cells. Figure 10A shows that PTK7CAR T cells have reduced efficacy in lysing Saos2 PTK7KO cells, indicating that CAR T cells are specific for target cells expressing PTK 7.

TABLE 9 PTK7 gRNA sequences

A498 cells overexpressing PTK7 were generated as follows: a498 renal cell carcinoma cells were plated at 60% -70% confluence in MEM1 α + 10% FBS medium supplemented with 10 μ g/ml polybrene. A498 cells (LPP-A6381-Lv225-200, Renergy Gene, Inc. (GeneCopoeia), Rokville, Md.) were transduced with lentiviruses expressing the hupTK 7cDNA the next day based on the desired multiplicity of infection (MOI). After 24-48 hours of lentiviral infection, fresh medium was replaced, containing 4 μ g/ml puromycin selection. Puromycin treatment was continued for 5-7 days after transduction until all untransduced cells were eliminated from the culture. Expression of the humPTK7cDNA lentiviral construct was assessed by flow cytometry using CTX181 mAb (fig. 10C). The in vitro efficacy of a498 cells expressing the humpkt 7cDNA was evaluated in a cytotoxicity assay as compared to a498 WT cells. Figure 10B shows increased efficacy of PTK7CAR T cells to lyse a498 cells expressing the humpttk 7cDNA compared to a498 WT cells, indicating that PTK7CAR T cells are specific for target cells expressing PTK7 antigen.

Example 6 in vitro potency of PTK7CAR T cells in solid tumor cell lines

Cell killing assay. To examine the efficacy in additional tumor cell lines, cell lines expressing PTK7 were selected from the broad cancer cell line database for high, medium, and low expression for breast, pancreatic, and NSCL cancers. Assessment of TRAC Using a cell killing assay^-/β2M^-anti-PTK 7CAR⁺T cells were introduced in adherent sarcoma cell lines expressing PTK7 (Saos-2), breast cancer cell lines expressing PTK7 to varying degrees (HCC1395, MCF7, HCC1419), pancreatic cell lines expressing PTK7 to varying degrees (Panc-1, Hs766T, Aspc1), and non-small cell lung cancer cell lines expressing PTK7 to varying degrees (NCI-H1975, NCI-H520, NCI-H460)Ability to act as a cell lysis. Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37 ℃. On the following day, T cells were added to wells containing target cells at a ratio of 0.125:1, 0.25:1, 1:1, or 4:1 effector T cells to target cells. Will TRAC^-/β2M^-T cells were used as negative controls. After approximately 24 hours, T cells were removed from the culture by aspiration and 100 μ L CellTiter-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted from each well was then quantified using a microplate reader. PTK7 protein expression was assessed by: target cells were stained with CTX181 antibody on ice for 30 min, then washed and stained with secondary APC fluorophore conjugated human IgG-Fc antibody for 15 min at room temperature. Staining of target cells was assessed on a Novocyte flow cytometer (Acea Biosciences). Figures 11A-11C show the in vitro efficacy of anti-PKT 7CAR T cells showing cytotoxicity against all cell lines tested and their tendency to have PTK7 expression levels in these tumor cell lines.

The functional activity of PTK7CAR T cells on cytokine γ (IFNg) and interleukin-2 (IL2) was further evaluated using a cytokine release assay. T cells of all genotypes tested were incubated with target cells for 24 hours at the cell ratios described above. After 24 hours, supernatant cultures around the cell samples were collected and levels of IFNg and IL2 were measured using ELISA (RD Systems) following the manufacturer's instructions. In the presence of cancer cell lines expressing PTK7 (Saos2, HCC1395, MCF7, Panc1, Hs766T, NCI-H520, and NCI-H1975), PTK7CAR T cells secreted IFNg and IL2 when used at 4:1 or 1:1T cell: target cell ratios. Control cells (TCR)^-/β2M^-Both (no AAV) and unedited (no RNP) showed no specific IFNg or IL2 secretory response in the presence of any of the cancer cell lines listed. Collectively, these functional assays demonstrated that anti-PTK 7CAR T cells were cytotoxic and secreted IFNg and IL2 in the presence of cells expressing PTK 7.

Example 7 functional Capacity of anti-PTK 7CAR T cells

TRAC^-/β2M^-anti-PTK 7-4CAR⁺T cells (also known as PTK7-4CAR T cells) were generated as described in example 5. Similarly to produce TRAC^-/β2M^-anti-CD 19CAR⁺T cells and TRACs^-/β2M^-A population of T cells was used as a control. After preparation of edited T cells by transfection, all edited cell types and unedited T cells were tested for their ability to proliferate over the course of 7 days. T cells for each genotype were plated 5x 10⁶And (4) cells. Notably, as shown in FIG. 12, TRAC^-/β2M^-anti-PTK 7-4CAR⁺T cells were able to proliferate at rates and levels comparable to all control experiments. Samples of each genotype injected into the mouse model were frozen in CyroStor10 and stored in liquid nitrogen 7 days after cell proliferation. The remaining cells of each genotype were allowed to continue to proliferate until day 14 after editing. Notably, as shown in fig. 13A-13B, the percentage of live cells with TCR, β 2M, and CAR genetic edits remained consistent from day 7 to day 14 post-editing. Figures 13C-13D show that in subsequent experiments, 70.9% of cells expressed the PTK7-4CAR construct, while 98% of cells had TRAC KO and 97% of cells had β 2M KO. Detection of expression of anti-CD 19CAR using biotinylated polyclonal anti-mouse FAB primary followed by streptavidin-APC conjugate; the expression of anti-PTK 7CAR was examined using an anti-PTK 7 antibody-PE conjugate (Miltenyi catalog number: 130-.

The functional activity of PTK7-4CAR T cells was verified using the adhesion cytotoxicity assay as described in example 5. PTK7-4CAR T cells (PTK7 CAR) were able to cause cytotoxicity of Saos-2 and MCF7 cells expressing PTK7 when used at 1:1 or 2:1T cell to target cell ratios. Control cells (TCR)^-/β2M^-(AAV-free); TCR with improved resistance to stress^-/β2M^-anti-CD 19CAR (CD19 CAR); unedited (no RNP)) showed no specific cytotoxicity against Saos-2 or MCF7 cells (fig. 14B and 14E).

The functional activity of PTK7-4CAR T cells was further assessed using a cytokine (interferon gamma/IFN gamma) release assay. T cells of all genotypes tested were incubated with target cells (Saos-2 and MCF7 cells) for 24 h at 1:1 and 2:1 cell ratiosThen (c) is performed. After 24 hours, supernatant cultures around the cell samples were collected and PTK7-4CAR T cells (PTK7 CAR) secreted IFN γ in the presence of Saos-2 and MCF7 cells expressing PTK7 when used at a 1:1 or 2:1T cell to target cell ratio using ELISA (RD systems company) following the manufacturer's instructions. Control cells (TCR)^-/β2M^-(AAV-free); TCR with improved resistance to stress^-/β2M^-anti-CD 19CAR⁺(CD19 CAR); unedited (no RNP)) showed no specific IFN γ secretion response in the presence of Saos-2 or MCF7 cells (fig. 14C and 14F).

Collectively, these functional assays demonstrate that anti-PTK 7CAR T cells are cytotoxic and secrete IFN γ in the presence of cells expressing PTK 7.

EXAMPLE 8 in vitro cytotoxicity assays

To evaluate the in vitro efficacy of anti-PTK 7CAR T cells on murine cells 3T3L1 fibroblasts and M158 breast cancer cells, a24 hour cytotoxicity assay was performed. The results were compared to the functional treatment of anti-PTK 7CAR T-cell lysis of human Saos2 osteosarcoma and human a498 renal cell carcinoma cell line. Adherent cells were seeded in 96-well plates at 50,000 cells per well and left overnight at 37 ℃. On the following day, T cells were added to wells containing target cells at a ratio of 0.125:1, 0.25:1, 1:1, or 4:1 effector T cells to target cells. Will TRAC^-/β2M^-T cells were used as negative controls. After approximately 24 hours, T cells were removed from the culture by aspiration and 100 μ L CellTiter-Glo (Promega) was added to each well of the plate to assess the number of remaining viable cells. The amount of light emitted from each well was then quantified using a microplate reader. anti-PKT 7CAR T cells showed equally good cytotoxicity against human Saos2 and murine 3T3L1 cell lines (fig. 15). In contrast, human a498 and murine M158 cells did not lyse in the presence of anti-PTK 7CAR T cells because of the low expression level of PTK7 in these 2 cell lines.

Example 9 tolerance against PTK7CAR T cells in a mouse model

Testing of tolerance in NOG mice by TRAC injection^-/β2M^-anti-PTK 7-4CAR⁺The ability of T cells to perform therapy. To two NOGMice were given 1000 ten thousand TRACs^-/β2M^-anti-PTK 7-4CAR T cells (generated as described previously); two additional mice were given 1000 million anti-CD 19CAR T cells. All CAR T cells were administered by tail vein injection.

Mice were weighed daily and monitored for distress or moribund. Mice treated with anti-PTK 7CAR T cells showed similar minimal weight loss as mice treated with anti-CD 19CAR T cells (figure 16). After a period of ten days post-injection, animals were sacrificed and spleen and blood samples were analyzed for the presence of human T cells (human CD45+ cells). In spleen and blood, mice treated with anti-PTK 7CAR T had higher levels of edited CAR T cells (huCD45+ cells) than mice treated with anti-CD 19CAR T cells (table 10), indicating that anti-PTK 7CAR T cells will expand in the presence of mouse antigens.

TABLE 10 percentage of humanCD45+ cells in PTK7 and CD19CAR T cell treated mice ((huCD45+/mucD45 +). 100)

Overall, transient weight loss and higher levels of CAR T cells in anti-PTK 7CAR treated mice indicate that anti-PTK 7CAR recognizes antigen in mice, which results in CAR T cell proliferation. The overall lack of significant toxicity is surprising, suggesting that the known on-target/off-tissue toxicity associated with targeting PTK7 may be tolerable in mice, and further may be tolerable in humans.

Example 10 in vivo efficacy of anti-PTK 7CAR-T cells in xenograft mouse models

The efficacy of anti-PTK 7CAR-T cells was tested in vivo in a SKOV-3 human ovarian, NCI-H1975 human non-small cell lung carcinoma and human pancreas Hs766T tumor xenograft mouse model. Fig. 17 shows the cell surface expression level of PTK7 in human cancer cell lines using CTX181 Ab. For each xenograft model, 5 female (5-8 weeks) NOG mice were single time point, single dose (5X 10)⁷Individual cells/ml) IV administration of TRAC^-/β2M^-anti-PTK 7CAR T cells (generated as described above). Body weight(2 x weekly) and tumor volume are endpoints measured during the course of the study. When the tumor (cell line injected subcutaneously into the right flank) reached 50mm, mice were given anti-PTK 7CAR T cells. The study was terminated when the tumors reached the terminal size or 90 days (whichever occurred first) (SKOV3 (ovary) 1000mm, NCI-H1975(NSCLC) and Hs766T (pancreas) 2000 mm). Mice were raised and monitored under pathogen-free conditions and IACUC standards. anti-PTK 7CAR-T cells were effective in all three xenograft models (NCI-H1975 non-small cell lung cancer xenograft model (fig. 18A), SKOV3 ovarian cancer xenograft model (fig. 18B), and Hs766T pancreatic cancer xenograft model (fig. 18C)).

Example 11 in vivo efficacy of anti-PTK 7CAR-T cells in xenograft mouse models

The efficacy of anti-PTK 7CAR-T cells was tested in OV90, OVCAR3, a2780 human ovary, MCF7, HCC70 human breast, HCT116 human colon and H209 human small cell lung cancer tumor xenograft mouse models. For each xenograft model, 5 female (5-8 weeks) NOG mice were single time point, single dose (5X 10)⁷Individual cells/ml) IV administration of TRAC^-/β2M^-anti-PTK 7CAR T cells (generated as described above). Body weight (2 x per week) and tumor volume were endpoints measured during the study. When the tumor (cell line injected subcutaneously into the right flank) reached 50mm, mice were given anti-PTK 7CAR-T cells. The study was terminated when the tumor reached the endpoint size (2000mm) or 90 days (whichever occurred first). Mice were raised and monitored under pathogen-free conditions and IACUC standards. Figure 19A shows the efficacy of anti-PTK 7CAR T cells on OV90 ovarian tumor xenograft model. Figure 19B shows the efficacy of anti-PTK 7CAR T cells on HCT116 colon tumor xenograft model. Figure 19C shows that anti-PTK 7CAR T cells were particularly effective in the MCF7 tumor xenograft model. Figure 20 shows the percent weight change of Hs-766T pancreatic tumor xenograft model treated with PTK7CAR T cells. Equivalent percent body weight change was observed in all xenograft studies measured. Potential toxicity showed variability in the xenograft model.

TABLE 11 CAR component

CAR structure:

CD8[ signal peptide ] -anti-Pkt 7[ scFV ] -CD8[ tm ] -CD28[ co-stimulatory domain ] -CD3 ζ; or

CD8[ signal peptide ] -anti-Pkt 7[ scFV ] -CD8[ tm ] -41BB [ co-stimulatory domain ] -CD3 zeta

TABLE 12 Donor Components

Donor structure: TRAC [ LHA ] -EF1a [ promoter ] -CAR-poly A-TRAC [ RHA ]

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to each subject matter cited, which in some cases may include the entire contents of the document.

As used herein, the indefinite article "a" or "an" as used herein in the specification and in the claims should be understood to mean "at least one" unless clearly indicated to the contrary.

It will also be understood that, unless explicitly stated to the contrary, in any methods claimed herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited.

In the claims, as well as in the specification above, all conjunctions such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" holding, "" consisting of … …, and the like are to be understood as being open-ended, i.e., to mean including but not limited to such. Only the transitional phrases "consisting of … …" and "consisting essentially of … …" should be individually closed or semi-closed transitional phrases, as described in the United States Patent Office Patent examination Manual of Patent Examing Procedures, section 2111.03.

The terms "about" and "substantially" preceding a numerical value refer to the mean ± 10% of the recited numerical value.

Where a range of values is provided, each value between the upper and lower end of the range is specifically contemplated and described herein.

Sequence listing

<110> Kries Per medical shares company (CRISPR Therapeutics AG)

<120> anti-PTK 7 immune cell cancer therapy

<130> 33165/CT111

<150> US 62/756,638

<151> 2018-11-07

<150> US 62/910,586

<151> 2019-10-04

<160> 113

<170> PatentIn 3.5 edition

<210> 1

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aagagcaaca aatctgact 19

<210> 2

<211> 58

<212> DNA

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<400> 2

aagagcaaca gtgctgtgcc tggagcaaca aatctgacta agagcaacaa atctgact 58

<210> 3

<211> 52

<212> DNA

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<400> 3

aagagcaaca gtgctggagc aacaaatctg actaagagca acaaatctga ct 52

<210> 4

<211> 53

<212> DNA

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aagagcaaca gtgcctggag caacaaatct gactaagagc aacaaatctg act 53

<210> 5

<211> 38

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aagagcaaca gtgctgacta agagcaacaa atctgact 38

<210> 6

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aagagcaaca gtgctgtggg cctggagcaa caaatctgac taagagcaac aaatctgact 60

<210> 7

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aagagcaaca gtgctggcct ggagcaacaa atctgactaa gagcaacaaa tctgact 57

<210> 8

<211> 60

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aagagcaaca gtgctgtgtg cctggagcaa caaatctgac taagagcaac aaatctgact 60

<210> 9

<211> 79

<212> DNA

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<400> 9

cgtggcctta gctgtgctcg cgctactctc tctttctgcc tggaggctat ccagcgtgag 60

tctctcctac cctcccgct 79

<210> 10

<211> 78

<212> DNA

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<400> 10

cgtggcctta gctgtgctcg cgctactctc tctttcgcct ggaggctatc cagcgtgagt 60

ctctcctacc ctcccgct 78

<210> 11

<211> 75

<212> DNA

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<400> 11

cgtggcctta gctgtgctcg cgctactctc tctttctgga ggctatccag cgtgagtctc 60

tcctaccctc ccgct 75

<210> 12

<211> 84

<212> DNA

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<400> 12

cgtggcctta gctgtgctcg cgctactctc tctttctgga tagcctggag gctatccagc 60

gtgagtctct cctaccctcc cgct 84

<210> 13

<211> 55

<212> DNA

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cgtggcctta gctgtgctcg cgctatccag cgtgagtctc tcctaccctc ccgct 55

<210> 14

<211> 82

<212> DNA

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<400> 14

cgtggcctta gctgtgctcg cgctactctc tctttctgtg gcctggaggc tatccagcgt 60

gagtctctcc taccctcccg ct 82

<210> 15

<211> 100

<212> DNA

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<220>

<221> features not yet classified

<222> (1)..(20)

<223> n is a, c, g, t or u

<400> 15

nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 16

<211> 96

<212> DNA

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<400> 16

nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugc 96

<210> 17

<211> 78

<212> DNA

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<222> (1)..(1)

<223> can be repeated 17-30 times

<220>

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<222> (1)..(1)

<223> n is a, c, g, t or u

<400> 17

nguuuuagag cuagaaauag caaguuaaaa uaaggcuagu ccguuaucaa cuugaaaaag 60

uggcaccgag ucggugcu 78

<210> 18

<211> 100

<212> DNA

<213> Artificial sequence

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<400> 18

agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 19

<211> 20

<212> DNA

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<400> 19

agagcaacag ugcuguggcc 20

<210> 20

<211> 100

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<400> 20

gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 21

<211> 20

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<400> 21

gcuacucucu cuuucuggcc 20

<210> 22

<211> 100

<212> DNA

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<400> 22

cugcagcuuc uccaacacau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 23

<211> 20

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<400> 23

cugcagcuuc uccaacacau 20

<210> 24

<211> 100

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<400> 24

gcuuuggucc cauuggucgc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 25

<211> 20

<212> DNA

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<400> 25

gcuuuggucc cauuggucgc 20

<210> 26

<211> 100

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<400> 26

gcccgcagga cgcacccaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 27

<211> 20

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<400> 27

gcccgcagga cgcacccaua 20

<210> 28

<211> 100

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<400> 28

agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 29

<211> 20

<212> DNA

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<400> 29

agagcaacag ugcuguggcc 20

<210> 30

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<400> 30

gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 31

<211> 20

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<400> 31

gcuacucucu cuuucuggcc 20

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<400> 32

cugcagcuuc uccaacacau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 33

<211> 20

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<400> 33

cugcagcuuc uccaacacau 20

<210> 34

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<400> 34

gcuuuggucc cauuggucgc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 35

<211> 20

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<400> 35

gcuuuggucc cauuggucgc 20

<210> 36

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<400> 36

gcccgcagga cgcacccaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 37

<211> 20

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<400> 37

gcccgcagga cgcacccaua 20

<210> 38

<211> 23

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<400> 38

gctttggtcc cattggtcgc ggg 23

<210> 39

<211> 23

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<400> 39

gcccgcagga cgcacccata ggg 23

<210> 40

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<400> 40

agagcaacag tgctgtggcc tgg 23

<210> 41

<211> 23

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<400> 41

gctactctct ctttctggcc tgg 23

<210> 42

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<400> 42

ctgcagcttc tccaacacat cgg 23

<210> 43

<211> 126

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<400> 43

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 44

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<212> PRT

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Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 45

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<400> 45

tcaaagcgga gtaggttgtt gcattccgat tacatgaata tgactcctcg ccggcctggg 60

ccgacaagaa aacattacca accctatgcc cccccacgag acttcgctgc gtacaggtcc 120

<210> 46

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<400> 46

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

1 5 10 15

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

20 25 30

Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 47

<211> 336

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<400> 47

cgagtgaagt tttcccgaag cgcagacgct ccggcatatc agcaaggaca gaatcagctg 60

tataacgaac tgaatttggg acgccgcgag gagtatgacg tgcttgataa acgccggggg 120

agagacccgg aaatgggggg taaaccccga agaaagaatc cccaagaagg actctacaat 180

gaactccaga aggataagat ggcggaggcc tactcagaaa taggtatgaa gggcgaacga 240

cgacggggaa aaggtcacga tggcctctac caagggttga gtacggcaac caaagatacg 300

tacgatgcac tgcatatgca ggccctgcct cccaga 336

<210> 48

<211> 112

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<400> 48

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 49

<211> 1530

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atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccgcaggtgc agctggtgga aagcggcggc ggcgtggtgc agccgggccg cagcctgcgc 120

ctgagctgcg cggcgagcgg ctttaccttt agcagctatg gcatgcattg ggtgcgccag 180

gcgccgggca aaggcctgga atgggtggcg gtgatttggg atgatggcag caacaaatat 240

tatgtggata gcgtgaaagg ccgctttacc attagccgcg ataacagcaa aaacaccctg 300

tatctgcaga tgaacagcct gcgcgcggaa gataccgcgg tgtattattg cgcgcgcgat 360

gattattatg gcagcggcag ctttaacagc tattatggca ccgatgtgtg gggccagggc 420

accaccgtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 480

ggcagcgaaa ttgtgctgac ccagagcccg gcgaccctga gcctgagccc gggcgaacgc 540

gcgaccctga gctgccgcgc gagccagagc gtgagcattt atctggcgtg gtatcagcag 600

aaaccgggcc aggcgccgcg cctgctgatt tatgatgcga gcaaccgcgc gaccggcatt 660

ccggcgcgct ttagcggcag cggcagcggc accgatttta ccctgaccat tagcagcctg 720

gaaccggaag attttgcggt gtattattgc cagcagcgca gcaactggcc gccgtttacc 780

tttggcccgg gcaccaaagt ggatattaaa agcgcggcgg cgtttgtgcc ggtgtttctg 840

ccggcgaaac cgaccaccac cccggcgccg cgcccgccga ccccggcgcc gaccattgcg 900

agccagccgc tgagcctgcg cccggaagcg tgccgcccgg cggcgggcgg cgcggtgcat 960

acccgcggcc tggattttgc gtgcgatatt tatatttggg cgccgctggc gggcacctgc 1020

ggcgtgctgc tgctgagcct ggtgattacc ctgtattgca accatcgcaa ccgcagcaaa 1080

cgcagccgcc tgctgcatag cgattatatg aacatgaccc cgcgccgccc gggcccgacc 1140

cgcaaacatt atcagccgta tgcgccgccg cgcgattttg cggcgtatcg cagccgcgtg 1200

aaatttagcc gcagcgcgga tgcgccggcg tatcagcagg gccagaacca gctgtataac 1260

gaactgaacc tgggccgccg cgaagaatat gatgtgctgg ataaacgccg cggccgcgat 1320

ccggaaatgg gcggcaaacc gcgccgcaaa aacccgcagg aaggcctgta taacgaactg 1380

cagaaagata aaatggcgga agcgtatagc gaaattggca tgaaaggcga acgccgccgc 1440

ggcaaaggcc atgatggcct gtatcagggc ctgagcaccg cgaccaaaga tacctatgat 1500

gcgctgcata tgcaggcgct gccgccgcgc 1530

<210> 50

<211> 510

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr

65 70 75 80

Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe

115 120 125

Asn Ser Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser

165 170 175

Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser

180 185 190

Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

195 200 205

Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe

210 215 220

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

225 230 235 240

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp

245 250 255

Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala

260 265 270

Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro

275 280 285

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

290 295 300

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

305 310 315 320

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

325 330 335

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

340 345 350

Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

355 360 365

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

370 375 380

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val

385 390 395 400

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

405 410 415

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

420 425 430

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

435 440 445

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

450 455 460

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

465 470 475 480

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

485 490 495

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 51

<211> 1536

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 51

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccgcaggtgc agctggtgga aagcggcggc ggcgtggtgc agccgggccg cagcctgcgc 120

ctgagctgcg cggcgagcgg ctttaccttt agcagctatg gcatgcattg ggtgcgccag 180

gcgccgggca aaggcctgga atgggtggcg gtgatttggg atgatggcag caacaaatat 240

tatgtggata gcgtgaaagg ccgctttacc attagccgcg ataacagcaa aaacaccctg 300

tatctgcaga tgaacagcct gcgcgcggaa gataccgcgg tgtattattg cgcgcgcgat 360

gattattatg gcagcggcag ctttaacagc tattatggca ccgatgtgtg gggccagggc 420

accaccgtga ccgtgagcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc 480

ggcagcgaaa ttgtgctgac ccagagcccg gcgaccctga gcctgagccc gggcgaacgc 540

gcgaccctga gctgccgcgc gagccagagc gtgagcattt atctggcgtg gtatcagcag 600

aaaccgggcc aggcgccgcg cctgctgatt tatgatgcga gcaaccgcgc gaccggcatt 660

ccggcgcgct ttagcggcag cggcagcggc accgatttta ccctgaccat tagcagcctg 720

gaaccggaag attttgcggt gtattattgc cagcagcgca gcaactggcc gccgtttacc 780

tttggcccgg gcaccaaagt ggatattaaa agcgcggcgg cgtttgtgcc ggtgtttctg 840

ccggcgaaac cgaccaccac cccggcgccg cgcccgccga ccccggcgcc gaccattgcg 900

agccagccgc tgagcctgcg cccggaagcg tgccgcccgg cggcgggcgg cgcggtgcat 960

acccgcggcc tggattttgc gtgcgatatt tatatttggg cgccgctggc gggcacctgc 1020

ggcgtgctgc tgctgagcct ggtgattacc ctgtattgca accatcgcaa ccgcaaacgc 1080

ggccgcaaaa aactgctgta tatttttaaa cagccgttta tgcgcccggt gcagaccacc 1140

caggaagaag atggctgcag ctgccgcttt ccggaagaag aagaaggcgg ctgcgaactg 1200

cgcgtgaaat ttagccgcag cgcggatgcg ccggcgtatc agcagggcca gaaccagctg 1260

tataacgaac tgaacctggg ccgccgcgaa gaatatgatg tgctggataa acgccgcggc 1320

cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 1380

gaactgcaga aagataaaat ggcggaagcg tatagcgaaa ttggcatgaa aggcgaacgc 1440

cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 1500

tatgatgcgc tgcatatgca ggcgctgccg ccgcgc 1536

<210> 52

<211> 512

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 52

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr

65 70 75 80

Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe

115 120 125

Asn Ser Tyr Tyr Gly Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr

130 135 140

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser

165 170 175

Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser

180 185 190

Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

195 200 205

Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe

210 215 220

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

225 230 235 240

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp

245 250 255

Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Ala

260 265 270

Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro

275 280 285

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

290 295 300

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

305 310 315 320

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

325 330 335

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

340 345 350

Cys Asn His Arg Asn Arg Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

355 360 365

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

370 375 380

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

385 390 395 400

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

405 410 415

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

420 425 430

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

435 440 445

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

450 455 460

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

465 470 475 480

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

485 490 495

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 53

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 53

caggtgcagc tggtggaaag cggcggcggc gtggtgcagc cgggccgcag cctgcgcctg 60

agctgcgcgg cgagcggctt tacctttagc agctatggca tgcattgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtggcggtg atttgggatg atggcagcaa caaatattat 180

gtggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcgatgat 300

tattatggca gcggcagctt taacagctat tatggcaccg atgtgtgggg ccagggcacc 360

accgtgaccg tgagcagcgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 420

agcgaaattg tgctgaccca gagcccggcg accctgagcc tgagcccggg cgaacgcgcg 480

accctgagct gccgcgcgag ccagagcgtg agcatttatc tggcgtggta tcagcagaaa 540

ccgggccagg cgccgcgcct gctgatttat gatgcgagca accgcgcgac cggcattccg 600

gcgcgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctggaa 660

ccggaagatt ttgcggtgta ttattgccag cagcgcagca actggccgcc gtttaccttt 720

ggcccgggca ccaaagtgga tattaaa 747

<210> 54

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 54

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly

100 105 110

Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val

130 135 140

Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala

145 150 155 160

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

180 185 190

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

210 215 220

Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr Phe

225 230 235 240

Gly Pro Gly Thr Lys Val Asp Ile Lys

245

<210> 55

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 55

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly

100 105 110

Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 56

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 56

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 57

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 57

Ser Tyr Gly Met His

1 5

<210> 58

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 58

Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210> 59

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 59

Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly Thr Asp

1 5 10 15

Val

<210> 60

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 60

Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu Ala

1 5 10

<210> 61

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 61

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 62

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 62

Gln Gln Arg Ser Asn Trp Pro Pro Phe Thr

1 5 10

<210> 63

<211> 4370

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 63

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840

cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900

gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960

gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020

ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080

gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140

tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200

tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260

tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320

tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380

tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440

ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500

ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560

gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620

agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680

aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740

gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800

ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860

agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920

cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980

accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040

aggccgcagg tgcagctggt ggagagcggc ggaggagtgg tgcaacccgg aaggtccctg 2100

aggctctcct gtgccgccag cggcttcacc ttctccagct acggtatgca ctgggtgaga 2160

caagcccccg gaaagggcct cgagtgggtg gccgtgatct gggatgatgg ctccaacaag 2220

tactacgtgg acagcgtcaa gggcagattc accatcagca gggacaacag caagaacacc 2280

ctgtacctgc agatgaactc cctgagagcc gaagacaccg ccgtgtacta ttgtgccagg 2340

gacgactact atggctccgg ctccttcaat agctactatg gcaccgacgt gtggggccag 2400

ggcaccacag tgacagtgag cagcggcgga ggaggatccg gaggaggagg aagcggagga 2460

ggaggaagcg agatcgtgct gacacagtcc cccgctaccc tgagcctgag ccccggcgag 2520

agagctaccc tgagctgcag agccagccag agcgtctcca tctacctggc ctggtaccag 2580

cagaagcctg gccaggcccc tagactgctg atctacgacg ccagcaacag ggccaccggc 2640

attcctgcca gattcagcgg ctccggctcc ggcaccgatt tcacactgac catcagctcc 2700

ctggagcctg aggacttcgc cgtgtattac tgccagcaga ggagcaactg gccccccttt 2760

accttcggcc ccggcaccaa ggtcgacatc aagagtgctg ctgcctttgt cccggtattt 2820

ctcccagcca aaccgaccac gactcccgcc ccgcgccctc cgacacccgc tcccaccatc 2880

gcctctcaac ctcttagtct tcgccccgag gcatgccgac ccgccgccgg gggtgctgtt 2940

catacgaggg gcttggactt cgcttgtgat atttacattt gggctccgtt ggcgggtacg 3000

tgcggcgtcc ttttgttgtc actcgttatt actttgtatt gtaatcacag gaatcgctca 3060

aagcggagta ggttgttgca ttccgattac atgaatatga ctcctcgccg gcctgggccg 3120

acaagaaaac attaccaacc ctatgccccc ccacgagact tcgctgcgta caggtcccga 3180

gtgaagtttt cccgaagcgc agacgctccg gcatatcagc aaggacagaa tcagctgtat 3240

aacgaactga atttgggacg ccgcgaggag tatgacgtgc ttgataaacg ccgggggaga 3300

gacccggaaa tggggggtaa accccgaaga aagaatcccc aagaaggact ctacaatgaa 3360

ctccagaagg ataagatggc ggaggcctac tcagaaatag gtatgaaggg cgaacgacga 3420

cggggaaaag gtcacgatgg cctctaccaa gggttgagta cggcaaccaa agatacgtac 3480

gatgcactgc atatgcaggc cctgcctccc agataataat aaaatcgcta tccatcgaag 3540

atggatgtgt gttggttttt tgtgtgtgga gcaacaaatc tgactttgca tgtgcaaacg 3600

ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccaggt aagggcagct 3660

ttggtgcctt cgcaggctgt ttccttgctt caggaatggc caggttctgc ccagagctct 3720

ggtcaatgat gtctaaaact cctctgattg gtggtctcgg ccttatccat tgccaccaaa 3780

accctctttt tactaagaaa cagtgagcct tgttctggca gtccagagaa tgacacggga 3840

aaaaagcaga tgaagagaag gtggcaggag agggcacgtg gcccagcctc agtctctcca 3900

actgagttcc tgcctgcctg cctttgctca gactgtttgc cccttactgc tcttctaggc 3960

ctcattctaa gccccttctc caagttgcct ctccttattt ctccctgtct gccaaaaaat 4020

ctttcccagc tcactaagtc agtctcacgc agtcactcat taacccacca atcactgatt 4080

gtgccggcac atgaatgcac caggtgttga agtggaggaa ttaaaaagtc agatgagggg 4140

tgtgcccaga ggaagcacca ttctagttgg gggagcccat ctgtcagctg ggaaaagtcc 4200

aaataacttc agattggaat gtgttttaac tcagggttga gaaaacagct accttcagga 4260

caaaagtcag ggaagggctc tctgaagaaa tgctacttga agataccagc cctaccaagg 4320

gcagggagag gaccctatag aggcctggga caggagctca atgagaaagg 4370

<210> 64

<211> 4376

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 64

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840

cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900

gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960

gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020

ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080

gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140

tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200

tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260

tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320

tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380

tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440

ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500

ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560

gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620

agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680

aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740

gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800

ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860

agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920

cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980

accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040

aggccgcagg tgcagctggt ggagagcggc ggaggagtgg tgcaacccgg aaggtccctg 2100

aggctctcct gtgccgccag cggcttcacc ttctccagct acggtatgca ctgggtgaga 2160

caagcccccg gaaagggcct cgagtgggtg gccgtgatct gggatgatgg ctccaacaag 2220

tactacgtgg acagcgtcaa gggcagattc accatcagca gggacaacag caagaacacc 2280

ctgtacctgc agatgaactc cctgagagcc gaagacaccg ccgtgtacta ttgtgccagg 2340

gacgactact atggctccgg ctccttcaat agctactatg gcaccgacgt gtggggccag 2400

ggcaccacag tgacagtgag cagcggcgga ggaggatccg gaggaggagg aagcggagga 2460

ggaggaagcg agatcgtgct gacacagtcc cccgctaccc tgagcctgag ccccggcgag 2520

agagctaccc tgagctgcag agccagccag agcgtctcca tctacctggc ctggtaccag 2580

cagaagcctg gccaggcccc tagactgctg atctacgacg ccagcaacag ggccaccggc 2640

attcctgcca gattcagcgg ctccggctcc ggcaccgatt tcacactgac catcagctcc 2700

ctggagcctg aggacttcgc cgtgtattac tgccagcaga ggagcaactg gccccccttt 2760

accttcggcc ccggcaccaa ggtcgacatc aagagtgctg ctgcctttgt cccggtattt 2820

ctcccagcca aaccgaccac gactcccgcc ccgcgccctc cgacacccgc tcccaccatc 2880

gcctctcaac ctcttagtct tcgccccgag gcatgccgac ccgccgccgg gggtgctgtt 2940

catacgaggg gcttggactt cgcttgtgat atttacattt gggctccgtt ggcgggtacg 3000

tgcggcgtcc ttttgttgtc actcgttatt actttgtatt gtaatcacag gaatcgcaaa 3060

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 3120

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 3180

ctgcgagtga agttttcccg aagcgcagac gctccggcat atcagcaagg acagaatcag 3240

ctgtataacg aactgaattt gggacgccgc gaggagtatg acgtgcttga taaacgccgg 3300

gggagagacc cggaaatggg gggtaaaccc cgaagaaaga atccccaaga aggactctac 3360

aatgaactcc agaaggataa gatggcggag gcctactcag aaataggtat gaagggcgaa 3420

cgacgacggg gaaaaggtca cgatggcctc taccaagggt tgagtacggc aaccaaagat 3480

acgtacgatg cactgcatat gcaggccctg cctcccagat aataataaaa tcgctatcca 3540

tcgaagatgg atgtgtgttg gttttttgtg tgtggagcaa caaatctgac tttgcatgtg 3600

caaacgcctt caacaacagc attattccag aagacacctt cttccccagc ccaggtaagg 3660

gcagctttgg tgccttcgca ggctgtttcc ttgcttcagg aatggccagg ttctgcccag 3720

agctctggtc aatgatgtct aaaactcctc tgattggtgg tctcggcctt atccattgcc 3780

accaaaaccc tctttttact aagaaacagt gagccttgtt ctggcagtcc agagaatgac 3840

acgggaaaaa agcagatgaa gagaaggtgg caggagaggg cacgtggccc agcctcagtc 3900

tctccaactg agttcctgcc tgcctgcctt tgctcagact gtttgcccct tactgctctt 3960

ctaggcctca ttctaagccc cttctccaag ttgcctctcc ttatttctcc ctgtctgcca 4020

aaaaatcttt cccagctcac taagtcagtc tcacgcagtc actcattaac ccaccaatca 4080

ctgattgtgc cggcacatga atgcaccagg tgttgaagtg gaggaattaa aaagtcagat 4140

gaggggtgtg cccagaggaa gcaccattct agttggggga gcccatctgt cagctgggaa 4200

aagtccaaat aacttcagat tggaatgtgt tttaactcag ggttgagaaa acagctacct 4260

tcaggacaaa agtcagggaa gggctctctg aagaaatgct acttgaagat accagcccta 4320

ccaagggcag ggagaggacc ctatagaggc ctgggacagg agctcaatga gaaagg 4376

<210> 65

<211> 1527

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 65

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccggaaattg tgctgaccca gagcccggcg accctgagcc tgagcccggg cgaacgcgcg 120

accctgagct gccgcgcgag ccagagcgtg agcatttatc tggcgtggta tcagcagaaa 180

ccgggccagg cgccgcgcct gctgatttat gatgcgagca accgcgcgac cggcattccg 240

gcgcgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctggaa 300

ccggaagatt ttgcggtgta ttattgccag cagcgcagca actggccgcc gtttaccttt 360

ggcccgggca ccaaagtgga tattaaaggc ggcggcggca gcggcggcgg cggcagcggc 420

ggcggcggca gccaggtgca gctggtggaa agcggcggcg gcgtggtgca gccgggccgc 480

agcctgcgcc tgagctgcgc ggcgagcggc tttaccttta gcagctatgg catgcattgg 540

gtgcgccagg cgccgggcaa aggcctggaa tgggtggcgg tgatttggga tgatggcagc 600

aacaaatatt atgtggatag cgtgaaaggc cgctttacca ttagccgcga taacagcaaa 660

aacaccctgt atctgcagat gaacagcctg cgcgcggaag ataccgcggt gtattattgc 720

gcgcgcgatg attattatgg cagcggcagc tttaacagct attatggcac cgatgtgtgg 780

ggccagggca ccaccgtgac cgtgagcagc gcggcggcgt ttgtgccggt gtttctgccg 840

gcgaaaccga ccaccacccc ggcgccgcgc ccgccgaccc cggcgccgac cattgcgagc 900

cagccgctga gcctgcgccc ggaagcgtgc cgcccggcgg cgggcggcgc ggtgcatacc 960

cgcggcctgg attttgcgtg cgatatttat atttgggcgc cgctggcggg cacctgcggc 1020

gtgctgctgc tgagcctggt gattaccctg tattgcaacc atcgcaaccg cagcaaacgc 1080

agccgcctgc tgcatagcga ttatatgaac atgaccccgc gccgcccggg cccgacccgc 1140

aaacattatc agccgtatgc gccgccgcgc gattttgcgg cgtatcgcag ccgcgtgaaa 1200

tttagccgca gcgcggatgc gccggcgtat cagcagggcc agaaccagct gtataacgaa 1260

ctgaacctgg gccgccgcga agaatatgat gtgctggata aacgccgcgg ccgcgatccg 1320

gaaatgggcg gcaaaccgcg ccgcaaaaac ccgcaggaag gcctgtataa cgaactgcag 1380

aaagataaaa tggcggaagc gtatagcgaa attggcatga aaggcgaacg ccgccgcggc 1440

aaaggccatg atggcctgta tcagggcctg agcaccgcga ccaaagatac ctatgatgcg 1500

ctgcatatgc aggcgctgcc gccgcgc 1527

<210> 66

<211> 509

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 66

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

85 90 95

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg

100 105 110

Ser Asn Trp Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

115 120 125

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

145 150 155 160

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

165 170 175

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

180 185 190

Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

195 200 205

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

210 215 220

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly

245 250 255

Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ala

260 265 270

Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

355 360 365

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

370 375 380

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys

385 390 395 400

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

405 410 415

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

420 425 430

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

435 440 445

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

450 455 460

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

465 470 475 480

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

485 490 495

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

<210> 67

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 67

gaaattgtgc tgacccagag cccggcgacc ctgagcctga gcccgggcga acgcgcgacc 60

ctgagctgcc gcgcgagcca gagcgtgagc atttatctgg cgtggtatca gcagaaaccg 120

ggccaggcgc cgcgcctgct gatttatgat gcgagcaacc gcgcgaccgg cattccggcg 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg 240

gaagattttg cggtgtatta ttgccagcag cgcagcaact ggccgccgtt tacctttggc 300

ccgggcacca aagtggatat taaaggcggc ggcggcagcg gcggcggcgg cagcggcggc 360

ggcggcagcc aggtgcagct ggtggaaagc ggcggcggcg tggtgcagcc gggccgcagc 420

ctgcgcctga gctgcgcggc gagcggcttt acctttagca gctatggcat gcattgggtg 480

cgccaggcgc cgggcaaagg cctggaatgg gtggcggtga tttgggatga tggcagcaac 540

aaatattatg tggatagcgt gaaaggccgc tttaccatta gccgcgataa cagcaaaaac 600

accctgtatc tgcagatgaa cagcctgcgc gcggaagata ccgcggtgta ttattgcgcg 660

cgcgatgatt attatggcag cggcagcttt aacagctatt atggcaccga tgtgtggggc 720

cagggcacca ccgtgaccgt gagcagc 747

<210> 68

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 68

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Asp

165 170 175

Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asp Tyr

210 215 220

Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly Thr Asp Val Trp Gly

225 230 235 240

Gln Gly Thr Thr Val Thr Val Ser Ser

245

<210> 69

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 69

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly

100 105 110

Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 70

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 70

Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu

1 5 10 15

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr Leu

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 71

<211> 4370

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 71

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840

cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900

gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960

gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020

ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080

gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140

tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200

tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260

tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320

tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380

tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440

ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500

ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560

gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620

agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680

aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740

gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800

ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860

agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920

cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980

accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040

aggccggaga tcgtgctgac ccagagccct gccacactga gcctgagccc cggagagagg 2100

gctaccctga gctgcagggc ctcccagtcc gtgagcatct acctggcctg gtaccagcag 2160

aaacctggcc aggcccccag gctgctgatc tacgacgcca gcaatagggc caccggaatc 2220

cctgccaggt ttagcggctc cggaagcggc accgacttca ccctgaccat ctcctccctg 2280

gagcccgagg atttcgccgt gtactactgc cagcagaggt ccaactggcc tccctttacc 2340

ttcggccccg gcaccaaggt ggatattaag ggcggcggcg gatccggagg aggaggcagc 2400

ggaggaggag gaagccaggt gcaactggtg gagtccggcg gaggcgtggt gcaacctggc 2460

agaagcctga ggctgagctg tgccgccagc ggcttcacct tcagcagcta cggtatgcac 2520

tgggtgaggc aggctcccgg aaagggcctg gaatgggtgg ccgtgatctg ggacgacggc 2580

tccaacaagt actacgtgga ctccgtgaag ggcaggttca ccatcagcag ggacaactcc 2640

aagaacacac tgtacctgca gatgaacagc ctgagggccg aggataccgc tgtgtattac 2700

tgcgccaggg acgattacta cggcagcggc agcttcaatt cctactacgg aaccgacgtc 2760

tggggccagg gaaccaccgt gaccgtgagc agcagtgctg ctgcctttgt cccggtattt 2820

ctcccagcca aaccgaccac gactcccgcc ccgcgccctc cgacacccgc tcccaccatc 2880

gcctctcaac ctcttagtct tcgccccgag gcatgccgac ccgccgccgg gggtgctgtt 2940

catacgaggg gcttggactt cgcttgtgat atttacattt gggctccgtt ggcgggtacg 3000

tgcggcgtcc ttttgttgtc actcgttatt actttgtatt gtaatcacag gaatcgctca 3060

aagcggagta ggttgttgca ttccgattac atgaatatga ctcctcgccg gcctgggccg 3120

acaagaaaac attaccaacc ctatgccccc ccacgagact tcgctgcgta caggtcccga 3180

gtgaagtttt cccgaagcgc agacgctccg gcatatcagc aaggacagaa tcagctgtat 3240

aacgaactga atttgggacg ccgcgaggag tatgacgtgc ttgataaacg ccgggggaga 3300

gacccggaaa tggggggtaa accccgaaga aagaatcccc aagaaggact ctacaatgaa 3360

ctccagaagg ataagatggc ggaggcctac tcagaaatag gtatgaaggg cgaacgacga 3420

cggggaaaag gtcacgatgg cctctaccaa gggttgagta cggcaaccaa agatacgtac 3480

gatgcactgc atatgcaggc cctgcctccc agataataat aaaatcgcta tccatcgaag 3540

atggatgtgt gttggttttt tgtgtgtgga gcaacaaatc tgactttgca tgtgcaaacg 3600

ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccaggt aagggcagct 3660

ttggtgcctt cgcaggctgt ttccttgctt caggaatggc caggttctgc ccagagctct 3720

ggtcaatgat gtctaaaact cctctgattg gtggtctcgg ccttatccat tgccaccaaa 3780

accctctttt tactaagaaa cagtgagcct tgttctggca gtccagagaa tgacacggga 3840

aaaaagcaga tgaagagaag gtggcaggag agggcacgtg gcccagcctc agtctctcca 3900

actgagttcc tgcctgcctg cctttgctca gactgtttgc cccttactgc tcttctaggc 3960

ctcattctaa gccccttctc caagttgcct ctccttattt ctccctgtct gccaaaaaat 4020

ctttcccagc tcactaagtc agtctcacgc agtcactcat taacccacca atcactgatt 4080

gtgccggcac atgaatgcac caggtgttga agtggaggaa ttaaaaagtc agatgagggg 4140

tgtgcccaga ggaagcacca ttctagttgg gggagcccat ctgtcagctg ggaaaagtcc 4200

aaataacttc agattggaat gtgttttaac tcagggttga gaaaacagct accttcagga 4260

caaaagtcag ggaagggctc tctgaagaaa tgctacttga agataccagc cctaccaagg 4320

gcagggagag gaccctatag aggcctggga caggagctca atgagaaagg 4370

<210> 72

<211> 1488

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 72

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccggaaattg tgctgaccca gagcccgggc accctgagcc tgagcccggg cgaacgcgcg 120

accctgagct gccgcgcgag ccagagcgtg agcagcagct atctggcgtg gtatcagcag 180

aaaccgggcc aggcgccgcg cctgctgatt tatggcgcga gcagccgcgc gaccggcatt 240

ccggatcgct ttagcggcag cggcagcggc accgatttta ccctgaccat tagccgcctg 300

gaaccggaag attttgcggt gtattattgc cagcagtatg gcagcagccc gatgtatacc 360

tttggccagg gcaccaaact ggaaattaaa ggcggcggcg gcagcggcgg cggcggcagc 420

ggcggcggcg gcagcgaagt gcagctggtg cagagcggcg gcggcctggt gcatccgggc 480

ggcagcctgc gcctgagctg cgcgggcagc ggctttacct ttagcaccta tctgatgtat 540

tgggtgcgcc aggcgccggg caaaaccctg gaatgggtga gcgcgattgg cagcggcggc 600

gatacctatt atgcggatag cgtgaaaggc cgctttacca ttagccgcga taacgcgaaa 660

aacagcctgt atctgcagat gaacagcctg cgcgcggaag atatggcggt gtattattgc 720

gcgcgcggcc tgggctattg gggccagggc accctggtga ccgtgagcag cgcggcggcg 780

tttgtgccgg tgtttctgcc ggcgaaaccg accaccaccc cggcgccgcg cccgccgacc 840

ccggcgccga ccattgcgag ccagccgctg agcctgcgcc cggaagcgtg ccgcccggcg 900

gcgggcggcg cggtgcatac ccgcggcctg gattttgcgt gcgatattta tatttgggcg 960

ccgctggcgg gcacctgcgg cgtgctgctg ctgagcctgg tgattaccct gtattgcaac 1020

catcgcaacc gcagcaaacg cagccgcctg ctgcatagcg attatatgaa catgaccccg 1080

cgccgcccgg gcccgacccg caaacattat cagccgtatg cgccgccgcg cgattttgcg 1140

gcgtatcgca gccgcgtgaa atttagccgc agcgcggatg cgccggcgta tcagcagggc 1200

cagaaccagc tgtataacga actgaacctg ggccgccgcg aagaatatga tgtgctggat 1260

aaacgccgcg gccgcgatcc ggaaatgggc ggcaaaccgc gccgcaaaaa cccgcaggaa 1320

ggcctgtata acgaactgca gaaagataaa atggcggaag cgtatagcga aattggcatg 1380

aaaggcgaac gccgccgcgg caaaggccat gatggcctgt atcagggcct gagcaccgcg 1440

accaaagata cctatgatgc gctgcatatg caggcgctgc cgccgcgc 1488

<210> 73

<211> 496

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 73

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile

65 70 75 80

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

Tyr Gly Ser Ser Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

115 120 125

Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly

145 150 155 160

Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Thr

165 170 175

Tyr Leu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Thr Leu Glu Trp

180 185 190

Val Ser Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val

195 200 205

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

210 215 220

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys

225 230 235 240

Ala Arg Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His

340 345 350

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

355 360 365

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

370 375 380

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

385 390 395 400

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

405 410 415

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

420 425 430

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 74

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 74

gaaattgtgc tgacccagag cccgggcacc ctgagcctga gcccgggcga acgcgcgacc 60

ctgagctgcc gcgcgagcca gagcgtgagc agcagctatc tggcgtggta tcagcagaaa 120

ccgggccagg cgccgcgcct gctgatttat ggcgcgagca gccgcgcgac cggcattccg 180

gatcgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag ccgcctggaa 240

ccggaagatt ttgcggtgta ttattgccag cagtatggca gcagcccgat gtataccttt 300

ggccagggca ccaaactgga aattaaaggc ggcggcggca gcggcggcgg cggcagcggc 360

ggcggcggca gcgaagtgca gctggtgcag agcggcggcg gcctggtgca tccgggcggc 420

agcctgcgcc tgagctgcgc gggcagcggc tttaccttta gcacctatct gatgtattgg 480

gtgcgccagg cgccgggcaa aaccctggaa tgggtgagcg cgattggcag cggcggcgat 540

acctattatg cggatagcgt gaaaggccgc tttaccatta gccgcgataa cgcgaaaaac 600

agcctgtatc tgcagatgaa cagcctgcgc gcggaagata tggcggtgta ttattgcgcg 660

cgcggcctgg gctattgggg ccagggcacc ctggtgaccg tgagcagc 708

<210> 75

<211> 236

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 75

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Thr Tyr Leu Met Tyr Trp

145 150 155 160

Val Arg Gln Ala Pro Gly Lys Thr Leu Glu Trp Val Ser Ala Ile Gly

165 170 175

Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala Arg Gly Leu Gly

210 215 220

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

225 230 235

<210> 76

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 76

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Leu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Thr Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 77

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 77

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 78

<211> 4331

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 78

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840

cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900

gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960

gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020

ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080

gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140

tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200

tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260

tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320

tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380

tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440

ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500

ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560

gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620

agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680

aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740

gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800

ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860

agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920

cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980

accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040

aggccggaga tcgtgctgac ccagagcccc ggaacactga gcctgtcccc cggagaaaga 2100

gccacactgt cctgcagggc cagccagagc gtgagcagct cctacctggc ctggtaccag 2160

cagaagcctg gacaggcccc caggctgctg atttacggcg ccagcagcag ggccaccggc 2220

atccccgaca gattcagcgg atccggcagc ggcaccgact tcaccctgac catcagcagg 2280

ctggagcccg aggacttcgc tgtgtactac tgccagcagt acggcagcag ccccatgtac 2340

accttcggcc agggcaccaa gctggagatc aagggaggag gaggatccgg aggaggcgga 2400

agcggaggag gaggaagcga ggtgcagctg gtgcagagcg gcggaggact ggtgcatccc 2460

ggaggatccc tgagactgag ctgtgccggc agcggattca cattctccac ctacctgatg 2520

tactgggtga ggcaggcccc tggcaagacc ctggagtggg tgtccgccat tggctccggc 2580

ggagacacct attatgccga ctccgtcaag ggcaggttca ccatcagcag agacaacgcc 2640

aagaactccc tgtacctgca gatgaacagc ctgagggccg aggatatggc tgtgtactat 2700

tgcgctaggg gcctgggata ctggggccag ggaaccctgg tgaccgtgag ctccagtgct 2760

gctgcctttg tcccggtatt tctcccagcc aaaccgacca cgactcccgc cccgcgccct 2820

ccgacacccg ctcccaccat cgcctctcaa cctcttagtc ttcgccccga ggcatgccga 2880

cccgccgccg ggggtgctgt tcatacgagg ggcttggact tcgcttgtga tatttacatt 2940

tgggctccgt tggcgggtac gtgcggcgtc cttttgttgt cactcgttat tactttgtat 3000

tgtaatcaca ggaatcgctc aaagcggagt aggttgttgc attccgatta catgaatatg 3060

actcctcgcc ggcctgggcc gacaagaaaa cattaccaac cctatgcccc cccacgagac 3120

ttcgctgcgt acaggtcccg agtgaagttt tcccgaagcg cagacgctcc ggcatatcag 3180

caaggacaga atcagctgta taacgaactg aatttgggac gccgcgagga gtatgacgtg 3240

cttgataaac gccgggggag agacccggaa atggggggta aaccccgaag aaagaatccc 3300

caagaaggac tctacaatga actccagaag gataagatgg cggaggccta ctcagaaata 3360

ggtatgaagg gcgaacgacg acggggaaaa ggtcacgatg gcctctacca agggttgagt 3420

acggcaacca aagatacgta cgatgcactg catatgcagg ccctgcctcc cagataataa 3480

taaaatcgct atccatcgaa gatggatgtg tgttggtttt ttgtgtgtgg agcaacaaat 3540

ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac accttcttcc 3600

ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct tcaggaatgg 3660

ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt ggtggtctcg 3720

gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc ttgttctggc 3780

agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga gagggcacgt 3840

ggcccagcct cagtctctcc aactgagttc ctgcctgcct gcctttgctc agactgtttg 3900

ccccttactg ctcttctagg cctcattcta agccccttct ccaagttgcc tctccttatt 3960

tctccctgtc tgccaaaaaa tctttcccag ctcactaagt cagtctcacg cagtcactca 4020

ttaacccacc aatcactgat tgtgccggca catgaatgca ccaggtgttg aagtggagga 4080

attaaaaagt cagatgaggg gtgtgcccag aggaagcacc attctagttg ggggagccca 4140

tctgtcagct gggaaaagtc caaataactt cagattggaa tgtgttttaa ctcagggttg 4200

agaaaacagc taccttcagg acaaaagtca gggaagggct ctctgaagaa atgctacttg 4260

aagataccag ccctaccaag ggcagggaga ggaccctata gaggcctggg acaggagctc 4320

aatgagaaag g 4331

<210> 79

<211> 1491

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 79

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccggaagtgc agctggtgca gagcggcggc ggcctggtgc atccgggcgg cagcctgcgc 120

ctgagctgcg cgggcagcgg ctttaccttt agcacctatc tgatgtattg ggtgcgccag 180

gcgccgggca aaaccctgga atgggtgagc gcgattggca gcggcggcga tacctattat 240

gcggatagcg tgaaaggccg ctttaccatt agccgcgata acgcgaaaaa cagcctgtat 300

ctgcagatga acagcctgcg cgcggaagat atggcggtgt attattgcgc gcgcggcctg 360

ggctattggg gccagggcac cctggtgacc gtgagcagcg gcggcggcgg cagcggcggc 420

ggcggcagcg gcggcggcgg cagcgaaatt gtgctgaccc agagcccggg caccctgagc 480

ctgagcccgg gcgaacgcgc gaccctgagc tgccgcgcga gccagagcgt gagcagcagc 540

tatctggcgt ggtatcagca gaaaccgggc caggcgccgc gcctgctgat ttatggcgcg 600

agcagccgcg cgaccggcat tccggatcgc tttagcggca gcggcagcgg caccgatttt 660

accctgacca ttagccgcct ggaaccggaa gattttgcgg tgtattattg ccagcagtat 720

ggcagcagcc cgatgtatac ctttggccag ggcaccaaac tggaaattaa aagcgcggcg 780

gcgtttgtgc cggtgtttct gccggcgaaa ccgaccacca ccccggcgcc gcgcccgccg 840

accccggcgc cgaccattgc gagccagccg ctgagcctgc gcccggaagc gtgccgcccg 900

gcggcgggcg gcgcggtgca tacccgcggc ctggattttg cgtgcgatat ttatatttgg 960

gcgccgctgg cgggcacctg cggcgtgctg ctgctgagcc tggtgattac cctgtattgc 1020

aaccatcgca accgcagcaa acgcagccgc ctgctgcata gcgattatat gaacatgacc 1080

ccgcgccgcc cgggcccgac ccgcaaacat tatcagccgt atgcgccgcc gcgcgatttt 1140

gcggcgtatc gcagccgcgt gaaatttagc cgcagcgcgg atgcgccggc gtatcagcag 1200

ggccagaacc agctgtataa cgaactgaac ctgggccgcc gcgaagaata tgatgtgctg 1260

gataaacgcc gcggccgcga tccggaaatg ggcggcaaac cgcgccgcaa aaacccgcag 1320

gaaggcctgt ataacgaact gcagaaagat aaaatggcgg aagcgtatag cgaaattggc 1380

atgaaaggcg aacgccgccg cggcaaaggc catgatggcc tgtatcaggg cctgagcacc 1440

gcgaccaaag atacctatga tgcgctgcat atgcaggcgc tgccgccgcg c 1491

<210> 80

<211> 497

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 80

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu

20 25 30

Val His Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe

35 40 45

Thr Phe Ser Thr Tyr Leu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Thr Leu Glu Trp Val Ser Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

85 90 95

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser

145 150 155 160

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

165 170 175

Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala

180 185 190

Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro

195 200 205

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

210 215 220

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr

225 230 235 240

Gly Ser Ser Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr

260 265 270

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

275 280 285

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

290 295 300

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

305 310 315 320

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

325 330 335

Thr Leu Tyr Cys Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu

340 345 350

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

355 360 365

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

370 375 380

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

385 390 395 400

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

405 410 415

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

420 425 430

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

435 440 445

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

450 455 460

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

465 470 475 480

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

485 490 495

Arg

<210> 81

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 81

gaagtgcagc tggtgcagag cggcggcggc ctggtgcatc cgggcggcag cctgcgcctg 60

agctgcgcgg gcagcggctt tacctttagc acctatctga tgtattgggt gcgccaggcg 120

ccgggcaaaa ccctggaatg ggtgagcgcg attggcagcg gcggcgatac ctattatgcg 180

gatagcgtga aaggccgctt taccattagc cgcgataacg cgaaaaacag cctgtatctg 240

cagatgaaca gcctgcgcgc ggaagatatg gcggtgtatt attgcgcgcg cggcctgggc 300

tattggggcc agggcaccct ggtgaccgtg agcagcggcg gcggcggcag cggcggcggc 360

ggcagcggcg gcggcggcag cgaaattgtg ctgacccaga gcccgggcac cctgagcctg 420

agcccgggcg aacgcgcgac cctgagctgc cgcgcgagcc agagcgtgag cagcagctat 480

ctggcgtggt atcagcagaa accgggccag gcgccgcgcc tgctgattta tggcgcgagc 540

agccgcgcga ccggcattcc ggatcgcttt agcggcagcg gcagcggcac cgattttacc 600

ctgaccatta gccgcctgga accggaagat tttgcggtgt attattgcca gcagtatggc 660

agcagcccga tgtatacctt tggccagggc accaaactgg aaattaaa 708

<210> 82

<211> 236

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 82

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Leu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Thr Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

130 135 140

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr

145 150 155 160

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

195 200 205

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Met

210 215 220

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 83

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 83

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Leu Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Thr Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 84

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 84

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 85

Thr Tyr Leu Met Tyr

1 5

<210> 86

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 86

Ala Ile Gly Ser Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 87

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 87

Gly Leu Gly Tyr

1

<210> 88

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 88

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 89

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 89

Gly Ala Ser Ser Arg Ala Thr

1 5

<210> 90

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 90

Gln Gln Tyr Gly Ser Ser Pro Met Tyr Thr

1 5 10

<210> 91

<211> 4331

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 91

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840

cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900

gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960

gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020

ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080

gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140

tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200

tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260

tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320

tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380

tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440

ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500

ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560

gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620

agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680

aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740

gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800

ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860

agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920

cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980

accatggcgc ttccggtgac agcactgctc ctccccttgg cgctgttgct ccacgcagca 2040

aggccggagg tccagctggt gcagagcgga ggcggactgg tgcatcctgg aggctccctg 2100

agactgtcct gtgccggcag cggcttcacc ttcagcacct acctgatgta ctgggtgaga 2160

caggcccccg gcaaaaccct ggagtgggtg agcgctatcg gcagcggcgg agacacatac 2220

tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacgccaa gaactccctg 2280

tacctgcaga tgaactccct gagggctgag gacatggccg tgtactactg cgctagaggc 2340

ctgggctact ggggacaggg cacactggtg acagtgagca gcggaggcgg cggcagcgga 2400

ggcggcggca gcggcggcgg aggcagcgag atcgtgctga cacagagccc tggcaccctg 2460

tccctgtccc ctggcgaaag ggccaccctg agctgtaggg ccagccagtc cgtgagcagc 2520

agctatctgg cctggtacca gcagaaaccc ggccaggccc ctagactgct gatctacggc 2580

gcctcctcca gagccaccgg aatccccgat agattcagcg gctccggcag cggcaccgat 2640

ttcacactga ccatcagcag gctggagccc gaggacttcg ccgtgtatta ctgccagcag 2700

tacggcagca gccctatgta cacattcggc cagggcacca agctggagat caagagtgct 2760

gctgcctttg tcccggtatt tctcccagcc aaaccgacca cgactcccgc cccgcgccct 2820

ccgacacccg ctcccaccat cgcctctcaa cctcttagtc ttcgccccga ggcatgccga 2880

cccgccgccg ggggtgctgt tcatacgagg ggcttggact tcgcttgtga tatttacatt 2940

tgggctccgt tggcgggtac gtgcggcgtc cttttgttgt cactcgttat tactttgtat 3000

tgtaatcaca ggaatcgctc aaagcggagt aggttgttgc attccgatta catgaatatg 3060

actcctcgcc ggcctgggcc gacaagaaaa cattaccaac cctatgcccc cccacgagac 3120

ttcgctgcgt acaggtcccg agtgaagttt tcccgaagcg cagacgctcc ggcatatcag 3180

caaggacaga atcagctgta taacgaactg aatttgggac gccgcgagga gtatgacgtg 3240

cttgataaac gccgggggag agacccggaa atggggggta aaccccgaag aaagaatccc 3300

caagaaggac tctacaatga actccagaag gataagatgg cggaggccta ctcagaaata 3360

ggtatgaagg gcgaacgacg acggggaaaa ggtcacgatg gcctctacca agggttgagt 3420

acggcaacca aagatacgta cgatgcactg catatgcagg ccctgcctcc cagataataa 3480

taaaatcgct atccatcgaa gatggatgtg tgttggtttt ttgtgtgtgg agcaacaaat 3540

ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac accttcttcc 3600

ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct tcaggaatgg 3660

ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt ggtggtctcg 3720

gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc ttgttctggc 3780

agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga gagggcacgt 3840

ggcccagcct cagtctctcc aactgagttc ctgcctgcct gcctttgctc agactgtttg 3900

ccccttactg ctcttctagg cctcattcta agccccttct ccaagttgcc tctccttatt 3960

tctccctgtc tgccaaaaaa tctttcccag ctcactaagt cagtctcacg cagtcactca 4020

ttaacccacc aatcactgat tgtgccggca catgaatgca ccaggtgttg aagtggagga 4080

attaaaaagt cagatgaggg gtgtgcccag aggaagcacc attctagttg ggggagccca 4140

tctgtcagct gggaaaagtc caaataactt cagattggaa tgtgttttaa ctcagggttg 4200

agaaaacagc taccttcagg acaaaagtca gggaagggct ctctgaagaa atgctacttg 4260

aagataccag ccctaccaag ggcagggaga ggaccctata gaggcctggg acaggagctc 4320

aatgagaaag g 4331

<210> 92

<211> 804

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 92

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 60

gacaccttct tccccagccc aggtaagggc agctttggtg ccttcgcagg ctgtttcctt 120

gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa tgatgtctaa aactcctctg 180

attggtggtc tcggccttat ccattgccac caaaaccctc tttttactaa gaaacagtga 240

gccttgttct ggcagtccag agaatgacac gggaaaaaag cagatgaaga gaaggtggca 300

ggagagggca cgtggcccag cctcagtctc tccaactgag ttcctgcctg cctgcctttg 360

ctcagactgt ttgcccctta ctgctcttct aggcctcatt ctaagcccct tctccaagtt 420

gcctctcctt atttctccct gtctgccaaa aaatctttcc cagctcacta agtcagtctc 480

acgcagtcac tcattaaccc accaatcact gattgtgccg gcacatgaat gcaccaggtg 540

ttgaagtgga ggaattaaaa agtcagatga ggggtgtgcc cagaggaagc accattctag 600

ttgggggagc ccatctgtca gctgggaaaa gtccaaataa cttcagattg gaatgtgttt 660

taactcaggg ttgagaaaac agctaccttc aggacaaaag tcagggaagg gctctctgaa 720

gaaatgctac ttgaagatac cagccctacc aagggcaggg agaggaccct atagaggcct 780

gggacaggag ctcaatgaga aagg 804

<210> 93

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 93

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 94

<211> 261

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 94

gctgctgcct ttgtcccggt atttctccca gccaaaccga ccacgactcc cgccccgcgc 60

cctccgacac ccgctcccac catcgcctct caacctctta gtcttcgccc cgaggcatgc 120

cgacccgccg ccgggggtgc tgttcatacg aggggcttgg acttcgcttg tgatatttac 180

atttgggctc cgttggcggg tacgtgcggc gtccttttgt tgtcactcgt tattactttg 240

tattgtaatc acaggaatcg c 261

<210> 95

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 95

Ser Ala Ala Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr

1 5 10 15

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

20 25 30

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

35 40 45

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

50 55 60

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

65 70 75 80

Leu Tyr Cys Asn His Arg Asn Arg

85

<210> 96

<211> 252

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 96

tttgtcccgg tatttctccc agccaaaccg accacgactc ccgccccgcg ccctccgaca 60

cccgctccca ccatcgcctc tcaacctctt agtcttcgcc ccgaggcatg ccgacccgcc 120

gccgggggtg ctgttcatac gaggggcttg gacttcgctt gtgatattta catttgggct 180

ccgttggcgg gtacgtgcgg cgtccttttg ttgtcactcg ttattacttt gtattgtaat 240

cacaggaatc gc 252

<210> 97

<211> 84

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 97

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His Arg Asn Arg

<210> 98

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 98

cgagtgaagt tttcccgaag cgcagacgct ccggcatatc agcaaggaca gaatcagctg 60

tataacgaac tgaatttggg acgccgcgag gagtatgacg tgcttgataa acgccggggg 120

agagacccgg aaatgggggg taaaccccga agaaagaatc cccaagaagg actctacaat 180

gaactccaga aggataagat ggcggaggcc tactcagaaa taggtatgaa gggcgaacga 240

cgacggggaa aaggtcacga tggcctctac caagggttga gtacggcaac caaagatacg 300

tacgatgcac tgcatatgca ggccctgcct cccaga 336

<210> 99

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 99

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 100

<211> 800

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 100

gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60

gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120

tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180

ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240

ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300

gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360

ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420

agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480

atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540

tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600

gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660

gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720

aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780

catgaggtct atggacttca 800

<210> 101

<211> 1178

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 101

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360

agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420

ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480

tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540

aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600

cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660

cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720

gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780

ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840

cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900

cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 960

agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020

agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080

tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140

tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178

<210> 102

<211> 49

<212> DNA

<213> Synthesis of polynucleotides

<400> 102

aataaaatcg ctatccatcg aagatggatg tgtgttggtt ttttgtgtg 49

<210> 103

<400> 103

000

<210> 104

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 104

agagcaacag tgctgtggcc 20

<210> 105

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 105

agagcaacag ugcuguggcc 20

<210> 106

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 106

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 107

<211> 84

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 107

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His Arg Asn Arg

<210> 108

<211> 456

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 108

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asp Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Tyr Tyr Gly Ser Gly Ser Phe Asn Ser Tyr Tyr Gly

100 105 110

Thr Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225 230 235 240

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

355 360 365

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

435 440 445

Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 109

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 109

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 110

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 110

ccgccgcgau gggagcugcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 111

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 111

ccgccgcgau gggagcugcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 112

<211> 1530

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 112

atggcgcttc cggtgacagc actgctcctc cccttggcgc tgttgctcca cgcagcaagg 60

ccgcaggtgc agctggtgga gagcggcgga ggagtggtgc aacccggaag gtccctgagg 120

ctctcctgtg ccgccagcgg cttcaccttc tccagctacg gtatgcactg ggtgagacaa 180

gcccccggaa agggcctcga gtgggtggcc gtgatctggg atgatggctc caacaagtac 240

tacgtggaca gcgtcaaggg cagattcacc atcagcaggg acaacagcaa gaacaccctg 300

tacctgcaga tgaactccct gagagccgaa gacaccgccg tgtactattg tgccagggac 360

gactactatg gctccggctc cttcaatagc tactatggca ccgacgtgtg gggccagggc 420

accacagtga cagtgagcag cggcggagga ggatccggag gaggaggaag cggaggagga 480

ggaagcgaga tcgtgctgac acagtccccc gctaccctga gcctgagccc cggcgagaga 540

gctaccctga gctgcagagc cagccagagc gtctccatct acctggcctg gtaccagcag 600

aagcctggcc aggcccctag actgctgatc tacgacgcca gcaacagggc caccggcatt 660

cctgccagat tcagcggctc cggctccggc accgatttca cactgaccat cagctccctg 720

gagcctgagg acttcgccgt gtattactgc cagcagagga gcaactggcc cccctttacc 780

ttcggccccg gcaccaaggt cgacatcaag agtgctgctg cctttgtccc ggtatttctc 840

ccagccaaac cgaccacgac tcccgccccg cgccctccga cacccgctcc caccatcgcc 900

tctcaacctc ttagtcttcg ccccgaggca tgccgacccg ccgccggggg tgctgttcat 960

acgaggggct tggacttcgc ttgtgatatt tacatttggg ctccgttggc gggtacgtgc 1020

ggcgtccttt tgttgtcact cgttattact ttgtattgta atcacaggaa tcgctcaaag 1080

cggagtaggt tgttgcattc cgattacatg aatatgactc ctcgccggcc tgggccgaca 1140

agaaaacatt accaacccta tgccccccca cgagacttcg ctgcgtacag gtcccgagtg 1200

aagttttccc gaagcgcaga cgctccggca tatcagcaag gacagaatca gctgtataac 1260

gaactgaatt tgggacgccg cgaggagtat gacgtgcttg ataaacgccg ggggagagac 1320

ccggaaatgg ggggtaaacc ccgaagaaag aatccccaag aaggactcta caatgaactc 1380

cagaaggata agatggcgga ggcctactca gaaataggta tgaagggcga acgacgacgg 1440

ggaaaaggtc acgatggcct ctaccaaggg ttgagtacgg caaccaaaga tacgtacgat 1500

gcactgcata tgcaggccct gcctcccaga 1530

<210> 113

<211> 747

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of polynucleotides

<400> 113

caggtgcagc tggtggaaag cggcggcggc gtggtgcagc cgggccgcag cctgcgcctg 60

agctgcgcgg cgagcggctt tacctttagc agctatggca tgcattgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtggcggtg atttgggatg atggcagcaa caaatattat 180

gtggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcgatgat 300

tattatggca gcggcagctt taacagctat tatggcaccg atgtgtgggg ccagggcacc 360

accgtgaccg tgagcagcgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 420

agcgaaattg tgctgaccca gagcccggcg accctgagcc tgagcccggg cgaacgcgcg 480

accctgagct gccgcgcgag ccagagcgtg agcatttatc tggcgtggta tcagcagaaa 540

ccgggccagg cgccgcgcct gctgatttat gatgcgagca accgcgcgac cggcattccg 600

gcgcgcttta gcggcagcgg cagcggcacc gattttaccc tgaccattag cagcctggaa 660

ccggaagatt ttgcggtgta ttattgccag cagcgcagca actggccgcc gtttaccttt 720

ggcccgggca ccaaagtgga tattaaa 747

Claims (65)

1. An engineered T cell comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular domain that specifically binds PTK 7.

2. The engineered T-cell of claim 1, further comprising a disrupted T-cell receptor alpha chain constant region (TRAC) gene.

3. The engineered T cell of claim 1 or 2, further comprising a disrupted β -2-microglobulin (β 2M) gene.

4. The engineered T cell of any one of claims 1-3, wherein the extracellular domain of the CAR comprises an anti-PTK 7 antibody.

5. The engineered T cell of claim 4, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

6. The engineered T-cell of claim 5, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Region (CDR) and the same light chain variable domain (VL) CDR as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84.

7. The engineered T cell of claim 6, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

8. The engineered T-cell of claim 6, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, or 82.

9. The engineered T cell of any one of claims 1-8, wherein the CAR further comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

10. The engineered T cell of claim 9, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

11. The engineered T cell of any one of claims 1-10, wherein the CAR is encoded by the nucleotide sequence of any one of SEQ ID NOs 49, 51, 65, 72, 79, or 112 or a nucleotide sequence comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NOs 49, 51, 65, 72, 79, or 112.

12. The engineered T cell of any one of claims 1-11, wherein the nucleic acid encoding the CAR is inserted into the disrupted TRAC gene.

13. The engineered T-cell of any one of claims 2-12, wherein the disrupted TRAC gene comprises a LHA and/or RHA encoding nucleotide sequence within any one of SEQ ID NOs 63, 64, 71, 78 or 91, a nucleotide sequence of SEQ ID NOs 92 or 100, and/or a nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78 or 91.

14. The engineered T cell of any one of claims 1-13, wherein the disrupted β 2M gene comprises at least one nucleotide sequence selected from any one of SEQ ID NOs 9-14.

15. An engineered T cell comprising:

(i) a disrupted TRAC gene;

(ii) a disrupted β 2M gene; and

(iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment.

16. The engineered T cell of claim 15, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta cytoplasmic signaling domain.

17. The engineered T cell of claim 15 or 16, wherein the disrupted TRAC gene comprises a nucleic acid encoding the CAR.

18. An engineered T cell comprising:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 co-stimulatory domain and a CD3 zeta cytoplasmic signaling domain; and

(ii) disrupted beta 2M gene.

19. An engineered T cell comprising:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising the amino acid sequence set forth in any one of SEQ ID NOs 50, 52, 66, 73, or 80; and

(ii) disrupted beta 2M gene. 8

20. An engineered T cell comprising:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112 and encodes a CAR comprising the amino acid sequence of SEQ ID NO 50, 52, 66, 73, or 80; and

(ii) disrupted beta 2M gene.

21. The engineered T cell of any one of claims 1-20, wherein the T cell is a human T cell.

22. A population of cells comprising the engineered T cell of any one of claims 1-21, wherein at least 25% or at least 50% of the engineered T cells of the population express the CAR.

23. The population of claim 22, wherein at least 70% of the engineered T cells of the population express the CAR.

24. The population of claim 22, wherein at least 25% of the engineered T cells of the population express the CAR after at least 7 days or at least 14 days of in vitro proliferation.

25. The population of any one of claims 22-24, wherein at least 50% of the engineered T cells of the population do not express detectable levels of a T Cell Receptor (TCR) protein.

26. The population of claim 25, wherein at least 90% of the engineered T cells of the population do not express detectable levels of TCR protein.

27. The population of any one of claims 22-26, wherein at least 50% of the engineered T cells of the population do not express detectable levels of β 2M protein.

28. The population of claim 27, wherein at least 70% of the engineered T cells of the population do not express detectable levels of β 2M protein.

29. The population of any one of claims 22-28, wherein the engineered T cells of the population induce cell lysis of at least 10%, at least 25%, or at least 50% of the cancer cells of the population when co-cultured in vitro with a population of cancer cells expressing PTK 7.

30. The population of claim 29, wherein the engineered T cells of the population induce cell lysis of at least 70%, at least 80%, or at least 90% of the population of cancer cells that express PTK7 when co-cultured in vitro with the population of cancer cells.

31. The population of claim 29 or 30, wherein the engineered T cells of the population secrete IFN γ when co-cultured in vitro with a population of cancer cells.

32. The population of any one of claims 29-31, wherein the ratio of engineered T cells to cancer cells is from 1:1 to 2: 1.

33. The population of any one of claims 29-32, wherein the cancer cells comprise sarcoma cells.

34. The population of any one of claims 29-32, wherein the cancer cells comprise breast cancer cells, ovarian cancer cells, small cell lung cancer cells, and/or colon cancer cells.

35. The population of any one of claims 22-34, which, when administered in vivo to a subject, does not induce toxicity in the subject.

36. A cell population comprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene;

(ii) a disrupted β 2M gene; and

(iii) a nucleic acid encoding a CAR comprising an anti-PTK 7 antigen-binding fragment.

37. The population of cells of claim 36, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 costimulatory domain and a CD3 zeta cytoplasmic signaling domain.

38. The population of cells of claim 36 or 37, wherein the disrupted TRAC gene comprises a nucleic acid encoding the CAR.

39. A cell population comprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR comprising (a) an extracellular domain comprising an antigen-binding fragment of anti-PTK 7, (b) a CD8 transmembrane domain, and (c) an intracellular domain comprising a CD28 co-stimulatory domain and a CD3 zeta cytoplasmic signaling domain; and

(ii) disrupted beta 2M gene.

40. A cell population comprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises a nucleic acid encoding a CAR, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO 49, 51, 65, 72, 79, or 112 and encodes a CAR of SEQ ID NO 50, 52, 66, 73, or 80; and

(ii) disrupted beta 2M gene.

41. A method comprising administering to a subject the population of engineered T cells of any one of claims 22-40.

42. The method of claim 41, wherein the subject is a human subject.

43. The method of claim 42, wherein the subject has cancer.

44. The method of claim 43, wherein the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma.

45. The method of claim 43 or 44, wherein the cancer comprises cancer cells expressing PTK 7.

46. A method of producing an engineered T cell, the method comprising

(a) Delivery to T cells

(i) An RNA-guided nuclease which is capable of inducing a DNA sequence,

(ii) gRNA targeting TRAC gene, and

(iii) a vector comprising a donor template, wherein the donor template comprises a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds PTK 7; and

(b) generating engineered T cells having a disrupted TRAC gene and expressing the CAR.

47. The method of claim 46, wherein the gRNA targeted to the TRAC gene comprises the nucleotide sequence of SEQ ID NO 18 or 19, or the nucleotide sequence targeted to SEQ ID NO 40.

48. The method of claim 46 or 47, further comprising: delivering a gRNA targeting the β 2M gene to the T cell.

49. The method of claim 48, wherein the gRNA targeted to the β 2M gene comprises the nucleotide sequence of SEQ ID NO 20 or 21, or the nucleotide sequence targeted to SEQ ID NO 41.

50. The method of any of claims 46-49, wherein the extracellular domain of the CAR comprises an anti-PTK 7 antibody.

51. The method of claim 50, wherein the anti-PTK 7 antibody is an anti-PTK 7 single chain variable fragment (scFv).

52. The method of claim 51, wherein the anti-PTK 7 scFv comprises the same heavy chain variable domain (VH) Complementarity Determining Region (CDR) and the same light chain variable domain (VL) CDR as a reference antibody, wherein the reference antibody comprises (i) the VH shown in SEQ ID NO:55 and the VL shown in SEQ ID NO:56, (ii) the VH shown in SEQ ID NO:69 and the VL shown in SEQ ID NO:70, (iii) the VH shown in SEQ ID NO:76 and the VL shown in SEQ ID NO:77, or (iv) the VH shown in SEQ ID NO:83 and the VL shown in SEQ ID NO: 84.

53. The method of claim 52, wherein the anti-PTK 7 scFv comprises the same VH and VL chains as the reference antibody.

54. The method of claim 52, wherein the anti-PTK 7 scFv comprises the amino acid sequence of any one of SEQ ID NOs 54, 68, 75, or 82.

55. The method of any of claims 46-54, wherein the CAR further comprises a CD28 co-stimulatory domain or a41 BB co-stimulatory domain.

56. The method of claim 55, wherein the CAR further comprises a CD3 ζ cytoplasmic signaling domain.

57. The method of any of claims 46-56, wherein the CAR is encoded by the nucleotide sequence of any of SEQ ID NOs 49, 51, 65, 72, 79, or 112 or a nucleotide sequence comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NOs 49, 51, 65, 72, 79, or 112.

58. The method of any of claims 46-57, wherein the nucleic acid encoding the CAR is flanked by the left and right homology arms of a TRAC gene.

59. The method of any one of claims 46-58, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs 63, 64, 71, 78 or 91.

60. The method of any one of claims 46-59, wherein the RNA-guided nuclease is Cas9 nuclease, optionally Streptococcus pyogenes (S.pyogenes) Cas9 nuclease.

61. An engineered T cell produced by the method of any one of claims 46-60.

62. A cell population comprising the engineered T-cell of claim 61.

63. A method of treating cancer in a subject, the method comprising administering to the subject the population of cells of any one of claims 22-40 or 62.

64. The method of claim 63, wherein the cancer is selected from the group consisting of: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung cancer (NSCLC), glioblastoma, neuronal tumors, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, glioblastoma, hepatocellular carcinoma, hepatobiliary cell carcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, renal cancer, and intrahepatic bile duct carcinoma.

65. The method of claim 63 or 64, wherein the cancer comprises cancer cells expressing PTK 7.