Composition of NY-ESO-1 specific T cell receptors restricted on multiple major histocompatibility complex molecules

Abstract

Tumor-specific T-cell receptor (TCR) gene transfer enables specific and potent immunotargeting of tumor antigens. The typical cancer-testis antigen NY-ESO-1 is not expressed in normal tissues but is aberrantly expressed in a wide range of cancer types. It has also been targeted by A2-restricted TCR gene therapy without adverse events or significant side effects. To achieve targeting of NY-ESO-1 across a broader range of HLA haplotypes, we isolated TCRs specific to the NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, HLA-B07, HLA-B18, and HLA-CO3. Using these TCRs, we have developed a pathway to extend NY-ESO-1-targeting TCR gene therapy to patient populations beyond those expressing HLA-A2.

Description

[0001] Cross-citation of related applications

[0002] This application claims the benefit of co-pending and co-assigned U.S. Provisional Patent Application Serial No. 62 / 727,485, filed September 5, 2018, entitled “COMPOSITION OF NY-ESO-1-SPECIFIC T CELL RECEPTORS RESTRICTED ON MULTIPLE MAJOR HISTO COMPPATIBILITY COMPLEX MOLECULES,” which is incorporated herein by reference. Statement regarding federally funded research and development.

[0003] This invention was developed with the support of government grant numbers CA132681 and CA197633 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention.

[0004] sequence list

[0005] This application contains a sequence list submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on August 28, 2019, is named 30435_364-WO-U1_SL.txt and is 101,375 bytes in size. Technical Field

[0006] This invention relates to methods and materials suitable for αβT cell receptor gene therapy. Background of the Invention

[0008] The αβ T cell receptor (TCR) determines the unique specificity of each naive T cell. After assembling with the CD3 signaling protein on the T cell surface, the TCR monitors peptide ligands presented by major histocompatibility complex (MHC) molecules on the surface of nucleated cells. For peptide-MHC complexes, TCR specificity is determined by both the presented MHC molecule and the presented peptide. MHC loci (also known as human leukocyte antigen (HLA) loci in humans) are the most numerous alleles in the human genome, containing >18,000 MHC class I and II alleles, with frequencies varying widely across subgroups (1,2). Ligands presented by MHC class I molecules primarily originate from proteasome cleavage of endogenously expressed antigens. Infected cancer cells present peptides that are recognized as foreign or aberrant by CD8+ T cells, leading to T cell-mediated killing of the presenting cells.

[0009] T cells can be engineered to kill tumor cells by transferring tumor-reactive αβTCR genes (3). The key to this approach is that the patient expresses a therapeutic TCR with its restricted MHC alleles, and the targeting peptide is derived from tumor-associated or tumor-specific antigens. Private (patient-specific) neoantigens generated by tumor-specific mutations are a potential source of such targets (4). However, the implementation of personalized TCR gene therapy is complicated by the need to identify mutations via sequencing, isolate mutation-reactive patient-specific TCRs, and genetically modify patient T cells on demand. This remains more challenging for tumors where sequencing is not possible and for tumors with low mutational burdens, such as those with little or no neoantigens (5). Especially for these final tumor types, targeting public (non-patient-specific) tumor-restricting antigens with existing TCRs remains an attractive option.

[0010] The first public antigen targeted by TCR gene therapy in clinical practice was the melanocyte antigen MART1 / Melan-A, which produced an objective response in 2 / 15 of patients with metastatic melanoma (6). Using a higher affinity MART1-responsive TCR (F5) can increase the response rate to 30%, but it also produces various side effects, including vitiligo, uveitis, and transient hearing loss, due to MART1 expression on healthy melanocytes in the skin, eyes, and middle ear (7). T-cell therapies targeting other public antigens have also produced dysmorphic or other serious adverse events due to on-target / extratumor reactivity. For example, targeting carcinoembryonic antigen has caused severe colitis in patients with metastatic colorectal cancer due to its reactivity with normal colorectal tissue (8). More seriously, T-cell therapies targeting ERBB2 or MAGE-A3 have resulted in death due to inaccurate expression of the target antigen (or similar variants) in vital organs (9,10). Therefore, these studies highlight the importance of rigorous identification of tumor-specific public antigens (11), especially when using high-affinity target receptors that are adequately expressed and necessary for therapeutic success (7,12).

[0011] The product of the CTAG1B gene, NY-ESO-1, is an attractive target for existing TCR gene therapies. As a prototypical onco-testis antigen, NY-ESO-1 is not expressed in normal non-germline tissues but is aberrantly expressed in many tumors (13). The frequency of aberrant expression in solid tumors ranges from 10-50%, in melanoma from 25-50%, and up to 80% in synovial sarcoma (13-18), with increased expression observed in higher-grade metastatic tumor tissues (14,15,19). Furthermore, NY-ESO-1 is highly immunogenic, targeting multiple epitopes presented by various MHC alleles, and eliciting spontaneous and vaccine-induced T-cell immune responses (20-23). Therefore, in gene therapy trials, the epitope NY-ESO-1157-165 (SLLMWITQC, (SEQ ID NO.36)) presented by HLA-A*02:01 has been targeted by homologous 1G4 TCRs, producing objective response rates of 55% and 61% in patients with metastatic melanoma and synovial sarcoma, respectively, without any target-related adverse events (24,25). Targeting this same A2-restricted epitope with lentivirus-mediated TCR gene therapy in patients with multiple myeloma also produced 70% complete or near-complete responses without significant safety concerns (26). However, regrettably, most patients who responded to the therapy reported relapse within months, and loss of heterozygosity at the MHCI locus is a mechanism by which tumors escape adoptive T-cell therapy targeting HLA-A*02:01 / NY-ESO-1157-165 (27). Therefore, NY-ESO-1 is a tumor-specific immunogenic public antigen that is expressed in multiple tumor types and is safely targeted in clinical practice, but it can easily escape when targeted by a single HLA subtype.

[0012] For the reasons mentioned above, there is a need in the art for alternative methods and materials suitable for NY-ESO-1TCR gene therapy. Summary of the Invention

[0013] As described above, T lymphocytes can be engineered to express tumor-specific T-cell receptor (TCR) genes, thereby killing cancer cells. This approach, known as TCR gene therapy, is effective, but can lead to serious adverse events if the target is also expressed in healthy, non-cancerous tissues. NY-ESO-1 is a tumor-specific antigen that has been successfully and safely targeted using TCR gene therapy for melanoma, synovial sarcoma, and myeloma. However, to date, trials have focused only on a single NY-ESO-1-derived epitope presented on HLA-A*02:01, limiting its application in patients expressing this allele. As disclosed below, novel TCRs have been developed that collectively recognize multiple NY-ESO-1-derived epitopes presented by multiple MHC alleles. Therefore, a universal approach to expanding targeted immunotherapy to a wider range of MHC haplotypes is provided.

[0014] Embodiments of the present invention include CD 8 for manufacturing and using modifications. + Methods and materials for T cell therapy, including the modified CD8 + T cells contain nucleic acids encoding certain αβT cell receptor polypeptides. Embodiments of the invention include, for example, polynucleotides disposed in a vector, wherein said polynucleotides encode VαT cell receptor polypeptides and / or VβT cell receptor polypeptides. In a typical embodiment, when a Vα / VβT cell receptor containing VαT cell receptor polypeptides and / or VβT cell receptor polypeptides is located on CD8... + When expressed in T cells, it is present in CD8. + Heterogeneous Vα / Vβ T cell receptors expressed on the surface of T cells recognize NY-ESO-1 peptides associated with human leukocyte antigen A2, human leukocyte antigen B07, human leukocyte antigen B18, or human leukocyte antigen CO3. In the illustrative working embodiments of the invention disclosed herein, the heterogeneous T cell receptors comprise Vα / Vβ T cell receptors named “3A1”, “4A2”, “5G6”, “9D2”, “1E4”, “2B8”, or “3C7”.

[0015] Embodiments of the present invention also include many different TCR nucleic acids and peptides disclosed herein (e.g., αβTCR nucleic acids and peptides encoding TCR, named “3A1”, “4A2”, “5G6”, “9D2”, “1E4”, “2B8”, and “3C7”). For example, embodiments of the present invention include a composition comprising one or more polynucleotides (typically polynucleotides housed in one or more carriers) encoding TCR Vα and / or TCR Vβ polynucleotides, including: a polynucleotide encoding at least 3A1 TCRVα peptide (SEQ ID NO:3); a polynucleotide encoding at least 3A1 TCR Vβ peptide (SEQ ID NO:4); a polynucleotide encoding at least 4A2 TCR Vα peptide (SEQ ID NO:7); a polynucleotide encoding at least 4A2 TCR Vβ peptide (SEQ ID NO:37); a polynucleotide encoding at least 5G6 TCR Vα peptide (SEQ ID NO:10); a polynucleotide encoding at least 5G6 TCR Vβ peptide (SEQ ID NO:11); and a polynucleotide encoding at least 9D2 TCR Vα peptide (SEQ ID NO:37). Polynucleotides encoding at least 9D2 TCR Vβ polypeptide (SEQ ID NO:14); polynucleotides encoding at least 1E4 TCR Vα polypeptide (SEQ ID NO:18); polynucleotides encoding at least 1E4 TCR Vβ polypeptide (SEQ ID NO:19); polynucleotides encoding at least 2B8 TCR Vα polypeptide (SEQ ID NO:22); polynucleotides encoding at least 2B8 TCR Vβ polypeptide (SEQ ID NO:23); polynucleotides encoding at least 3C7 TCR Vα polypeptide (SEQ ID NO:26); or polynucleotides encoding at least 3C7 TCR Vβ polypeptide (SEQ ID NO:27). In a typical embodiment of the invention, these polynucleotides also encode additional amino acids, such as constant regions, TM domains, short cytoplasmic tails, etc., of α and / or β polypeptides. In an illustrative embodiment of the invention, the composition comprises a combination of polynucleotides encoding TCR Vα polypeptides encoding TCR Vα polypeptides. Vβ polypeptides are polynucleotides, wherein such polynucleotides are placed within one or more vectors such that Vα / VβTCR can be transduced in mammalian cells (e.g., CD8) using said vectors. + The heterologous Vα / VβTCR expressed on the surface of T cells recognizes the NY-ESO-1 peptide, which is associated with human leukocyte antigen.

[0016] On the other hand, the present invention includes CD8 for generating modifications. +A method for using T cells, the method comprising introducing a nucleic acid encoding a TCR polypeptide disclosed herein into T cells (e.g., CD8 cells obtained from an individual diagnosed with cancer expressing the NY-ESO-1 antigen). + In T cells. On the other hand, the present invention includes a composition comprising modified CD8 generated according to the methods described herein. + T cells. On the other hand, the present invention includes a method for treating diseases or symptoms characterized by NY-ESO-1 expression. The treatment method includes administering an effective amount of the modified CD8 described herein to a subject in need of this treatment. + T cells. In a typical embodiment of the invention, the subject has cancer. In some embodiments of the invention, the cancer cells form a solid tumor. In some embodiments of the invention, the cancer is melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

[0017] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. However, it should be understood that while the detailed description and specific embodiments indicate some implementations of the invention, they are given by way of example and not limitation. Many changes and modifications can be made within the scope of the invention without departing from its spirit, and the invention includes all such modifications. Attached Figure Description

[0018] Figure 1A -1E shows disclosures related to the expansion and isolation of NY-ESO-1 specific T cell clones. PBMCs were obtained from a metastatic melanoma patient. Representative HLA-A2 is shown. + HLA-Cw3 + Donor T-cell cloning strategy. Figure 1(A) provides a schematic diagram outlining the expansion and testing strategy for identifying NY-ESO-1 reactive T-cell clones. PBMCs were cultured with 28 NY-ESO-1 18-mer peptides (overlapping 12 amino acids) and then expanded for 10 days, followed by restimulation with individual peptides in the presence of BFA. Among those peptides containing full epitope sequences, epitopes presented by patient MHC alleles were stained red, blue, and green. Figure 1(B) provides a representative flow cytometry measurement of intracellular IFN-γ staining in expanded PBMCs restimulated with individual NY-ESO-1-derived 18-mer peptides. Figure 1(C) provides a schematic diagram outlining the re-expansion strategy using individual 9-mer or 10-mer peptides that have been proven to elicit T-cell responses. Figure 1(D) provides an illustration of CD3 prior to sorting. + CD8 +Representative flow cytometry data of the NY-ESO-1 reactive subset of T cells. Sorted cells expanded in the presence of IL-2 and irradiated autologous PBMCs. Figure 1(E) shows the CD3+ distribution after sorting. + CD8 + Representative flow cytometry data of the NY-ESO-1 reactive subset of T cells.

[0019] Figure 2A-2D This document provides disclosures related to the cloning and functional screening of the NY-ESO-1 specific T cell receptor. Figure 2(A) provides a schematic diagram of the functional TCR cloning strategy. For each TCR, two constructs incorporating human or murine TCR constant domains were prepared. Figure 2(B) provides the protein sequence (SEQ ID NO: 28) of NY-ESO-1 with the described epitopes relevant to this study. Figure 2(C) provides flow cytometry histograms comparing HLA-A2 / NY-ESO-1 specific TCRs transfected with HEK 293T cells transfected with only the vector backbone, the previously reported 1G4 TCR, and the novel A2-restricted NY-ESO-1 specific TCR. 157-165 Binding of the dextramer. Figure 2(D) provides flow cytometry histograms comparing the binding of HEK 293T cells transfected with either the vector backbone only or the novel NY-ESO-1-specific TCR shown, which restricts MHC alleles other than HLA-A2, to the dextramer of the peptide shown. Transfection experiments were performed twice, in duplicate each time. Representative histograms are shown.

[0020] Figures 3A-3F Public information relating to the function of A2-restricted NY-ESO-1-specific TCRs is provided. Figure 3(A) provides an overlay of representative flow cytometry plots comparing Jurkat and CD8 expressing A2-restricted TCRs with human or murine constant domains. + Jurkat cells against A2 / NY-ESO-1 157-165 Binding of the dextrorotatory isomer. Figure 3(B) shows the mean fluorescence intensity measurements of dextrorotatory isomer binding from two independent experiments as in A. Figure 3(C) shows the ratio of the mean fluorescence intensity measurements of dextrorotatory isomer binding from two independent experiments as in B. Figure 3(D) shows the ELISA, which measures and expresses A2 / MART. 26-35 Or A2 / NY-ESO-1 157-165 IL-2 secreted from TCR-transduced Jurkat cells after co-culturing K562 target cells with single-stranded trimers for 48 hours. The experiment was repeated three times, with two technical replicas per cell. Mean ± SD of representative experiments are shown. Figure 3(E) shows the ELISA measurements with melanoma cell lines M257 or A2.+ IFN-γ secreted from TCR-transduced PBMCs was co-cultured for 48 hours. Experiments were repeated at least three times, with two technical replicas per experiment. Mean ± SD of representative experiments are shown. Figure 3(F) shows the total area of ​​the green target over time as an IncuCyte measurement as GFP. + A2 + A measure of T cell-mediated killing transduced by the TCR in M257 cells. The mean ± SD of four technology replicas is shown.

[0021] Figure 4A-4J Public information relating to the in vivo antitumor efficacy of NY-ESO-1TCR-engineered human T cells is provided. Figures 4 (A and B) illustrate the following experimental designs: Figure 4 (A) generation of NY-ESO-1TCR-engineered human T cells, and Figure 4 (B) investigation of the antitumor efficacy of these engineered T cells in a NSG mouse xenograft model of human prostate tumor. PBMCs: peripheral blood mononuclear cells; NSG: immunodeficient NOD / SCID / γc - / - Mouse. Figure 4(C) shows a representative flow cytometry plot characterizing engineered human T cells present in the peripheral blood of experimental mice on day 14 following adoptive T cell transfer. Figure 4(D) provides an illustration of engineered human T cells (gated to LNGFR). + hCD45 + The persistence time course of TCR in peripheral blood of experimental mice. Figure 4 (E and F) provides the following mean fluorescence intensity measurements: Figure 4 (E) mouse TCR and Figure 4 (F) HLA-A2 / NY-ESO-1 dextrorotatory isomers targeting engineered human T cells in peripheral blood of experimental mice on day 14 after adoptive T cell transfer. Figure 4 (G and H) provides the following cross-sectional area measurements: Figure 4 (G) PC-3 / HLA-A2 and Figure 4 (H) PC-3 / HLA-A2 / NY-ESO-1 tumors. Figure 4 (I) provides immunohistochemical images showing representative tumor sections. CD3 + Cells were stained red. Top image scale bar: 500 μm; Bottom image scale bar: 50 μm. Figure 4(J) shows CD3. + Percentage of cell area to total tumor slice area. Representative from two experiments. Data are expressed as mean ± SEM (n = 4–5). ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA.

[0022] Figure 5A-5E provides disclosures relating to the function of NY-ESO-1-specific TCRs restricted in MHC alleles other than HLA-A2. Figure 5(A) provides an overlay of representative flow cytometry plots comparing Jurkat and CD8 TCRs expressing novel TCRs with human or murine constant domains. + Jurkat cells bind to the designated dextrorotatory isomer. Figure 5(B) shows the measurement results of the mean fluorescence intensity of the dextrorotatory isomer binding from two independent experiments as in (A). Figure 5(C) shows the measurement results of the corresponding ratio of the mean fluorescence intensity of the dextrorotatory isomer binding from two independent experiments as in (B). Figures 5(D) and E provide ELISA measurements of: IL-2 secreted from TCR-transduced Jurkat cells in Figure 5(D) or IFN-γ secreted from TCR-transduced PBMCs after co-culturing with K562 target cells expressing the indicated single-stranded trimer for 48 hours in Figure 5(E). The experiments were repeated three times, with two technical replicas each time. Mean ± SD of representative experiments are shown.

[0023] Figures 6A-6D provide disclosures related to the following: Targeting the NY-ESO-1 epitope, which is restricted at multiple MHC alleles, broadens the application of TCR gene therapy and makes it robust against loss of heterozygosity at MHC loci. T cells transduced with LNGFR-only, A2-restricted 3A1 TCR, or B7-restricted 1E4 TCR, or a 1:1 mixture of 3A1 and 1E4, were then treated with HLA-A2... + eGFP + Target cells, HLA-B7 + eGFP + Target cells or a 1:1 target cell mixture were co-cultured for 48 hours. Figures 6 (A and B) provide ELISA measurements of IFN-γ secreted from TCR-transduced PBMCs after co-culturing for 48 hours with PC-3 tumor cell lines engineered to express eGFP and HLA-A*02:01 or HLA-B*07:02, as shown in Figure 6 (A) M257 or Figure 6 (B). The PC-3 line was additionally engineered to express NY-ESO-1. The M257 line expressed endogenous NY-ESO-1. Experiments were repeated three times, with 4 or 8 replicates each time. Mean ± SD of representative experiments are shown. Figures 6 (C and D) show T cell-mediated killing of T cell derivatives of the Figure 6 (C) M257 and Figure 6 (D) PC-3 tumor cell lines as measured over time using IncuCyte live cell analysis. The total area of ​​green targets (indicating tumor cell density) measured at each time point within 48 hours for each treatment was normalized relative to treatment with LNGFR-transduced T cells. The experiment was repeated three times, with either four or eight replicas each time. Results from a representative eight-replica experiment are shown.

[0024] Figures 7A-7E Disclosures related to the determination of EC50 in NY-ESO-1 specific TCRs are provided. Figures 7 (A and B) show ELISA measurements of IFN-γ secreted from TCR-transduced PBMCs after co-culturing with K562 cells engineered to express HLA-A*02:01 and pulsed with varying concentrations of the peptides in Figure 7 (A) MART126-35 or (B) NYESO1157-165 for 48 hours. Figure 7 (CE) shows ELISA measurements of IFN-γ secreted from TCR-transduced PBMCs after co-culturing with K562 cells engineered to express HLA-B*07:02, HLA-B*18:01, or HLA-C*03:04 and pulsed with varying concentrations of the indicator peptides in Figure 7 (C), HLA-B*07:02, HLA-B*18:01, or HLA-C*03:04 for 48 hours. Mean ± SD of the two technical replicas are shown. EC50 values ​​and associated errors determined by nonlinear curve fitting are indicated.

[0025] Figures 8A-8C Disclosures relating to the establishment of xenograft tumor lines and the function of introduced T cells for in vivo experiments are provided. Figure 8(A) presents an ELISA measuring IFN-γ secreted from TCR-transduced PBMCs after co-culturing for 48 hours with derivatives of PC-3 prostate cancer cell lines engineered to express (left) HLA-A*02:01 and NY-ESO-1 whole proteins, (middle) HLA-A*02:01 alone, or (right) NY-ESO-1 whole protein alone. Mean ± SD of the two technical replicas is shown. Figure 8(B) presents an ELISA comparing IFN-γ secreted from TCR-transduced PBMCs after co-culturing for 48 hours with the indicated M257 or PC-3 target cells. On day 4 post-transduction, TCR-transduced PBMCs were sorted for CD3+ / LNGFR+, then amplified for 13 days before co-culture / ELISA assays and in vivo experiments. Mean ± SD of representative experiments with two technical replicas is shown. Figure 8(C) provides flow cytometry contour plots comparing the transduction (LNGFR+) levels of PBMCs transduced by TCR for in vivo experiments. Detailed Implementation

[0026] In the description of the embodiments, reference may be made to the accompanying drawings, which form a part thereof, and are illustrated by way of example of specific embodiments in which the invention may be practiced. It should be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. Many techniques and procedures described or referenced herein are fully understood and commonly used by those skilled in the art. Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this invention pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or convenience of reference, and the inclusion of such definitions herein is not necessarily to be construed as a considerable difference from the general understanding in the art.

[0027] NY-ESO-1 is a prime example of a cancer-testis antigen, whose expression is restricted in germ cells and placental cells, but is re-expressed in tumor cells. Expression of NY-ESO-1 has been reported in a wide variety of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer. Its ability to elicit spontaneous humoral and cellular immune responses, along with its restricted expression pattern, makes it a promising candidate target for cancer immunotherapy. See, for example, Thomas et al., FrontImmunol. 2018; 9:947. doi:10.3389 / fimmu.2018.00947.

[0028] The disclosure in this paper demonstrates the achievement of two important goals related to methods and materials applicable to NY-ESO-1 TCR gene therapy. First, due to the greater effectiveness of TCRs with higher strength and affinity, the aim is to identify targets targeting A2 / NY-ESO-1. 157-165 The novel TCRs exhibited sensitivity comparable to or better than the clinically used 1G4 TCRs. Because the enhanced affinity TCRs can exhibit cross-reactivity (28-30), a protocol was developed for the direct isolation of antigen-reactive TCRs from patient blood. In tumor killing assays, two of these novel TCRs showed sensitivity comparable to or higher than 1G4, both in vitro and in vivo. Secondly, to broaden the clinical utility of NY-ESO-1 as a TCR gene therapy target, an isolation protocol was used to identify TCRs targeting NY-ESO-1 epitopes presented by common MHC alleles other than HLA-A*02:01. Targeting multiple NY-ESO-1 epitopes will enable the treatment of a larger patient population and make treatment more robust against tumor escape.

[0029] As described herein, the present invention provides methods and materials for preparing and using modified T cells containing nucleic acids encoding certain T cell receptor polypeptides. As used herein, the term "T cell receptor" or "TCR" refers to a membrane protein complex that participates in T cell activation in response to antigen presentation. The TCR is responsible for recognizing antigens that bind to major histocompatibility complex molecules. The TCR consists of heterodimers of alpha (α) and beta (β) chains, although in some cells, the TCR consists of γ and δ chains. The TCR can exist in α / β and γ / δ forms, which are structurally similar but have different anatomical locations and functions. Each chain consists of two extracellular domains: a variable domain and a constant domain. Embodiments of the present invention include many different TCR α / β nucleic acids and their encoded polypeptides (e.g., TCR nucleic acids and TCR-encoding polypeptides are named "3A1", "4A2", "5G6", "9D2", "1E4", "2B8", and "3C7").

[0030] Embodiments of the present invention include material compositions comprising one or more vectors containing the TCR polynucleotides disclosed herein. A “vector” is a material composition comprising isolated nucleic acids and usable for delivering the isolated nucleic acids into cells. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0031] Typically, the vector is an expression vector. As used herein, the term "expression" is defined as transcription and / or translation of a specific nucleotide sequence driven by its promoter. In this context, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operatively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating recombinant polynucleotides.

[0032] Embodiments of the present invention include, for example, polynucleotides disposed in an expression vector, wherein said polynucleotides encode VαT cell receptor polypeptides and / or VβT cell receptor polypeptides. In such embodiments, when a Vα / VβT cell receptor comprising VαT cell receptor polypeptides and / or VβT cell receptor polypeptides is expressed on CD8... + When expressed in T cells, the Vα / Vβ T cell receptor recognizes the NY-ESO-1 peptide associated with human leukocyte antigen A2, human leukocyte antigen B07, human leukocyte antigen B18, or human leukocyte antigen CO3. In the working embodiments of the invention disclosed herein, the modified CD8... + T cell receptors include 3A1 T cell receptor, 4A2 T cell receptor, 5G6 T cell receptor, 9D2 T cell receptor, 1E4 T cell receptor, 2B8 T cell receptor, or 3C7 T cell receptor.

[0033] In a typical embodiment of the present invention, the vector comprises at least one of the following: a polynucleotide encoding a 3A1 TCR Vα polypeptide (SEQ ID NO:3); a polynucleotide encoding a 3A1 TCR Vβ polypeptide (SEQ ID NO:4); a polynucleotide encoding a 4A2 TCR Vα polypeptide (SEQ ID NO:7); a polynucleotide encoding a 4A2 TCR Vβ polypeptide (SEQ ID NO:37); a polynucleotide encoding a 5G6 TCR Vα polypeptide (SEQ ID NO:10); a polynucleotide encoding a 5G6 TCR Vβ polypeptide (SEQ ID NO:11); a polynucleotide encoding a 9D2 TCR Vα polypeptide (SEQ ID NO:14); a polynucleotide encoding a 9D2 TCR Vβ polypeptide (SEQ ID NO:15); a polynucleotide encoding a 1E4 TCR Vα polypeptide (SEQ ID NO:18); a polynucleotide encoding a 1E4 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B8 TCR Vα polypeptide (SEQ ID NO:22); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:22); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B8 TCR Vα polypeptide (SEQ ID NO:22); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B2 TCR Vα polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B2 ... The polynucleotide encoding SEQ ID NO:23; the polynucleotide encoding 3C7 TCR Vα polypeptide (SEQ ID NO:26); or the polynucleotide encoding 3C7 TCR Vβ polypeptide (SEQ ID NO:27). Illustrative polynucleotide sequences encoding these TCR polypeptides are disclosed in Table 1 below.

[0034] Typically, the compositions of the present invention comprise one or more Vα / Vβ polynucleotides, such as polynucleotides encoding TCR Vα polypeptides combined with polynucleotides encoding TCR Vβ polypeptides, so that Vα / VβTCR can be expressed in mammalian cells (e.g., CD8) transduced with one or more vectors. +The Vα / VβTCR is expressed on the surface of T cells, where it recognizes the HLA-associated NY-ESO-1 peptide. As used herein, the terms “transduced,” “transfected,” or “converted” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “converted,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary subject cells and their progeny.

[0035] In another aspect, the present invention includes a method for generating modified T cells, the method comprising introducing one or more nucleic acids encoding the TCR disclosed herein (e.g., nucleic acids housed within a lentiviral vector) into T cells (e.g., CD8 cells obtained from an individual diagnosed with cancer expressing the NY-ESO-1 antigen). + The invention also includes T cells with modified gene expression that is downregulated or knocked out (e.g., modified T cells with a knocked-out endogenous T cell receptor and an exogenous / introduced T cell receptor that recognizes an HLA-associated NY-ESO-1 peptide). As used herein, the term "knockout" refers to a reduction in the expression of one or more genes.

[0036] The modified T cells described herein can be included in compositions used in treatment regimens. The compositions may comprise pharmaceutical compositions and also pharmaceutically acceptable carriers. A therapeutically effective amount of the pharmaceutical composition comprising modified T cells can be administered. The pharmaceutical compositions of the present invention may comprise modified T cells as described herein combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0037] Adoptive immunotherapy using T cells carrying antigen-specific TCRs shows therapeutic potential in cancer treatment. CD8+ cells with specific TCRs... + Genetic engineering of T cells has the advantage of redirecting T cells to selected antigens (such as the NY-ESO-1 antigen). In this context, in one aspect, the invention includes a method for stimulating a T cell-mediated immune response against target cells or tissues in a subject, the method comprising administering an effective amount of modified CD8 to the subject. + T cells. In this implementation, CD8 is modified as described elsewhere herein. +T cells. Embodiments of the invention also include administration of multiple modified CD8 cells targeting multiple NY-ESO-1 epitopes. + T cells. For example, embodiments of the present invention include the administration of at least two different modified CD8 cells. + T cells, for example, target the first modification of the NY-ESO-1 peptide associated with the first human leukocyte antigen (HLA) on CD8. + T-cell combination contains a second CD8 peptide that targets NY-ESO-1, which is associated with second human leukocyte antigen (2HLA). + T cells.

[0038] Embodiments of the present invention include methods for treating diseases or symptoms characterized by the expression of NY-ESO-1 (i.e., the proto-cancer-testis antigen). The treatment method includes administering to a subject in need an effective amount of a pharmaceutical composition comprising T cells modified as described herein. The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, cattle, pigs, dogs, cats, and rodents. Preferably, the subject is a human. In a typical embodiment of the invention, the human has cancer expressing the NY-ESO-1 antigen. In some embodiments of the invention, the cancer cells form a solid tumor. In illustrative embodiments of the invention, the cancer cells are neuroblastoma cells, myeloma cells, metastatic melanoma cells, synovial sarcoma cells, bladder cancer cells, esophageal cancer cells, hepatocellular carcinoma cells, head and neck cancer cells, non-small cell lung cancer cells, ovarian cancer cells, prostate cancer cells, or breast cancer cells.

[0039] One related embodiment of the present invention includes a method for preventing and / or treating an individual diagnosed with, suspected of having, or at risk of developing or recurring cancer, wherein the cancer comprises cancer cells expressing the NY-ESO-1 antigen. This method includes administering to the individual modified human T cells comprising a recombinant polynucleotide encoding a TCR, wherein the T cells are capable of directly recognizing cancer cells expressing the NY-ESO-1 antigen, and wherein the direct recognition of cancer cells involves HLA class II-restricted binding of the TCR to the NY-ESO-1 antigen expressed by the cancer cells.

[0040] Regarding the engineering CD8 of this invention + The use of T cells, the method of which typically involves administering an effective dose (e.g., via intravenous or intraperitoneal injection) of CD8-containing T cells to an individual in need. +Compositions for T cells. Suitable pharmaceutical compositions are adapted for administration via any suitable route, such as parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or nasal routes. Such compositions can be prepared by any method known in the pharmaceutical field, for example by mixing the active ingredient with one or more carriers or excipients under aseptic conditions.

[0041] On the other hand, the present invention includes the polynucleotide or modified CD8 described herein. + Use of T cells in the manufacture of agents for treating subjects in need of treatment who are characterized by the expression of NY-ESO-1. In an illustrative embodiment of the invention, the disease is a cancer expressing the NY-ESO-1 antigen, such as melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

[0042] The technology in this field is quite advanced, and many methods and materials known in the art can be adapted for use with the inventions disclosed herein. Such methods and materials are disclosed in, for example, U.S. Patent Publications 20190247432, 20190119350, 20190002523, 20190002522, 20180371050, 20180057560, 20170029483, 20160024174, and 20150141347, the contents of which are incorporated herein by reference.

[0043] Other aspects and embodiments of the invention are provided in the following examples.

[0044] Example

[0045] Example 1: Expansion and isolation of NY-ESO-1 specific T cell clones.

[0046] We previously reported the presence of T cells in the blood of patients with metastatic melanoma that respond to various NY-ESO-1-derived epitopes (22). To enrich these reactive T cells, peripheral blood mononuclear cells (PBMCs) of patients were stimulated with 28 overlapping 18-mers, which together constitute the complete NY-ESO-1 protein sequence ( Figure 1A Then, the expanded cells were restimulated with individual peptides, intracellular staining was performed against IFN-γ to determine which peptides drove the expansion, and predictive algorithms were used to analyze the stimulating peptides to identify minimal epitopes associated with each patient's MHC haplotype (31). Figure 1BReactive T cells were re-expanded in the presence of individual 9-10-meric peptides corresponding to immunostimulatory epitopes (Fig. 1C) and sorted using homologous peptide-MHC tetramers via fluorescence-activated cell sorting (FACS) (Fig. 1D). Cell lines grown from these single-cell sorts were asexual and reactive to their homologous epitopes (Fig. 1E). A total of HLA-A*02:01 / NY-ESO-1 were selected. 157-165 Further studies were conducted on four reactive cell lines and four cell lines that were reactive to epitopes presented by the HLA-B and HLA-C alleles.

[0047] Example 2: Cloning and screening of NY-ESO-1 specific TCRs

[0048] Using a commercially available RT-PCR kit, paired TCRα and TCRβ genes were cloned from sorted single cells using custom multiplex primers targeting all human TRAV and TRBV gene regions. The resulting V... α and V β cDNA subcloning was performed into a retroviral vector backbone containing the human or murine TCR constant region. Figure 2A To verify the specificity of the cloned TCR, CD3 was transfected with each intact human TCR. + HEK 293T cells were transfected and stained with a peptide-MHC dextrorotatory isomer reagent for each targeted NY-ESO-1 epitope (Fig. 2B). All four HLA-A2-restricted TCRs showed the expected reactivity (Fig. 2C). Despite similar levels of gated transfection at the analysis events, the novel TCRs exhibited highly variable dextrorotatory isomer binding. The 9D2 TCR showed almost indistinguishable dextrorotatory isomer binding from the background, while the 3A1 TCR showed stronger dextrorotatory isomer binding compared to the clinically used 1G4 TCR. The dextrorotatory isomer binding of the 4A2 and 5G6 TCRs was intermediate between that of 9D2 and 1G4.

[0049] Furthermore, three of the four TCRs restricted on MHC alleles other than HLA-A2 have been shown to bind specifically to their targets. Figure 2D ). Expression B7 / NY-ESO-1 60-72 -Specific 1E4 TCR, B18 / NY-ESO-1 88-96 Specific 2B8TCR or Cw3 / NY-ESO-1 96-104 293T cells transfected with the specific 3C7 TCR each bound their corresponding dextrorotatory isoforms, while untransfected cells did not. (Using Cw3 / NY-ESO-1) 92-100Cells transfected with the cloned 9G2 TCR in the responding T cells did not detectably bind the homologous dextrorotatory isomer relative to untransfected cells. A possible reason is that HEK 293T cells do not express the CD8 co-receptor. CD8 increases the affinity of the TCR-pMHC interaction by directly binding to MHC1, thereby engaging the lower-affinity TCR (32). Therefore, this TCR was included to further analyze CD8 dependence in Jurkat T cells.

[0050] Example 3: Functional characterization of A2-restricted NY-ESO-1 specific TCR

[0051] The sensitivity of TCR-transduced T cells is determined by the monomeric affinity (Kb) between the TCR and its homologous peptide-MHC. d ~0.1-400 μM)(33) and the density of TCRs on the cell surface as a function (12). Transduced TCRs are expressed at a wide range of different levels on the surface of T cells due to variations in their folding, dimerization, and efficiency in competing with endogenous TCRs to assemble a limited number of CD3 chains (a property known as TCR “strength”) (34,35). Therefore, optimal cytotoxicity of TCR-transduced T cells is associated with TCR affinity and surface expression (3,12), highlighting the importance of selecting high-affinity, efficiently exported TCRs for gene therapy (7).

[0052] Since high-affinity TCR-pMHC interactions are less dependent on CD8 involvement, it was inferred that high-affinity TCRs could be identified by comparing the binding of the dextrorotatory isomer of Jurkat T cells transduced with or without CD8 co-expression. Furthermore, since the surface expression intensity of human TCRs can be enhanced by substitution with murine constant domains (36), each TCR was expressed as a fully human or murine derivative to assess the intensity of each TCR. Cells transduced with the mediator alone or with a mismatched TCR (MART1-specific F5 TCR) did not show strong affinity for A2 / NY-ESO-1. 157-165 Any combination of dextrorotatory bodies ( Figure 3A ,3B). In contrast, regardless of whether 1G4 is fully human or mouse-ified and whether CD8 is present, with a fully established 1G4 TCR(K D Cells transduced with 1G4 (9.3 μM) (37) all bound the homologous dextrorotatory isoform. Mouse-like 1G4 resulted in a 1.4-fold higher binding strength of the muTCR dextrorotatory isoform compared to the parental huTCR, indicating a moderate increase in binding strength. Figure 3B The presence of CD8 increased the dextral binding of 1G4 muTCR by 3.8 times. The dextral binding of the novel TCRs 4A2 and 5G6 was similar to that of 1G4 in both intensity and comparison index. Figures 3A-3CIn the presence of CD8, the binding of the dextrorotatory isomer of the 3A1 TCR increased only 1.9-fold, indicating that this TCR has a higher affinity for A2 / NY-ESO-1 than 1G4. 157-165 Combined. Compared to CD8 transduced using 1G4, 4A2, and 5G6 muTCR. + Cells, the dextrorotatory isotope binds to CD8 transduced by 3A1 muTCR. + The decreased dependence of CD8 levels in cells further supports this (comparison). Figure 3A (Slope of the green population). Ultimately, in the absence of CD8, 9D2 did not show detectable binding to the dextrorotatory isoform on Jurkat cells, while only weak binding was shown with CD8 co-expression. Monotization of 9D2 did not increase its binding to the dextrorotatory isoform.

[0053] To compare the functional sensitivity of T cells expressing the novel A2 / NY-ESO-1 specific TCR, Jurkat T cells transduced with TCR were compared with those expressing A*02:01 / NY-ESO-1. 157-165 Or A*02:01 / MART1 27-35 K562 cells with single-chain trimers were co-cultured (38) and secreted interleukin-2 (IL-2) was measured. All TCRs exhibited their expected peptide specificity: the control MART1-specific F5 TCR responded only to MART1-presented IL-2 release, and all NY-ESO-1-specific TCRs responded only to NY-ESO-1-presented IL-2 release (Fig. 3D). Mutation improved the functional sensitivity of all TCRs except 1G4. Consistent with the results of dextral isomeric staining, 1G4 and 3A1 muTCRs were superior to 4A2 and 5G6 muTCRs. In contrast, although 9D2 had weaker binding to the dextral isomeric form, it exhibited high functional sensitivity to homologous ligands, comparable to 3A1. To quantify this observation, different concentrations of NY-ESO-1 were used. 157-165 Or MART1 27-35 Peptide A2 + K562 cells were pulsed, and then IFN-γ secreted by primary T cells transduced from TCR cells co-cultured with the target cells of the peptide pulse was measured. Figure 7A , 7B). As observed using single-chain trimer targets, 3A1, 9D2, and 1G4 affect NY-ESO-1 157-165 The peptide exhibited the highest sensitivity. Although the MFI of 4A2 binding to the dextrorotatory isomer was 18 times higher than that of 9D2, the functional sensitivity of 9D2 was 10 times higher than that of 4A2. Figure 3A (3B). To assess the response to endogenous processing and antigen presentation, TCR-transduced primary T cells were compared with the human melanoma cell line A2. +M257 co-culture (Figure 3E). Again, T cells transduced with 3A1, 9D2, and 1G4 responded comparably to each other and showed higher sensitivity than T cells transduced with 5G6 and 4A2. TCR-transduced T cells did not respond to the M257 line lacking HLA-A*02:01. Finally, in vitro cytotoxicity was closely related to cytokine release: T cells expressing 9D2 or 3A1 most effectively killed A2 cells. + M257 tumor cells, followed by T cells transduced by 1G4, 5G6, and the least efficient 4A2 cells. Figure 3F ).

[0054] To assess TCR function in a tumor xenograft model, the PC-3 human prostate cancer cell line was engineered to express NY-ESO-1 and HLA-A*02:01, and then validated that this line elicited TCR-transduced T cell functional responses in an antigen-dependent and MHC-restricted manner. Figure 8A The figure shows the reaction of the novel NY-ESO-1 reactive TCR with respect to A2. + NY + The relative reaction of PC-3 with A2 + The reaction induced by M257 was consistent (Figure 3E and...). Figure 8B Based on these results, 1G4, 3A1, and 9D2muTCRs were selected for further in vivo functional characterization. Activated human PBMCs were transduced using vectors encoding each murine TCR and transduction markers (low-affinity nerve growth factor receptor (LNGFR)). Figure 4A ). For transduction (CD3) + LNGFR + T cells are sorted. Figure 8C These T cells were then intraorally injected retro-orbitally into irradiated NOD / SCID / γc tumors that had been pre-seeded bilaterally with PC-3 / HLA-A2 (control) and PC-3 / HLA-A2 / NYESO (target). - / - (NSG) mice) in ( Figure 4B Subsequently, T-cell implantation and tumor size were monitored until the experiment ended two weeks after T-cell injection.

[0055] T cells transduced with 1G4 or 9D2 TCR persisted or expanded minimally in peripheral blood, while T cells transduced with 3A1 expanded significantly. Figure 4C and 4D Conversely, T cells transduced solely with LNGFR contracted during the experiment, suggesting that the expansion of TCR-transduced T cells was antigen-driven. The expression level of murine TCRβ (mTCRβ) remained stable throughout the experiment and was comparable between T cells transduced with different murine TCRs. Figure 4CAnd 4E). Each with A*02:01 / NY 157-165 Dextral body + The corresponding staining levels in the T cell cohorts transduced with TCRs also stabilized over time, but as expected from in vitro results, significant differences existed between TCRs. Approximately 90% of human T cells transduced with 1G4 or 3A1 were dextrorotatory isomers with high MFI. + In contrast, only about 1% of 9D2-transduced T cells are dextrorotatory. + Furthermore, there was no significant difference between the stained MFI and the LNGFR-transduced control. Figure 4C And 4F). Nevertheless, T cells transduced with 1G4, 3A1, or 9D2 reduced tumor size equally in an antigen-specific manner, while T cells transduced with LNGFR failed to control tumor growth (and 4F). Figure 4G and 4H ).

[0056] At the end of the experiment, mice were sacrificed and tumor T-cell infiltration was analyzed by immunohistochemistry. Immunohistochemical staining showed that in all cohorts of T cells transduced by TCR, antigen-specific T cells infiltrated only the target tumor. Figure 4I and 4J Mice receiving 3A1-transduced T cells showed significantly higher infiltration rates compared to mice receiving 1G4 or 9D2-transduced T cells.

[0057] Example 4: Functional characterization of NY-ESO-1-specific TCRs restricted in HLA-B and HLA-C alleles

[0058] Most immunotherapies targeting NY-ESO-1 focus on A2-restricted NY-ESO-1. 157-165 On the epitope. To broaden the application of NY-ESO-1-targeted immunotherapy, TCRs were cloned from four non-A2-restricted T cell clones and transfected with CD3. + The reactivity of NY-ESO-1 in three of them was verified in 293T. Figure 2D ). From Cw3 / NY-ESO-1 92-100 The fourth TCR-9G2 cloned in reactive T cells, even with co-expression of CD8, did not confer Cw3 / NY-ESO-1 on transduced Jurkat T cells. 92-100 With specificity ( Figure 5A ,5B), and no further research was conducted. Through Jurkat or CD8 + A comparison of the binding strength and affinity of three validated TCRs expressing human or rodent TCRs in Jurkat to the dextral isotope showed differences. Figure 5A(5B, 5C). B7 / NY-ESO-1 60-72 The specific 1E4 TCR exhibits high intensity but low affinity, and is expressed equally as huTCR or muTCR on the surface of Jurkat cells, but only on CD8. + It only binds to the dextrin when it is present. The dextrin combines with these CD8 groups. + The binding of 1E4-transduced cells is strongly dependent on the level of expressed CD8. In contrast, in the absence of CD8... + In the case of B18 / NY-ESO-1 88-96 The 2B8 TCR specifically binds to the dextrorotatory isomer, but binding to the murine TCR is generally higher. Ultimately, Cw3 / NY-ESO-1 96-104 The specific 3C7 TCR exhibited moderate surface expression intensity and an affinity index comparable to 2B8.

[0059] These differences in TCR strength and affinity were reflected in functional assays. For all three TCRs, murine morphology of the TCR constant region increased IL-2 production in Jurkat cells transduced with the TCR co-cultured with homologous target cells. However, this increase was only 1.6-fold and 3.0-fold compared to the corresponding fully human TCRs of 1E4 and 3C7, respectively, while the increase for 2B8 was 18.6-fold, consistent with the latter's lower strength (Fig. 5D). In peptide titration assays, the 1E4 TCR showed increased affinity for transduced CD8+. + The sensitivity of homologous peptides on T cells is lower than that of 3C7 or 2B8. Figure 7C (7D, 7E), which is consistent with the low affinity of 1E4 inferred strictly from CD8-dependent dextral binding.

[0060] Primary PBMCs transduced with each TCR responded to the presentation of NY-ESO-1-derived epitopes in a peptide-specific and MHC-restricted manner (Fig. 5E). Therefore, TCR gene therapy using NY-ESO-1-specific TCRs restricted on multiple MHCs is expected to have broader applicability to patient haplotypes and be more robust against tumor escape via loss of heterozygosity at the MHCI locus. To test this, human cancer cells expressing NY-ESO-1 were transduced with HLA-A2 or HLA-B7. One or both of these tumor targets were then co-cultured with human T cells transduced with A2-restricted 3A1 TCRs, T cells transduced with B7-restricted 1E4 TCRs, or a mixture of 3A1 and 1E4 transduced T cells (Fig. 6). As expected, co-targeting with a mixture of 3A1 and 1E4 transduced T cells was able to recognize tumor cell populations expressing both MHC alleles or expressing either MHC allele alone (Fig. 6A, 6B). In contrast, T cells targeting a single NY-ESO-1 epitope did not respond to tumor cells expressing NY-ESO-1 lacking the homologous MHC allele. Furthermore, when the tumor target contained a mixture of cells expressing different MHC alleles (mimicking tumor heterogeneity caused by haplotype deficiency), T cells targeting two NY-ESO-1 epitopes were more effective at killing tumor cells than T cells targeting either single epitope. Figure 6C ,6D).

[0061] discuss

[0062] T-cell-mediated immunotherapy is bringing previously refractory cancers into the clinic. Two of the most successful immunotherapy modalities are checkpoint blockade and adoptive transfer of cancer-specific T cells. Checkpoint blockade elicits better clinical responses with increasing tumor mutational burden (39-41), suggesting that the immune system cannot detect nonsynonymous mutations unless, by chance, they generate novel epitopes presented by the patient's MHC complement. Recent findings support this explanation: because checkpoint blockade leads to higher overall survival in patients with heterozygous melanoma at HLA-A, HLA-B, and HLA-C loci, they present more diverse epitopes compared to those homozygous at one or more of these MHC loci (42). Results from adoptive T-cell therapy similarly support the importance of multi-targeted antitumor immune responses, with studies suggesting that loss of heterozygosity is a mechanism by which tumors evade single-specific immune recognition while continuing to express another immunogenic antigen (43). Therefore, a prominent narrative emerging from these studies is that successful immunotherapy requires multi-targeted treatment of multiple epitopes presented by multiple MHC alleles. The second key point is that for cancers with low mutation burden, targeting multiple epitopes derived from tumor-specific public antigens may be a promising alternative to targeting novel epitopes.

[0063] It has been demonstrated that it is difficult to identify common tumor-associated antigens that mediate tumor regression without exhibiting severe morbidity or death resulting from on-target extratumor T-cell reactivity. NY-ESO-1 was selected as a common antigen target based on the following criteria: 1) it is expressed only in cancer cells and immune privileged germ cells; 2) it is expressed in many patients across various tumor types; 3) it carries a high-affinity ligand with multiple common MHC alleles; 4) it has been thoroughly reviewed and has generated objective responses in patients across several tumor types without specificity-related adverse events; and 5) since most studies have focused on A2-restricted NY-ESO-1 only. 157-165 Epitopes are used to mobilize T cell responses and therefore have not been fully utilized.

[0064] NY-ESO-1 reactive T cells were isolated from peripheral blood of patients with metastatic melanoma using an antigen-specific amplification protocol. Several HLA-A2-restricted TCRs were cloned using this method, and their surface expression intensity, affinity (i.e., CD8-dependent target binding), and function (antigen-induced cytokine release and tumor-target killing) were compared. From four candidates, two were identified as capable of recognizing and killing NY-ESO-1-expressing cancer cells with equivalent or superior efficacy to the clinically used 1G4 TCR. This amplification-based TCR candidate identification method is well-suited for targeting public epitopes because the rate of isolation is not a critical parameter; once identified, these TCRs can be used as existing target receptors for any patient expressing the required MHC allele. Antigen-specific amplification of neoantigen-reactive T cells from peripheral blood was also demonstrated (44,45). However, methods for on-demand isolation of proprietary TCRs targeting novel epitopes would be much faster than those used here (e.g., direct capture of antigen-specific T cells from the blood or amplification protocols optimized for speed). Since IFN-γ release is closely associated with cytotoxicity (46), it can be used as an alternative method to more involved tumor xenograft assays to accelerate candidate evaluation.

[0065] One isolated HLA-A2 / NY-ESO-1 reactive TCR (9D2) stained poorly for its homopolymer but exhibited high functional affinity for homoantigen-presenting target cells. This is consistent with observations that polymer staining underestimates a subset of functional T cells (47) and can be explained by the higher affinity threshold for polymer binding relative to that for T cell activation (48). However, another isolated A2-restricted TCR – 4A2 – showed robust polymer staining but poor function in cell-based assays, seemingly inconsistent with this affinity threshold explanation. While neither result provides an explanation for the latter, both remind us not to rely excessively on polymer staining when selecting immunotherapy candidates.

[0066] The HLA-A*02:01 allele is the most prevalent MHCI allele in the Caucasian (45%) and Hispanic (41%) U.S. populations, but less common in the Asian (15%) and African (16%) U.S. populations (2). This latter population will be particularly well served by expanding the targeting scope of TCR gene therapy from HLA-A2 to a broader range of targetable MHC alleles. In addition to HLA-A2-restricted TCRs, NY-ESO-1-specific TCRs restricted on various HLA-B and HLA-C alleles were isolated and functionally characterized. By doing so, it was demonstrated in principle that TCR gene therapy can be extended to a larger patient / haplotype subset, and that TCRs recognizing multiple epitopes from the same antigen, when used in combination, can more powerfully kill tumors with heterogeneous MHC expression (e.g., caused by somatic loss of heterozygosity). More than 80% of the population express at least one allele from three MHCI supertypes (A2, A3, and B7, two of which are shown here), and >99% express at least one allele from nine MHCI supertypes (49). Therefore, obtaining a set of publicly available antigen-specific TCR agents that enable comprehensive application of TCR gene therapy is a limited but surmountable challenge.

[0067] Materials and methods

[0068] Material

[0069] Peptides were purchased from Anaspec (Fremont, CA), Thermo Fisher Scientific (Waltham, MA), and Mimotopes (Victoria, Australia). Fluorescent antibodies and 7-AAD for flow cytometry were purchased from BDBiosciences (San Jose, CA), BioLegend (San Diego, CA), or eBioscience (San Diego, CA). Fluorescent peptide-MHC multimers were purchased from TCMetrex (Epalinges, Switzerland) or prepared internally as described in (50) from biotinylated monomers (obtained from NIH Tetramer Core, Atlanta, GA, or heterologously expressed in E. coli, refolded, and internally biotinylated as described in (51). Primers were purchased from Integrated DNA Technologies (Coralville, IA). KOD polymerase premix and polyglobulin were purchased from EMD Millipore (Darmstadt, Germany). Sequencing was performed by Retrogen Inc (San Diego, CA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) activating antibodies were purchased from eBioscience. Cytokines were purchased from Peprotech, Inc. (Rocky Hill, NJ). BioT transfection reagent was purchased from Bioland Scientific (Paramount, CA). Cell culture medium, antibiotics, and fetal bovine serum were purchased from Corning (Corning, NY). Human AB serum was purchased from Omega Scientific (Tarzana, CA). Poly-L-lysine and PHA-L (phytohemagglutinin-L) were purchased from Sigma (St. Louis, MO).

[0070] cell

[0071] Cell lines (293T / 17, Jurkat E6-1, and K562) were purchased from the American Culture Collection (Manassas, VA). 293T cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with antibiotics (penicillin / streptomycin) and 10% (v / v) fetal bovine serum (FBS). Jurkat and K562 cells were grown in RPMI 1640 medium supplemented with antibiotics, 10% (v / v) FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 1x MEM NEAA, and 1 mM sodium pyruvate. Cells were divided every 2–3 days to maintain a density of adherent subconfluenced cells or non-adhesive cells <10-1. 6Cells / mL. Jurkat and K562 cells were transduced with non-replicating viral vectors, analyzed by flow cytometry, and used directly in cell assays or sorted by FACS to establish the derived cell lines shown. Primary human PBMCs used in functional assays were purchased from the CFAR Virology Core Lab at the UCLA AIDS Institute and stimulated, transduced, and cultured as previously described (52). T cells were derived from PBMCs in fresh T cell culture medium supplemented with cytokines (AIM-V medium supplemented with 5% heat-inactivated human AB serum, 55 μM β-mercaptoethanol, and 4 mM L-glutamine). All cells were grown and analyzed at 37°C and 5% atmospheric CO2.

[0072] NY-ESO-1 specific CD8 + Generation and culture of T lymphocyte clones

[0073] CD8 specific to epitopes from NY-ESO-1 with various HLA restrictions (157-165 / HLA-A*02:01(53), 60-72 / HLA-B*07:02(21), 88-96 / HLA-B*18:01(23), 92-100 / HLA-C*03:04(54), 96-104 / HLA-C*03:04(22), 124-133 / HLA-C*03:04(22)). + T lymphocyte clones were generated from patients with HLA-type melanoma. All selected patients had grade III / IV metastatic melanoma and had previously documented responses to NY-ESO-1 in vitro to associated T lymphocyte epitopes (55). Patient PBMCs were stimulated in the presence of 1 μM pooling peptide (Mimotopes), which contains 28x18 polymers overlapping 12 amino acids that co-span the NY-ESO-1 protein sequence, and then these cells were cultured for 10 days in the presence of 25 IU / ml IL-2 (Peprotech).

[0074] On day 10, in the presence of brefidobacterium A, cells were restimulated with 1 μM of each individual peptide, and CD8 responses to each peptide were determined by intracellular cytokine staining (ICS). +T cell activation. In short, following the manufacturer's instructions, label cells with a live / dead fixable purple stain (Invitrogen) and then incubate at 4°C for 15 minutes with antibodies against CD3 and CD8. Wash the sample and fix with a fixation / permeabilization reagent (BD Biosciences) at 4°C for 20 minutes. Stain cells with anti-IFNγ (eBiosciences) at 4°C for 25 minutes in permeabilization / washing solution (BD Biosciences). Gating strategy: SSC / LD. - CD3 + / CD8 + CD8 + / IFNγ + Data from at least 100,000 stained cells were collected on FACSCanto and analyzed using FlowJo software. Data collection and analysis were performed according to the MIATA guidelines (56).

[0075] NY-ESO-1 reactive T cells were expanded in the presence of their identified homologous 9-10-meric epitopes, then labeled with fluorescent tetramers containing related peptides and HLA molecules (TCMetrix, Epalinges, Switzerland), and single-cell sorted using a MoFlo cell sorter. Collected allogeneic healthy donor PBMCs were used as feeder cells at 1 μg / ml. PHA The clones were further expanded using 1–10 x 10⁻⁶ IU / ml IL-2 (Cetus) in the presence of allogeneic PBMCs as feeder cells, PHA-L, and IL-2, as described above. 3 Each clone was restimulated. Clone specificity was confirmed by tetramer staining.

[0076] T lymphocyte clones / lines were cultured in RPMI 1640 medium supplemented with 2 mM Glutamax, 100 IU / ml penicillin, 100 μg / ml streptomycin, 20 mM HEPES, 1% non-essential amino acids, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, and 10% human serum (TCRPMI). IL-2 (100 IU / ml) was added and the medium was changed every 3 days.

[0077] Cloning TCR construct

[0078] Single NY-ESO-1 reactive T cells were sorted for antigen specificity on FACS Aria II and thawed in the presence of an RNase inhibitor. A novel TCR variable gene was cloned from the single-sorted T cells using a Qiagen OneStep RT-PCR kit (Redwood City, CA) with a custom set of human TCR variable region-specific primers, followed by nested PCR amplification. The amplified variable gene was integrated into a TCR expression cassette containing a human or mouse TCR constant domain and a 2A ribosomal jumping peptide linking the α and β genes via assembly PCR and restriction enzyme-mediated cloning. A truncated form of the P2A-linked gene encoding the low-affinity nerve growth factor receptor (LNGFR) was also included in the cassette as a transfection / transduction-independent marker. As previously described, the antigen specificity and MHC restriction of the cloned TCR were evaluated in 293T cells co-transfected with the TCR and CD3 genes (52).

[0079] Assess TCR output and dextrorotatory isotope binding on Jurkat T cells

[0080] Jurkat T cells were transduced with an MSGV-based retrovirus encoding each novel TCR in the form of LNGFRΔ-P2A-TCRα-F2A-TCRβ. The virus was generated in 293T cells as described (52). For transduction, Jurkat T cells were centrifuged (1350 x g, 30 °C for 90 min) with unconcentrated viral supernatant supplemented with 5 μg / mL polybrene. TCR-transduced Jurkat cells were stained with the homologous pMHC dextrorotatory isomer for 15 min at room temperature, followed by co-staining with antibodies against LNGFR and CD8α for 15 min at 4 °C. The stained cells were analyzed by flow cytometry using a FACSCanto analyzer. Data shown in LNGFR... + (Transduction) Gated cells. Transduction efficiency >95%.

[0081] PBMC activation and transduction

[0082] Primary human PBMCs were purchased from the CFAR Virology Core Lab at the UCLA AIDS Institute. The same PBMC donor was used in all reported experiments. Primary human PBMCs were transduced with a retrovirus encoding a novel TCR as described (52). Briefly, two days prior to viral transduction, 1–2 x 10⁻⁶ T cells were activated per well in 24-well plates coated with anti-CD3 (clone OKT3) medium containing 1 μg / mL soluble anti-CD28 (clone CD28.2) and 300 U / mL IL-2. 6Thawed PBMCs were collected. Forty-eight hours after activation, most of the culture medium was replaced with unconcentrated retroviral supernatant supplemented with 10 μg / mL polybrene, and the cells were centrifuged at 1350 x g for 90 minutes at 30°C. After centrifugation and transfection, most of the retroviral supernatant was replaced with fresh culture medium containing 300 U / mL IL-2 and 1 μg / mL anti-CD28. Transduction was repeated after 24 hours, followed by washing the cells with 1xPBS, returning them to fresh culture medium containing the final 300 U / mL IL-2, and culturing for another 3 to 4 days before use in antigen stimulation assays. PBMCs were analyzed by FACS on the day before or the day of co-culture to assess the expression levels of LNGFR, TCR, and / or pMHC multimer binding.

[0083] Functional co-culture assay – cytokine ELISA

[0084] When Jurkat T cells were used as effectors, they were co-cultured in RPMI supplemented with 10% FBS, 100 IU / ml penicillin, 100 μg / ml streptomycin, and 4 mM L-glutamine. Effector cells (50,000 TCR-transduced Jurkat T cells) were co-cultured with target cells (50,000 K562 cells transduced with homologous or control single-stranded trimers) in 96-well plates. Supernatants from duplicate wells were collected 44–48 h after co-culture and analyzed by enzyme-linked immunosorbent assay (ELISA) as described below.

[0085] When using primary PBMCs as effectors, co-culture was performed in T cell culture medium containing 300 U / mL IL-2. Effector cells (50,000 TCR-transduced PBMCs) were co-cultured with target cells (50,000 M257, PC-3, or K562 cells) in 96-well plates. In some experiments, the target cells were pulsed with peptides. Supernatants from 2-8 times replication wells under each condition were collected 44-48 hours after co-culture and analyzed by enzyme-linked immunosorbent assay (ELISA) as described below.

[0086] For experiments involving the titration of target cells with pulsed peptides, the lyophilized peptides were dissolved in DMSO to 10 mM and then further diluted in water to a 2 mM working stock. Before use, the 2 mM stock was diluted to 250 μM in cell culture medium, and then serially diluted 5-fold from 250 μM to 3.2 nM. Target cells were pulsed by adding 25 μL of each serial dilution to each well in a 96-well U-shaped plate, followed by adding 50,000 target cells to 100 μL of culture medium, resulting in a final peptide concentration ranging from 50 μM to 0.64 nM. Cells were pulsed with the peptide at 37°C for 2 hours. At the end of the culture, each well was diluted with 100 μL of culture medium, centrifuged, and the supernatant was removed. Cells were washed with 200 μL of culture medium and then resuspended in 100 μL of medium. Subsequently, 50,000 PBMCs prepared in 100 μL of culture medium were added to each well for co-culture.

[0087] Typically, ELISA results are converted to concentrations (ng / mL) by interpolation relative to a standard curve, and the average concentrations obtained from replicate ELISA measurements are taken. The supernatant is diluted 50–100-fold for ELISA analysis. Sometimes, higher dilutions are required to bring the signal within the standard curve range. All reagents used for ELISA analysis were from BD Biosciences: OptEIA Reagent Set B (550534) was used for diluents and washes, and the OptEIA Human IFN-γ ELISA Kit (555142) and OptEIA Human IL-2 ELISA Kit (555190) were used to measure IFN-γ and IL-2 release, respectively.

[0088] Functional co-culture assay - IncuCyte cell killing assay

[0089] Before co-culturing for the IncuCyte killing assay, 96-well plates were coated with 100 μl of 0.001% poly-L-lysine in PBS for 1 hour at 37°C, washed twice with 200 μl of PBS each time, and briefly air-dried. Target cells were added and allowed to stand at room temperature for 3 hours, after which effector cells were added. Co-culturing typically used 25,000 PBMCs and 25,000 target cells per well of the 96-well plate. In assays that combined multiple effector populations (loaded with different TCRs) or multiple targets (loaded with different MHCs), 25,000 cells were used per cell type, resulting in a total of 75,000 or 100,000 cells per well (for single / mixed or mixed / mixed, respectively). The total volume of all wells was adjusted to 200 μL. The total area of ​​green targets (μm²) was quantified. 2 / pore), and its disappearance is interpreted as a response to GFP. +Target cell killing. Cells were imaged at two locations in each well every 2 hours, and the two images were combined as a single data point. Data points obtained from 4–8 replicate co-cultures of each effector / target combination were used to plot curves and calculate the standard deviation.

[0090] animal

[0091] NOD.Cg-PrkdcSCIDIL-2rgtm1Wjl / SzJ(NOD / SCID / IL-2Rg - / - NSG mice were purchased from the Jackson Laboratory and are held in the animal facility at the University of California, Los Angeles (UCLA). Adult (16-week-old) male mice were used for in vivo tumor stimulation experiments. All animal experiments were approved by the UCLA Institutional Animal Care and Use Committee.

[0092] Human prostate tumor xenograft mouse model

[0093] For xenograft tumor implantation, 10×10 6 PC-3 / HLA-A2 cells (PC-3 cell line overexpressing HLA-A2) were subcutaneously injected on one side of each mouse, while 10 × 10⁶ cells were subcutaneously injected on the other side. 6 PC-3 / HLA-A2 / NY-ESO-1 cells (PC-3 cell line overexpressing HLA-A2 and NYESO). Mice were allowed to develop solid tumors over a 1-week period. On day 8 post-tumor injection, mice were irradiated (100 rads) followed by intravenous injection of 8 × 10⁸ cells into the retroorbital vein. 6 Purified T cells were engineered to express LNGFR alone or together with NY-ESO-1 specific TCRs (1G4, 3A1, or 9D2). Mice were exsanguinated on days 3, 7, 10, and 14 for flow cytometry analysis. On day 14, mice were euthanized and tumors were collected for immunohistochemical analysis.

[0094] Immunohistochemistry

[0095] Solid tumors excised from laboratory mice were fixed in 10% neutral buffered formalin and embedded in paraffin for sectioning (4 mm thick). Hematoxylin and eosin (H / E) staining or antibody staining (for human CD3ε) was then performed using standard procedures (UCLA Translational Pathology Core Laboratory). The sections were imaged at 4x and 40x magnification using an Olympus BX51 upright microscope equipped with an Optronics Macrofire CCD camera (AU Optronics). Images were analyzed using Optronics PictureFrame software (AU Optronics) and ImageJ software (version 1.51J8). CD3ε was measured using ImageJ by setting a color threshold. + The area of ​​the slide stained with human CD3 antibody was used for quantification. The parameters used were as follows: threshold method: default value; threshold color: red; color space: HSB; brightness: 168-215.

[0096] Statistical analysis

[0097] Statistical analysis of tumor xenograft experiments was performed using one-way ANOVA followed by Tukey's multiple comparison test. Data are expressed as mean ± SEM. P < 0.05 was considered significant. ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All statistical analyses were performed using GraphPad PRISM software (version 6.0).

[0098] Table 1: TCRα / β polynucleotide and polypeptide sequences

[0099] The following disclosure provides polynucleotide sequences of various embodiments of the present invention and the variable region TCR protein sequences they encode (e.g., the polynucleotide sequence of SEQ ID NO:1 encodes the variable region TCR protein sequence of SEQ ID NO:3).

[0100] 3A1 TCRVα DNA sequence

[0101]

[0102] 3A1 TCR VβDNA sequence

[0103]

[0104] 3A1 TCR Vα protein sequence

[0105]

[0106] 3A1 TCR Vβ protein sequence

[0107]

[0108] 4A2 TCR VαDNA sequence

[0109]

[0110] 4A2 TCR VβDNA sequence

[0111]

[0112] 4A2 TCR Vα protein sequence

[0113]

[0114] 4A2 TCR Vβ protein sequence

[0115]

[0116] 5G6 TCR VαDNA sequence

[0117]

[0118] 5G6 TCR VβDNA sequence

[0119]

[0120] 5G6 TCR Vα protein sequence

[0121]

[0122] 5G6 TCR Vβ protein sequence

[0123]

[0124] 9D2 TCR VαDNA sequence

[0125]

[0126] 9D2 TCR VβDNA sequence

[0127]

[0128] 9D2 TCR Vα protein sequence

[0129]

[0130] 9D2 TCR Vβ protein sequence

[0131]

[0132] 1E4 TCR VαDNA sequence

[0133]

[0134] 1E4 TCR VβDNA sequence

[0135]

[0136] 1E4 TCR Vα protein sequence

[0137]

[0138] 1E4 TCR Vβ protein sequence

[0139]

[0140] 2B8 TCR VαDNA sequence

[0141]

[0142] 2B8 TCR VβDNA sequence

[0143]

[0144] 2B8 TCR Vα protein sequence

[0145]

[0146] 2B8 TCR Vβ protein sequence

[0147] 3C7 TCR VαDNA sequence

[0148]

[0149] 3C7 TCR VβDNA sequence

[0150]

[0151] 3C7 TCR Vα protein sequence

[0152]

[0153] 3C7 TCR Vβ protein sequence

[0154]

[0155] NY-ESO-1 protein (Humans): GenBank: CAA05908.1

[0156]

[0157] Terms used in this disclosure, such as “A2 / NY-ESO-1” 157-165 "This refers to HLA A2 associated with the NY-ESO-1 peptide containing amino acids 157-165 (i.e., SLLMWITQC (SEQ ID NO:36)) of the protein sequence described above.

[0158] The following sequences contain polynucleotide embodiments of the present invention disposed in a vector. pMTB1328(MSGV- LNGFR-P2A-GB4A2TCR mice (constant)

[0159]

[0160]

[0161] pMTB1329 (MSGV-LNGFR-P2A-GB5G6TCR mouse constant)

[0162]

[0163]

[0164]

[0165] PMTB1330(MSGV-LNGR-P2A+HHD2 TCR mouse constant

[0166]

[0167]

[0168] pMTB1331 (MSGV-LNGFR-P2A-B07NYTCR mouse constant)

[0169]

[0170]

[0171] pMTB1332 (MSGV-LNGFR-P2A-B18NYTCR mouse constant)

[0172]

[0173]

[0174] pMTB1333 (MSGV-LNGFR-P2A-C03NY96TCR mouse constant)

[0175]

[0176]

[0177]

[0178] pMTB1289 (MSGV-LNGFR-P2A-GBA1TCR mouse constant)

[0179]

[0180]

[0181]

[0182] publication

[0183] All publications mentioned herein (e.g., those listed numerically) are incorporated herein by reference, thereby disclosing and describing methods and / or materials relevant to the content cited in those publications. Publications cited herein are cited for their disclosure prior to the filing date of this application. Nothing herein shall be construed as an admission that the inventor is not entitled to claim priority by an earlier date or prior to a publication. Furthermore, actual publication dates may differ from those shown and require independent verification. The following references include descriptions of methods and materials in this art.

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[0241] in conclusion

[0242] This concludes the description of illustrative embodiments of the present invention. For purposes of illustration and description, one or more embodiments of the invention have been described above. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. In view of the above teachings, many modifications and variations are possible. sequence list <110> University of California Board of Trustees Caltech Olivia Newton-John Cancer Institute <120> Composition of NY-ESO-1 specific T cell receptors restricted on multiple major histocompatibility complex molecules <130> 30435.364-WO-U1 <140> <141> <150> 62 / 727,485 <151> 2018-09-05 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 336 <212> DNA <213> Homo sapiens <400> 1 ggtcaacagc tgaatcagag tcctcaatct atgtttatcc aggaaggaga agatgtctcc 60 atgaactgca cttcttcaag catatttaac acctggctat ggtacaagca ggaccctggg 120 gaaggtcctg tcctcttgat agccttatat aaggctggtg aattgacctc aaatggaaga 180 ctgactgctc agtttggtat aaccagaaag gacagcttcc tgaatatctc agcatccata 240 cctagtgatg taggcatcta cttctgtgct ggatttctgg atagcaacta tcagttaatc 300 tggggcgctg ggaccaagct aattataaag ccagat 336 <210> 2 <211> 345 <212> DNA <213> Homo sapiens <400> 2 gaagcccaag tgacccagaa cccaagatac ctcatcacag tgactggaaa gaagttaaca 60 gaagcccaag tgacccagaa cccaagatac ctcatcacag tgactggaaa gaagttaaca 60 gtgacttgtt ctcagaatat gaaccatgag tatatgtcct ggtatcgaca agacccaggg 120 gtgacttgtt ctcagaatat gaaccatgag tatatgtcct ggtatcgaca agacccaggg 120 ctgggcttaa ggcagatcta ctattcaatg aatgttgagg tgactgataa gggagatgtt 180 ctgggcttaa ggcagatcta ctattcaatg aatgttgagg tgactgataa gggagatgtt 180 cctgaagggt acaaagtctc tcgaaaagag aagaggaatt tccccctgat cctggagtcg 240 cctgaagggt acaaagtctc tcgaaaagag aagaggaatt tccccctgat cctggagtcg 240 cccagcccca accagacctc tctgtacttc tgtgccagcg ctagcgggta ccgcacagat 300 cccagcccca accagacctc tctgtacttc tgtgccagcg ctagcgggta ccgcacagat 300 acgcagtatt ttggcccagg cacccggctg acagtgctcg aggac 345 acgcagtatt ttggcccagg cacccggctg acagtgctcg aggac 345 <210> 3 <211> 112 <212> PRT <213> Homo sapiens <400> 3 Gly Gln Gln Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln Glu Gly Gly Gln Gln Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln Glu Gly 1 5 10 15 Glu Asp Val Ser Met Asn Cys Thr Ser Ser Ser Ile Phe Asn Thr Trp Glu Asp Val Ser Met Asn Cys Thr Ser Ser Ser Ile Phe Asn Thr Trp 20 25 30 Leu Trp Tyr Lys Gln Asp Pro Gly Glu Gly Pro Val Leu Leu Ile Ala Leu Trp Tyr Lys Gln Asp Pro Gly Glu Gly Pro Val Leu Leu Ile Ala 35 40 45 Leu Tyr Lys Ala Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr Ala Gln Leu Tyr Lys Ala Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr Ala Gln 50 55 60 Phe Gly Ile Thr Arg Lys Asp Ser Phe Leu Asn Ile Ser Ala Ser Ile 65 70 75 80 Pro Ser Asp Val Gly Ile Tyr Phe Cys Ala Gly Phe Leu Asp Ser Asn 85 90 95 Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys Pro Asp 100 105 110 <210> 4 <211> 115 <212> PRT <213> Homo sapiens <400> 4 Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr Val Thr Gly 1 5 10 15 Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln Ile Tyr Tyr 35 40 45 Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro Glu Gly Tyr 50 55 60 Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile Leu Glu Ser 65 70 75 80 Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser Ala Ser Gly 85 90 95 Tyr Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Leu Glu Asp 115 <210> 5 <211> 354 <212> DNA <213> Homo sapiens <400> 5 gctcagtcag tggctcagcc ggaagatcag gtcaacgttg ctgaagggaa tcctctgact 60 gtgaaatgca cctattcagt ctctggaaac ccttatcttt tttggtatgt tcaatacccc 120 aaccgaggcc tccagttcct tctgaaatac atcacagggg ataacctggt taaaggcagc 180 tatggctttg aagctgaatt taacaagagc caaacctcct tccacctgaa gaaaccatct 240 gcccttgtga gcgactccgc tttgtacttc tgtgctgtga gagacagtcg gtctggggct 300 gggagttacc aactcacttt cgggaagggg accaaactct cggtcatacc aaat 354 <210> 6 <211> 357 <212> DNA <213> Homo sapiens <400> 6 ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60 atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120 aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180 ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240 gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctcc ccaaggttat 300 gggggcacag atacgcagta ttttggccca ggcacccggc tgacagtgct cgaggac 357 <210> 7 <211> 118 <212> PRT <213> Homo sapiens <400> 7 Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val Ala Glu Gly 1 5 10 15 Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly Asn Pro Tyr 20 25 30 Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln Phe Leu Leu 35 40 45 Lys Tyr Ile Thr Gly Asp Asn Leu Val Lys Gly Ser Tyr Gly Phe Glu 50 55 60 Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys Lys Pro Ser 65 70 75 80 Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val Arg Asp Ser 85 90 95 Arg Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys 100 105 110 Leu Ser Val Ile Pro Asn 115 <210> 8 <211> 339 <212> DNA <213> Homo sapiens <400> 8 gatgctaaga ccacacagcc aaattcaatg gagagtaacg aagaagagcc tgttcacttg 60 ccttgtaacc actccacaat cagtggaact gattacatac attggtatcg acagcttccc 120 tcccagggtc cagagtacgt gattcatggt cttacaagca atgtgaacaa cagaatggcc 180 tctctggcaa tcgctgaaga cagaaagtcc agtaccttga tcctgcaccg tgctaccttg 240 agagatgctg ctgtgtacta ctgcatcctg agaacctctg gggctgggag ttaccaactc 300 actttcggga aggggaccaa actctcggtc ataccaaat 339 <210> 9 <211> 351 <212> DNA <213> Homo sapiens <400> 9 agtgctgtca tctctcaaaa gccaagcagg gatatctgtc aacgtggaac ctccctgacg 60 atccagtgtc aagtcgatag ccaagtcacc atgatgttct ggtaccgtca gcaacctgga 120 cagagcctga cactgatcgc aactgcaaat cagggctctg aggccacata tgagagtgga 180 tttgtcattg acaagtttcc catcagccgc ccaaacctaa cattctcaac tctgactgtg 240 agcaacatga gccctgaaga cagcagcata tatctctgca gcgcgggagg agcgggagcg 300 tcagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga c 351 <210> 10 <211> 113 <212> PRT <213> Homo sapiens <400> 10 Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu Glu 1 5 10 15 Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp Tyr 20 25 30 Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val Ile 35 40 45 His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala Ile 50 55 60 Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr Leu 65 70 75 80 Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Arg Thr Ser Gly Ala Gly 85 90 95 Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile Pro 100 105 110 Asn <210> 11 <211> 117 <212> PRT <213> Homo sapiens <400> 11 Ser Ala Val Ile Ser Gln Lys Pro Ser Arg Asp Ile Cys Gln Arg Gly 1 5 10 15 Thr Ser Leu Thr Ile Gln Cys Gln Val Asp Ser Gln Val Thr Met Met 20 25 30 Phe Trp Tyr Arg Gln Gln Pro Gly Gln Ser Leu Thr Leu Ile Ala Thr 35 40 45 Ala Asn Gln Gly Ser Glu Ala Thr Tyr Glu Ser Gly Phe Val Ile Asp 50 55 60 Lys Phe Pro Ile Ser Arg Pro Asn Leu Thr Phe Ser Thr Leu Thr Val 65 70 75 80 Ser Asn Met Ser Pro Glu Asp Ser Ser Ile Tyr Leu Cys Ser Ala Gly 85 90 95 Gly Ala Gly Ala Ser Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Leu Glu Asp 115 <210> 12 <211> 324 <212> DNA <213> Homo sapiens <400> 12 cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60 ctcaactgca cttacagtga ccgaggttcc cagtccttct tctggtacag acaatattct 120 gggaaaagcc ctgagttgat aatgttcata tactccaatg gtgacaaaga agatggaagg 180 tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240 cccagtgatt cagccaccta cctctgtgcc gtagatgaca agatcatctt tggaaaaggg 300 acacgacttc atattctccc caat 324 <210> 13 <211> 351 <212> DNA <213> Homo sapiens <400> 13 gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60 ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120 cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180 cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240 ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttggg acagccaagc 300 acagatacgc agtattttgg cccaggcacc cggctgacag tgctcgagga c 351 <210> 14 <211> 108 <212> PRT <213> Homo sapiens <400> 14 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asp Asp Lys Ile Ile 85 90 95 Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn 100 105 <210> 15 <211> 117 <212> PRT <213> Homo sapiens <400> 15 Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly 1 5 10 15 Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu 20 25 30 Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr 35 40 45 Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg 50 55 60 Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln 65 70 75 80 Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu 85 90 95 Gly Gln Pro Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Thr Val Leu Glu Asp 115 <210> 16 <211> 351 <212> DNA <213> Homo sapiens <400> 16 aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60 ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120 gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180 agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240 cagcctggtg actcagccac ctacctctgt gctgtgagta ctgcgtattc aggaggaggt 300 gctgacggac tcacctttgg caaagggact catctaatca tccagcccta t 351 <210> 17 <211> 351 <212> DNA <213> Homo sapiens <400> 17 gatactggag tctcccagaa ccccagacac aagatcacaa agaggggaca gaatgtaact 60 ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120 cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180 ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240 cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagcccccc gactgttcgg 300 gtctatggct acaccttcgg ttcggggacc aggttaaccg ttgtagagga c 351 <210> 18 <211> 117 <212> PRT <213> Homo sapiens <400> 18 Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn 20 25 30 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu 35 40 45 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala 50 55 60 Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser 65 70 75 80 Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Ser Thr Ala Tyr 85 90 95 Ser Gly Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu 100 105 110 Ile Ile Gln Pro Tyr 115 <210> 19 <211> 117 <212> PRT <213> Homo sapiens <400> 19 Asp Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr Lys Arg Gly 1 5 10 15 Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu 20 25 30 Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr 35 40 45 Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg 50 55 60 Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln 65 70 75 80 Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro 85 90 95 Pro Thr Val Arg Val Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu 100 105 110 Thr Val Val Glu Asp 115 <210> 20 <211> 336 <212> DNA <213> Homo sapiens <400> 20 ggacaacagg taatgcaaat tcctcagtac cagcatgtac aagaaggaga agacttcacc 60 acgtactgca attcctcaac tactttaagc aatatacagt ggtataagca aaggcctggt 120 ggacatcccg tttttttgat acagttagtg aagagtggag aagtgaagaa gcagaaaaga 180 ctgacatttc agtttggaga agcaaaaaag aacagctccc tgcacatcac agccacccag 240 actacagatg taggaaccta cttctgtgcg gaccctaact ttggaaatga gaaattaacc 300 tttgggactg gaacaagact caccatcata cccaat 336 <210> 21 <211> 351 <212> DNA <213> Homo sapiens <400> 21 gaagcccaag tgacccagaa cccaagatac ctcatcacag tgactggaaa gaagttaaca 60 gtgacttgtt ctcagaatat gaaccatgag tatatgtcct ggtatcgaca agacccaggg 120 ctgggcttaa ggcagatcta ctattcaatg aatgttgagg tgactgataa gggagatgtt 180 cctgaagggt acaaagtctc tcgaaaagag aagaggaatt tccccctgat cctggagtcg 240 cccagcccca accagacctc tctgtacttc tgtgccagca gtttgaatcc ctttgcaact 300 aatgaaaaac tgttttttgg cagtggaacc cagctctctg tcttggagga c 351 <210> 22 <211> 112 <212> PRT <213> Homo sapiens <400> 22 Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln Glu Gly 1 5 10 15 Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser Asn Ile 20 25 30 Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu Ile Gln 35 40 45 Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr Phe Gln 50 55 60 Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala Thr Gln 65 70 75 80 Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Asp Pro Asn Phe Gly Asn 85 90 95 Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro Asn 100 105 110 <210> 23 <211> 117 <212> PRT <213> Homo sapiens <400> 23 Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr Val Thr Gly 1 5 10 15 Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln Ile Tyr Tyr 35 40 45 Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro Glu Gly Tyr 50 55 60 Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile Leu Glu Ser 65 70 75 80 Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser Ser Leu Asn 85 90 95 Pro Phe Ala Thr Asn Glu Lys Leu Phe Phe Gly Ser Gly Thr Gln Leu 100 105 110 Ser Val Leu Glu Asp 115 <210> 24 <211> 327 <212> DNA <213> Homo sapiens <400> 24 ggacaaaaca ttgaccagcc cactgagatg acagctacgg aaggtgccat tgtccagatc 60 aactgcacgt accagacatc tgggttcaac gggctgttct ggtaccagca acatgctggc 120 gaagcaccta catttctgtc ttacaatgtt ctggatggtt tggaggagaa aggtcgtttt 180 tcttcattcc ttagtcggtc taaagggtac agttacctcc ttttgaagga gctccagatg 240 aaagactctg cctcttacct ctgtgctgtg agaggcgact acaagctcag ctttggagcc 300 ggaaccacag taactgtaag agcaaat 327 <210> 25 <211> 354 <212> DNA <213> Homo sapiens <400> 25 gattctggag tcacacaaac cccaaagcac ctgatcacag caactggaca gcgagtgacg 60 ctgagatgct cccctaggtc tggagacctc tctgtgtact ggtaccaaca gagcctggac 120 cagggcctcc agttcctcat tcagtattat aatggagaag agagagcaaa aggaaacatt 180 cttgaacgat tctccgcaca acagttccct gacttgcact ctgaactaaa cctgagctct 240 ctggagctgg gggactcagc tttgtatttc tgtgccagca gctcgataca cggtgtctct 300 ggggccaacg tcctgacttt cggggccggc agcaggctga ccgtgctgga ggac 354 <210> 26 <211> 109 <212> PRT <213> Homo sapiens <400> 26 Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly Ala 1 5 10 15 Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly Leu 20 25 30 Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser Tyr 35 40 45 Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe Leu 50 55 60 Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln Met 65 70 75 80 Lys Asp Ser Ala Ser Tyr Leu Cys Ala Val Arg Gly Asp Tyr Lys Leu 85 90 95 Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg Ala Asn 100 105 <210> 27 <211> 118 <212> PRT <213> Homo sapiens <400> 27 Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly 1 5 10 15 Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val 20 25 30 Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln 35 40 45 Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe 50 55 60 Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser 65 70 75 80 Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Ile 85 90 95 His Gly Val Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg 100 105 110 Leu Thr Val Leu Glu Asp 115 <210> 28 <211> 180 <212> PRT <213> Homo sapiens <400> 28 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110 Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175 Gly Gln Arg Arg 180 <210> 29 <211> 8347 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotides <400> 29 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc aggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 1320 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag 1500 agttactaac agcccctctc tccaagctca cttacaggct ctctacttag tccagcacga 1560 agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc tcgctggaa ggaccttaca cagtcctgct gaccacccc accgccctca aagtagcgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctggggcgctg cgaggcgtgc cggcgtgtgcg aggcgggctc gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc 2340 cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac ccaggagcct 2460 gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaaccccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag cagctcagtc agtggctcag 2820 ccggaagatc aggtcaacgt tgctgaaggg aatcctctga ctgtgaaatg cacctattca 2880 gtctctggaa acccttatct tttttggtat gttcaatacc ccaaccgagg cctccagttc 2940 cttctgaaat acatcacagg ggataacctg gttaaaggca gctatggctt tgaagctgaa 3000 tttaacaaga gccaaacctc cttccacctg aagaaaccat ctgcccttgt gagcgactcc 3060 gctttgtact tctgtgctgt gagagacagt cggtctgggg ctgggagtta ccaactcact 3120 ttcgggaagg ggaccaaact ctcggtcata ccaaatatcc agaaccccga gcccgccgtg 3180 taccagctga aggaccccag aagccaggac agcaccctgt gcctgttcac cgacttcgac 3240 agccagatca acgtgcccaa gaccatggag agcggcacct tcatcaccga caagaccgtg 3300 ctggacatga aggccatgga cagcaagagc aacggcgcca tcgcctggtc caaccagacc 3360 agcttcacat gccaggacat cttcaaggag accaacgcca cctaccccag cagcgacgtg 3420 ccctgcgacg ccaccctgac cgagaagagc ttcgagaccg acatgaacct gaacttccag 3480 aacctgagcg tgatgggcct gagaatcctg ctgctgaagg tggccggctt caacctgctg 3540 atgaccctga ggctgtggag cagcagggca aaacgttcgg gttcgggtgc gccagtaaag 3600 cagacattaa actttgattt gctgaaactt gcaggtgatg tagagtcaaa tccaggtcca 3660 atggcaacag ggagccgaac ctctctgctc cttgctttcg ggctcctttg cctaccgtgc 3720 ctgcaggagg gctcggcagg tgctgtcgtc tctcaacatc cgagctgggt tatctgtaag 3780 agtggaacct ctgtgaagat cgagtgccgt tccctggact ttcaggccac aactatgttt tggtatcgtc agttcccgaa acagagtctc atgctgatgg caacttccaa tgagggctcc aaggccacat acgagcaagg cgtcgagaag gacaagtttc tcatcaacca tgcaagcctg accttgtcca ctctgacagt gaccagtgcc catcctgaag acagcagctt ctacatctgc agtgctcccc aaggttatgg gggcacagat acgcagtatt ttggcccagg cacccggctg 4080 acagtgctcg aggacctgag gaacgtgacc ccccccaagg tgtccctgtt cgagcccagc 4140 aaggccgaga tcgccaacaa gcagaaggcc accctggtgt gcctggccag gggcttcttc cccgaccacg tggagctgtc ttggtgggtg aacggcaagg aggtgcacag cggcgtgagc 4260 accgacccc aggcctacaa ggagagcaac tacagctact gcctgagcag caggctgaga gtgagcgcca ccttctggca caaccccagg aaccacttcc gctgtcaggt gcagttccac ggcctgagcg aggagacaa gtggcccgag ggcagcccca agcccgtgac ccagaacatc 4440 agcgccgagg cctggggcag agccgactgc ggcatcacca gcgccagcta ccaccagggc 4500 gtgctgtccg ccaccatcct gtacgagatc ctgctgggca aggccacact gtacgccgtg 4560 ctggtgtccg gcctggtgct gatggccatg gtgaagaaga agaacagcta aggatccga 4620 taaaataaaa gatttatttt agtctccaga aaaagggggg aatgaaagac cccacctgta 4680 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata cataactgag 4740 atagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg gccaaacagg 4800 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg 4860 cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac 4920 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg 4980 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gcgcgccagt 5040 cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca 5100 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc 5160 agcgggggtc tttcatgggt aacagtttct tgaagttgga gaacaacatt ctgagggtag 5220 gagtcgaata ttaagtaatc ctgactcaat tagccactgt tttgaatcca catactccaa 5280 tactcctgaa atccatcgat ggagttcatt atggacagcg cagaaagagc tggggagaat 5340 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 5400 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 5460 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 5520 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5580 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 5640 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 5700 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 5760 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 5820 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 5880 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc ttaggttatc 5940 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 6000 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 6060 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 6120 ctacagagtt cttgaagtgg tggcctaact acggctcac tagaaggaca gtatttggta 6180 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6240 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6300 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6360 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aggatcttc acctagatcc 6420 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 6480 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 6540 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 6600 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 6660 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6720 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 6780 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 6840 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6900 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6960 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 7020 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 7080 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 7140 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7200 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7260 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7320 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7380 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7440 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 7500 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg 7560 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 7620 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 7680 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 7740 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc 7800 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 7860 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 7920 gttgtaaaac gacggccagt gccacgctct cccttatgcg actcctgcat taggaagcag 7980 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag 8040 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg 8100 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg 8160 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggcga 8220 tttaaagaca ggatatcagt ggtccaggct ctagttttga ctcaacaata tcaccagctg 8280 aagcctatag agtacgagcc atagataaaa taaaagattt tatttagtct ccagaaaaag 8340 gggggaa 8347 <210> 30 <211> 8326 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 30 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggaggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 1320 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag 1500 agttactaac agcccctctc tccaagctca cttacaggct ctctacttag tccagcacga 1560 agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca 1620 cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc 1680 tcgctggaaa ggaccttaca cagtcctgct gaccaccccc accgccctca aagtagacgg 1740 catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctggggcgctg cgaggcgtgc cggcgtgtgcg aggcgggctc gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2340. gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attackcggt ccacaccccc agggggctcg cccccagcc ccaggagcct 2460 gaggcacctc cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaacccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag cagatgctaa gaccacacag 2820 ccaaattcaa tggagagataa cgaagaagag cctgttcact tgccttgtaa ccactccaca 2880 atcagtggaa ctgattacat acattggtat cgacagcttc cctcccaggg tccagagtac 2940 gtgattcatg gtcttacaag caatgtgaac aacagaatgg cctctctggc aatcgctgaa 3000 gacagaaagt ccagtacctt gatcctgcac cgtgctacct tgagagatgc tgctgtgtac 3060 tactgcatcc tgagaacctc tggggctgggg agttaccaac tcactttcgg gaaggggacc 3120 aaactctcgg tcataccaaa tatccagaac cccgagcccg ccgtgtacca gctgaaggac 3180 cccagaagcc aggacagcac cctgtgcctg ttcaccgact tcgacagcca gatcaacgtg cccaagacca tggagagcgg caccttcatc accgacaaga ccgtgctgga catgaaggcc atggacagca agagcaacgg cgccatcgcc tggtccaacc agaccagctt cacatgccag 3420. gacatcttca aggagacca cgccacctac cccagcagcg acgtgccctg cgacgccacc ctgaccgaga agagcttcga gaccgacatg aacctgaact tccagaacct gagcgtgatg ggcctgagaa tcctgctgct gaaggtggcc ggcttcaacc tgctgatgac cctgaggctg tggagcagca gggcaaaacg ttcgggttcg ggtgcgccag taaagcagac attaaacttt gatttgctga aacttgcagg tgatgtagg tcaaatccag gtccaatggc aacagggagc cgaacctctc tgctccttgc tttcgggctc ctttgcctac cgtgcctgca ggagggctcg 3720 gcaagtgctg tcatctctca aaagccaagc agggatatct gtcaacgtgg aacctccctg accatccagt gtcaagtcga tagccaagtc accatgatgt tctggtaccg tcagcaacct ggacagagcc tgacactgat cgcaactgca aatcagggct ctgaggccac atatgagagt ggatttgtca ttgacaagtt tcccatcagc cgcccaaacc taacattctc aactctgact 3960 gtgagcaaca tgagccctga agacagcagc atatatctct gcagcgcggg aggagcggga 4020 gcgtcagata cgcagtattt tggcccaggc acccggctga cagtgctcga ggacctgagg 4080 aacgtgaccc cccccaaggt gtccctgttc gagcccagca aggccgagat cgccaacaag 4140 cagaaggcca ccctggtgtg cctggccagg ggcttcttcc ccgaccacgt ggagctgtct 4200 tggtgggtga acggcaagga ggtgcacagc ggcgtgagca ccgaccccca ggcctacaag 4260 gagagcaact acagctactg cctgagcagc aggctgagag tgagcgccac cttctggcac 4320 aaccccagga accacttccg ctgtcaggtg cagttccacg gcctgagcga ggaggacaag 4380 tggcccgagg gcagccccaa gcccgtgacc cagaacatca gcgccgaggc ctggggcaga 4440 gccgactgcg gcatcaccag cgccagctac caccagggcg tgctgtccgc caccatcctg 4500 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgg cctggtgctg 4560 atggccatgg tgaagaagaa gaacagctaa aggatccgat aaaataaaag attttattta 4620 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 4680 taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag 4740 gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc 4800 tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt 4860 ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta 4920 tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tcccgagct 4980 caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc 5040 cggtacccg tgtatccaat aaaccctt gcagttgcat ccgacttgtg gtctcgctgt 5100 tccttgggag ggtctcctct gagtgattga ctaccccgtca gcgggggtct ttcatgggta 5160 acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc 5220 tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa tccatcgatg 5280 gagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca 5340 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5400 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5460 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5520 gctcttcgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5580 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5640 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5700 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5760 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5820 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5880 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5940 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 6000 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 6060 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 6120 ggcctaacta cggctacact agaagcag tatttggtat ctgcgctctg ctgaagccag 6180 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 6240 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 6300 ctttgatctt ttctacgggg tctgacgctc agtgggaacga aaactcacgt taagggattt 6360 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6420 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 6480 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 6540 tcgtgtagat aactacgata cgggaggct taccatctgg ccccagtgct gcaatgatac 6600 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaccagcca gccggaaggg 6660 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 6720 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 6780 caggcatcgt gtgtcacgc tcgtcgttg gtatggcttc attcagctcc ggttcccac 6840 gatcaggcg agttacatga tccccatgt tgtgcaaaa agcggttagc tccttcggtc 6900 ctccgatcgt tgtcagaagt aagttggccg cagtgttac actcatggtt atggcagcac 6960 tgcataattc tcttactgtc atgccatccg taagatgctt tctgtgact ggtgagtact 7020 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgcttgc ccggcgtcaa 7080 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 7140 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 7200 ctcgtgcacc caacgatct tcagcatctt ttacttcac cagcgtttct gggtgagcaa 7260 aaaaaaaag gcaaatgcc gcaaaaagg gaatagggc gacacgaaa tgttgaatac 7320 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 7380 gatacatatt tgaatgtatt tagaaaata aaaatagg ggttccgcgc acatttcccc 7440 gaaaagtgcc acctgacgtc windowaaacca ttatcat gatacacc taaaaaata 7500 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac 7560 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 7620 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat 7680 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 7740 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 7800 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 7860 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 7920 ccacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg 7980 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg 8040 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga 8100 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg 8160 ccggtgatgc cggccacgat gcgtccggcg tagaggcgat ttaaagacag gatatcagtg 8220 gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga gtacgagcca 8280 tagataaaat aaaagatttt atttagtctc cagaaaaagg ggggaa 8326 <210> 31 <211> 8311 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 31 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggaggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggctttttg acccccctcc ctggggtcaag ccctttgtac accctaagcc 1320. tccgcctcct cttcctccat ccgccccgtc tctccccctt gaaccctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag agttactac agccctctc tccaagctca cttacaggct ctctacttag tccagcacga agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc tcgctggaa ggaccttaca cagtcctgct gaccacccc accgccctca aagtagcgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag 1980 ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac 2040 gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg 2100 tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc gggcctcgtg 2220 ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2280 gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc 2340 cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac ccaggagcct 2460 gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaaccccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag cacagaagga ggtggagcag 2820 aattctggac ccctcagtgt tccagaggga gccattgcct ctctcaactg cacttacagt 2880 gaccgaggtt cccagtcctt cttctggtac agacaatatt ctgggaaaag ccctgagttg 2940 ataatgttca tatactccaa tggtgacaaa gaagatggaa ggtttacagc acagctcaat 3000 aaagccagcc agtatgtttc tctgctcatc agagactccc agcccagtga ttcagccacc 3060 tacctctgtg ccgtagatga caagatcatc tttggaaaag ggacacgact tcatattctc 3120 cccaatatcc agaaccccga gcccgccgtg taccagctga aggaccccag aagccaggac 3180 agcaccctgt gcctgttcac cgacttcgac agccagatca acgtgcccaa gaccatggag 3240 agcggcacct tcatcaccga caagaccgtg ctggacatga aggccatgga cagcaagagc 3300 aacggcgcca tcgcctggtc caaccagacc agcttcacat gccaggacat cttcaaggag 3360 accaacgcca cctaccccag cagcgacgtg ccctgcgacg ccaccctgac cgagaagagc 3420 ttcgagaccg acatgaacct gaacttccag aacctgagcg tgatgggcct gagaatcctg 3480 ctgctgaagg tggccggctt caacctgctg atgaccctga ggctgtggag cagcagggca 3540 aaacgttcgg gttcgggtgc gccagtaaag cagacattaa actttgattt gctgaaactt 3600 gcaggtgatg tagagtcaaa tccaggtcca atggcaacag ggagccgaac ctctctgctc 3660 cttgctttcg ggctcctttg cctaccgtgc ctgcaggagg gctcggcaga tgctggagtt 3720 atccagtcac cccggcacga ggtgacagag atgggacaag aagtgactct gagatgtaaa 3780 ccaatttcag gacacgacta ccttttctgg tacagacaga ccatgatgcg gggactggag 3840 ttgctcattt actttaacaa caacgttccg atagatgatt cagggatgcc cgaggatcga 3900 ttctcagcta agatgcctaa tgcatcattc tccactctga agatccagcc ctcagaaccc 3960 agggactcag ctgtgtactt ctgtgccagc agtttgggac agccaagcac agatacgcag 4020 tattttggcc caggcacccg gctgacagtg ctcgaggacc tgaggaacgt gacccccccc 4080 aaggtgtccc tgttcgagcc cagcaaggcc gagatcgcca aaagcagaa ggccaccctg 4140 gtgtgcctgg ccaggggctt cttccccgac cacgtggagc tgtcttggtg ggtgaacggc 4200 aaggaggtgc acagcggct gagcaccgac cccaggcct acaaggagag caactacagc 4260 tactgcctga gcagcaggct gagagtgagc gccaccttct ggcacaaccc caggaaccac 4320 ttccgctgtc aggtgcagtt ccacggcctg agcgaggagg acaagtggcc cgagggcagc 4380 cccaagcccg tgacccagaa catcagcgcc gaggcctggg gcagagccga ctgcggcatc 4440 accagcgcca gctaccacca gggcgtgctg tccgccacca tcctgtacga gatcctgctg 4500 ggcaaggcca cactgtacgc cgtgctggtg tccggcctgg tgctgatggc catggtgaag 4560 aaaagaaca gctaaaggat ccgataaaat aaaagatttt atttagtctc cagaaaaagg 4620 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca 4680 aggcatggaa aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag 4740 acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg 4800 ccaagaacag atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag 4860 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat 4920 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca 4980 caacccctca ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtgtat 5040 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct 5100 cctctgagtg attgactacc cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt 5160 tggagaacaa cattctgagg gtaggagtcg aatattaagt aatcctgact caattagcca 5220 ctgttttgaa tccacatact ccaatactcc tgaaatccat cgatggagtt cattatggac 5280 agcgcagaaa gagctgggga gaattgtgaa attgttatcc gctcacaatt ccacacaaca 5340 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 5400 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 5460 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 5520 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 5580 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 5640 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 5700 tccgcccccc tgacgagcat cacaaaaaatc gacgctcaag tcagaggtgg cgaaacccga 5760 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 5820 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 5880 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctggggct 5940 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6000 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6060 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6120 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6180 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt tttttgttt 6240 gcaagcagca gattacgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6300 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6360 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 6420 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 6480 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 6540 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 6600 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 6660 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 6720 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 6780 cacgctcgtc gtttggtatg gcttcattca gctccggttc caacgatca aggcgagtta 6840 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 6900 gaagtaagtt ggccgcagtg tttcactca tggttatggc agcactgcat aattctctta 6960 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7020 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7080 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7140 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7200 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7260 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7320 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7380 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 7440 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 7500 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 7560 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 7620 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 7680 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 7740 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 7800 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 7860 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccacg ctctccctta 7920 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc 7980 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc 8040 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca 8100 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc 8160 acgatgcgtc cggcgtagag gcgatttaaa gacaggatat cagtggtcca ggctctagtt 8220 ttgactcaac aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag 8280 attttattta gtctccagaa aaagggggga a 8311 <210> 32 <211> 8338 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 32 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggaggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 1320 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag agttactac agccctctc tccaagctca cttacaggct ctctacttag tccagcacga agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc tcgctggaa ggaccttaca cagtcctgct gaccacccc accgccctca aagtagcgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc gggcctcgtg 2220 ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2280 gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc 2340 cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac ccaggagcct 2460 gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaaccccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag caaaacagga ggtgacgcag 2820 attcctgcag ctctgagtgt cccagaagga gaaacttgg ttctcaactg cagtttcact 2880 gatagcgcta tttacaacct ccagtggttt aggcaggacc ctgggaaagg tctcacatct 2940 3000 gataaatcat caggacgtag tactttatac attgcagctt ctcagcctgg tgactcagcc 3060 acctacctct gtgctgtgag tactgcgtat tcaggaggag gtgctgacgg actcaccttt 3120 ggcaaaggga ctcatctaat catccagccc tatatccaga accccgagcc cgccgtgtac 3180 cagctgaagg accccagaag ccaggacagc accctgtgcc tgttcaccga cttcgacagc 3240 cagatcaacg tgcccaagac catggagagc ggcaccttca tcaccgacaa gaccgtgctg 3300 gacatgaagg ccatggacag caagagcaac ggcgccatcg cctggtccaa ccagaccagc 3360 ttcacatgcc aggacatctt caaggagacc aacgccacct accccagcagcag cgacgtgccc 3420 tgcgacgcca ccctgaccga gaagcttc gagaccgaca tgaacctgaa cttccagaac 3480 ctgagcgtga tgggcctgag aatcctgctg ctgaaggtgg ccggcttcaa cctgctgatg 3540 accctgaggc tgtggagcag cagggcaaaa cgttcgggtt cgggtgcgcc agtaaagcag 3600 acattaaact ttgatttgct gaaacttgca ggtgatgtag agtcaaatcc aggtccaatg 3660 gcaacaggga gccgaacctc tctgctcctt gctttcgggc tcctttgcct accgtgcctg 3720 caggagggct cggcagatac tggagtctcc cagaacccca gacacaagat cacaaagagg 3780 ggacagaatg taactttcag gtgtgatcca atttctgaac acaaccgcct ttattggtac 3840 cgacagaccc tggggcaggg cccagagttt ctgacttact tccagaatga agctcaacta 3900 gaaaaatcaa ggctgctcag tgatcggttc tctgcagaga ggcctaaggg atctttctcc 3960 accttggaga tccagcgcac agagcagggg gactcggcca tgtatctctg tgccagcagc 4020 cccccgactg ttcgggtcta tggctacacc ttcggttcgg ggaccaggtt aaccgttgta 4080 gaggacctga ggaacgtgac cccccccaag gtgtccctgt tcgagcccag caaggccgag 4140 atcgccaaca agcagaaggc caccctggtg tgcctggcca ggggcttctt ccccgaccac 4200 gtggagctgt cttggtgggt gaacggcaag gaggtgcaca gcggcgtgag caccgacccc 4260 caggcctaca aggagagcaa ctacagctac tgcctgagca gcaggctgag agtgagcgcc 4320. accttctggc acaaccccag gaaccacttc cgctgtcagg tgcagttcca cggcctgagc gaggagaca agtggcccga gggcagcccc aagcccgtga cccagaacat cagcgccgag gcctggggca gagccgactg cggcatcacc agcgccagct accaccaggg cgtgctgtcc 4500. gccaccatcc tgtacgagat cctgctgggc aaggccacac tgtacgccgt gctggtgtcc 4560 ggcctggtgc tgatggccat ggtgaagaag aagaacagct aaaggatccg ataaataaa agttttatt tagtctccag aaaaaggggg ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaat acataactga catgaga gttcagatca aggttaggaa cagagagaca gcagaattg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agacagatg gtccccagat gcggtcccgc 4860. cctcagcagt ttctagagaa cctcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata 5040 gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg 5100 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt 5160 ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat 5220 attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga 5280 aatccatcga tggagttcat tatggacagc gcagaaagag ctggggagaa ttgtgaaatt 5340 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 5400 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 5460 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 5520 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 5580 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 5640 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 5700 ccgcgttgct ggggttttc cataggctcc gccccctga cgagcatcac aaaaatcgac 5760 gctcaagtca gaggtggcga aacccgacag gacttaaag ataccaggcg tttccccctg 5820 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 5880 ttctccctc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 5940 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgacc cccgttcag cccgaccgct 6000 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6060 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6120 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 6180 tgctgaagcc agttaccttc ggaaaagag ttggtagctc ttgatccggc aaacaaacca 6240 cggctgtag cggtggtttt ttgttttgca agcagcagat tacgcgcaga aaaaaaggat 6300 ctcaagaaga tccttgatc tttctacgg ggtctgacgc tcagtggaac gaaaactcac 6360 gttaagggat ttggtcatg agattatca aaaggatctt cacctagatc cttttaaatt 6420 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 6480 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 6540 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 6600 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 6660 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 6720 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 6780 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 6840 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 6900 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 6960 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 7020 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 7080 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 7140 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 7200 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 7260 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 7320 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 7380 gtctcatgag cggatacata tttgaatgta tttagaaaaa taacaaata ggggttccgc 7440 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 7500 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg 7560 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 7620 gggcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta 7680 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaataccgc 7740 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact 7800 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaaggggat 7860 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 7920 cgacggccag tgccacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt 7980 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc 8040 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc 8100 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc 8160 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggcg atttaaagac 8220 aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata 8280 gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaa 8338 <210> 33 <211> 8323 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 33 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc aggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 1320 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag 1500 agttactaac agcccctctc tccaagctca cttacaggct ctctacttag tccagcacga 1560 agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc tcgctggaa ggaccttaca cagtcctgct gaccacccc accgccctca aagtagcgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctggggcgctg cgaggcgtgc cggcgtgtgcg aggcgggctc gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2340. gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attackcggt ccacaccccc agggggctcg cccccagcc ccaggagcct 2460 gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt gaccacagtg atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580. tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccgcggc cgccatggcg acggggttcaa gaacttccct acttcttgca tttggcctgc tttgtttgcc gtggttacag gaagcctcag caggacaaca ggtaatgcaa attcctcagt accagcatgt acaagaagga gaagacttca ccacgtactg caattcctca actactttaa gcaatataca gtggtataag caaaggcctg gtggacatcc cgtttttttg atacagttag tgaagagtgg agaagtgaag aagcagaaaa gactgacatt tcagtttgga gaagcaaaaa agaacagctc cctgcacatc acagccaccc agactacaga tgtaggaacc 3060 tacttctgtg cggaccctaa ctttggaaat gagaaattaa cctttgggac tggaacaaga 3120 ctcaccatca tacccaatat ccagaacccc gagcccgccg tgtaccagct gaaggacccc 3180 agaagccagg acagcaccct gtgcctgttc accgacttcg acagccagat caacgtgccc 3240 aagaccatgg agagcggcac cttcatcacc gacaagaccg tgctggacat gaaggccatg 3300 gacagcaaga gcaacggcgc catcgcctgg tccaaccaga ccagcttcac atgccaggac 3360 atcttcaagg agaccaacgc cacctacccc agcagcgacg tgccctgcga cgccaccctg 3420 accgagaaga gcttcgagac cgacatgaac ctgaacttcc agaacctgag cgtgatgggc 3480 ctgagaatcc tgctgctgaa ggtggccggc ttcaacctgc tgatgaccct gaggctgtgg 3540 agcagcaggg caaaacgttc gggttcgggt gcgccagtaa agcagacatt aaactttgat 3600 ttgctgaaac ttgcaggtga tgtagagtca aatccaggtc caatggcaac agggagccga 3660 acctctctgc tccttgcttt cgggctcctt tgcctaccgt gcctgcagga gggctcggca 3720 gaagcccaag tgacccagaa cccaagatac ctcatcacag tgactggaaa gaagttaaca 3780 gtgacttgtt ctcagaatat gaaccatgag tatatgtcct ggtatcgaca agaccaggg 3840 ctgggcttaa ggcagatcta ctattcaatg aatgttgagg tgactgataa gggagatgtt 3900 cctgaagggt acaaagtctc tcgaaaagag aagaggaatt tccccctgat cctggagtcg 3960 cccagcccca accagacctc tctgtacttc tgtgccagca gtttgaatcc ctttgcaact 4020 aatgaaaaac tgttttttgg cagtggaacc cagctctctg tcttggagga cctgaggaac 4080 gtgacccccc ccaaggtgtc cctgttcgag cccagcaagg ccgagatcgc caacaagcag 4140 aaggccaccc tggtgtgcct ggccaggggc ttcttccccg accacgtgga gctgtcttgg 4200 tgggtgaacg gcaaggaggt gcacagcggc gtgagcaccg acccccaggc ctacaaggag 4260 agcaactaca gctactgcct gagcagcagg ctgagagtga gcgccacctt ctggcacaac 4320 cccaggaacc acttccgctg tcaggtgcag ttccacggcc tgagcgagga ggaaagtgg 4380 cccgagggca gccccaagcc cgtgacccag aacatcagcg ccgaggcctg gggcagagcc 4440 gactgcggca tcaccagcgc cagctaccac cagggcgtgc tgtccgccac catcctgtac 4500 gagatcctgc tgggcaaggc cacactgtac gccgtgctgg tgtccggcct ggtgctgatg 4560 gccatggtga agaagaagaa cagctaaagg atccgataaa ataaaagatt ttatttagtc 4620 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 4680 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt 4740 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc 4800 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta 4860 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt 4920 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa 4980 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg 5040 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc 5100 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca 5160 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga 5220 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatcc atcgatggag 5280 ttcattatgg acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa 5340 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 5400 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 5460 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 5520 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 5580 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 5640 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 5700 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 5760 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 5820 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 5880 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 5940 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 6000 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 6060 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 6120 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 6180 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 6240 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 6300 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 6360 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 6420 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 6480 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 6540 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 6600 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 6660 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 6720 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 6780 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 6840 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 6900 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 6960 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 7020 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 7080 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 7140 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 7200 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 7260 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 7320 tactcttcct ttttcaatat tattgaagca tttatcaggg ttatgtctc atgagcggat 7380 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 7440 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 7500 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 7560 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 7620 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 7680 7740 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat 7800 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 7860 taagttgggt aacgccaggg ttttcccagt caggacgttg taaacgacg gccagtgcca 7920 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg 7980 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc 8040 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc 8100 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg 8160 gtgatgccgg ccacgatgcg tccggcgtag aggcgattta aagacaggat atcagtggtc 8220 caggctctag ttttgactca acaatatcac cagctgaagc ctatagagta cgagccatag 8280 ataaaataaa agattttatt tagtctccag aaaaaggggg gaa 8323 <210> 34 <211> 8317 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polynucleotide <400> 34 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggaggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac accctaagcc 1320 tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag 1500 agttactaac agcccctctc tccaagctca cttacaggct ctctacttag tccagcacga 1560 agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca 1620 cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc 1680 tcgctggaaa ggaccttaca cagtcctgct gaccaccccc accgccctca aagtagacgg 1740 catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctggggcgctg cgaggcgtgc cggcgtgtgcg aggcgggctc gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2340. gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attackcggt ccacaccccc agggggctcg cccccagcc ccaggagcct 2460 gaggcacctc cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaacccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag caggacaaaa cattgaccag 2820 cccactgaga tgacagctac ggaaggtgcc attgtccaga tcaactgcac gtaccagaca 2880 tctgggttca acgggctgtt ctggtaccag caacatgctg gcgaagcacc tacatttctg 2940 tcttcaatg ttctggatgg tttgggagg aaaggtcgtt tttcttcatt ccttagtcgg 3000 tctaaagggt acagttacct ccttttgaag gagctccaga tgaaagactc tgcctcttac 3060 3120 agagcaata tccagaaccc cgagcccgcc gtgtaccagc tgaaggaccc cagaagccag 3180 gacagcaccc tgtgcctgtt caccgacttc gacagccaga tcaacgtgcc caagaccatg 3240 gagagcggca ccttcatcac cgacaagacc gtgctggca tgaaggccat ggacagcaag 3300 agcaacggcg ccatcgcctg gtccaaccag accagcttca catgccagga catcttcaag 3360 gagaccaacg ccacctaccc cagcagcgac gtgccctgcg acgccaccct gaccgagaag 3420 3480 ctgctgctga aggtggccgg cttcaacctg ctgatgaccc tgaggctgtg gagcagcagg 3540 gcaaaacgtt cgggttcggg tgcgccagta aagcagacat taaactttga tttgctgaaa 3600 cttgcaggtg atgtagagtc aaatccaggt ccaatggcaa cagggagccg aacctctctg 3660 ctccttgctt tcgggctcct ttgcctaccg tgcctgcagg agggctcggc agattctgga 3720 gtcacaaa ccccaaagca cctgatcaca gcaactggac agcgagtgac gctgagatgc 3780 tcccctaggt ctggagacct ctctgtgtac tggtaccaac agagcctgga ccagggcctc 3840 cagttcctca ttcagtatta taatggagaa gagagagcaa aaggaaacat tcttgaacga 3900 ttctccgcac aacagttccc tgacttgcac tctgaactaa acctgagctc tctggagctg 3960 ggggactcag ctttgtattt ctgtgccagc agctcgatac acggtgtctc tggggccaac 4020 gtcctgactt tcggggccgg cagcaggctg accgtgctgg aggacctgag gaacgtgacc 4080 ccccccaagg tgtccctgtt cgagcccagc aaggccgaga tcgccaacaa gcagaaggcc 4140 accctggtgt gcctggccag gggcttcttc cccgaccacg tggagctgtc ttggtgggtg 4200 aacggcaagg aggtgcacag cggcgtgagc accgaccccc aggcctacaa ggagagcaac 4260 tacagctact gcctgagcag caggctgaga gtgagcgcca ccttctggca caaccccagg 4320 aaccacttcc gctgtcaggt gcagttccac ggcctgagcg aggaggacaa gtggcccgag 4380 ggcagcccca agcccgtgac ccagaacatc agcgccgagg cctggggcag agccgactgc 4440 ggcatcacca gcgccagcta ccaccagggc gtgctgtccg ccaccatcct gtacgagatc 4500 ctgctgggca aggccacact gtacgccgtg ctggtgtccg gcctggtgct gatggccatg 4560 gtgaagaaga agaacagcta aaggatccga taaaataaaa gattttattt agtctccaga 4620 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat 4680 tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac 4740 agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc 4800 tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac 4860 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta 4920 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag 4980 agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc 5040 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga 5100 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct 5160 tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat 5220 tagccactgt tttgaatcca catactccaa tactcctgaa atccatcgat ggagttcatt 5280 atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac 5340 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 5400 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 5460 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 5520 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5580 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5640 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5700 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5760 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5820 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5880 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5940 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 6000 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 6060 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 6120 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 6180 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6240 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6300 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6360 gattatcaaa aggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6420 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6480 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6540 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6600 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6660 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6720 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6780 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6840 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6900 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6960 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 7020 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 7080 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7140 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7200 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7260 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7320 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7380 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7440 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 7500 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 7560 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 7620 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 7680 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 7740 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 7800 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 7860 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccacgctct 7920 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 7980 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg 8040 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 8100 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 8160 ccggccacga tgcgtccggc gtagaggcga tttaaagaca ggatatcagt ggtccaggct 8220 ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa 8280 taaaagattt tatttagtct ccagaaaaag gggggaa 8317 <210> 35 <211> 8317 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polynucleotide <400> 35 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat 420 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc 480 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag 540 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc 600 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg 660 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttcgga 720 acacccggcc gcaaccctgg gagacgtccc agggacttcg ggggccgttt ttgtggcccg 780 acctgagtcc taaaatcccg atcgtttagg actctttggt gcacccccct tagaggaggg 840 atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt 900 gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc tgctgcagca tcgttctgtg 960 ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat atgggcccgg gctagcctgt 1020 taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga tcgctcacaa 1080 ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag aatggccaac 1140 ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca cccaggttaa 1200 gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct acatcgtgac 1260 ctgggaagcc ttggctttttg acccccctcc ctggggtcaag ccctttgtac accctaagcc 1320. tccgcctcct cttcctccat ccgccccgtc tctccccctt gaaccctcctc gttcgacccc 1380 gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccccca tatggccata 1440 tgagatctta tatggggcac ccccgcccct tgtaaacttc cctgaccctg acatgacaag agttactac agccctctc tccaagctca cttacaggct ctctacttag tccagcacga agtctggaga cctctggcgg cagcctacca agaacaactg gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac cagactaaga acctagaacc tcgctggaa ggaccttaca cagtcctgct gaccacccc accgccctca aagtagcgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc gaccccgggg gtggaccatc ctctagaccg ccatgtcgggg ggcaggtgcc accggccgcg ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca aggaggcatg ccccacaggc 1920 ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc tgggcgaggg tgtggcccag 1980 ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg acagcgtgac gttctccgac 2040 gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt gcgtggggct ccagagcatg 2100 tcggcgccat gcgtggaggc cgacgacgcc gtgtgccgct gcgcctacgg ctactaccag 2160 gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg aggcgggctc gggcctcgtg 2220 ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt gccccgacgg cacgtattcc 2280 gacgaggcca accacgtgga cccgtgcctg ccctgcaccg tgtgcgagga caccgagcgc 2340 cagctccgcg agtgcacacg ctgggccgac gccgagtgcg aggagatccc tggccgttgg 2400 attacacggt ccacaccccc agagggctcg gacagcacag cccccagcac ccaggagcct 2460 gaggcacctc cagaacaaga cctcatagcc agcacggtgg caggtgtggt gaccacagtg 2520 atgggcagct cccagcccgt ggtgacccga ggcaccaccg acaacctcat ccctgtctat 2580 tgctccatcc tggctgctgt ggttgtgggt cttgtggcct acatagcctt caagaggtgg 2640 aacagctccg gctccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag 2700 gagaaccccg gccccgcggc cgccatggcg acgggttcaa gaacttccct acttcttgca 2760 tttggcctgc tttgtttgcc gtggttacag gaagcctcag caggtcaaca gctgaatcag 2820 agtcctcaat ctatgtttat ccaggaagga gaagatgtct ccatgaactg cacttcttca 2880 agcatattta acacctggct atggtacaag caggaccctg gggaaggtcc tgtcctcttg 2940 atagccttat ataaggctgg tgaattgacc tcaaatggaa gactgactgc tcagtttggt 3000 ataaccagaa aggacagctt cctgaatatc tcagcatcca tacctagtga tgtaggcatc 3060 tacttctgtg ctggatttct ggatagcaac tatcagttaa tctggggcgc tgggaccaag 3120 ctaattataa agccagatat ccagaacccc gagcccgccg tgtaccagct gaaggacccc 3180 agaagccagg acagcaccct gtgcctgttc accgacttcg acagccagat caacgtgccc 3240 aagaccatgg agagcggcac cttcatcacc gacaagaccg tgctggacat gaaggccatg 3300 gacagcaaga gcaacggcgc catcgcctgg tccaaccaga ccagcttcac atgccaggac 3360 atcttcaagg agaccaacgc cacctacccc agcagcgacg tgccctgcga cgccaccctg 3420 accgaagaga gcttgagac cgacatgaac ctgaacttcc agaacctgag cgtgatgggc 3480 ctgagaatcc tgctgctgaa ggtggccggc ttcaacctgc tgatgaccct gaggctgtgg 3540 agcagcaggg caaaacgttc gggttcgggt gcgccagtaa agcagacatt aaactttgat 3600 ttgctgaaac ttgcaggtga tgtagagtca aatccaggtc caatggcaac agggagccga 3660 acctctctgc tccttgcttt cgggctcctt tgcctaccgt gcctgcagga gggctcggca 3720 gaagcccaag tgacccagaa cccaagatac ctcatcacag tgactggaaa gaagttaaca 3780 gtgacttgtt ctcagaatat gaaccatgag tatatgtcct ggtatcgaca agaccaggg 3840 ctgggcttaa ggcagatcta ctattcaatg aatgttgagg tgactgataa gggagatgtt 3900 cctgaagggt acaaagtctc tcgaaaagag aagaggaatt tccccctgat cctggagtcg 3960 cccagcccca accagacctc tctgtacttc tgtgccagcg ctagcgggta ccgcacagat 4020 acgcagtatt ttggcccagg caccccggctg acagtgctcg aggacctgag gaacgtgacc 4080 ccccccaagg tgtccctgtt cgagcccagc aaggccgaga tcgccaacaa gcagaaggcc 4140 accctggtgt gcctggccag gggcttcttc cccgaccacg tggagctgtc ttggtgggtg 4200 aacggcaagg aggtgcacag cggcgtgagc accgaccccc aggcctacaa ggagagcaac 4260 tacagctact gcctgagcag caggctgaga gtgagcgcca ccttctggca caaccccagg 4320 aaccacttcc gctgtcaggt gcagttccac ggcctgagcg aggaggacaa gtggcccgag 4380 ggcagcccca agcccgtgac ccagaacatc agcgccgagg cctggggcag agccgactgc 4440 ggcatcacca gcgccagcta ccaccagggc gtgctgtccg ccaccatcct gtacgagatc 4500 ctgctgggca aggccacact gtacgccgtg ctggtgtccg gcctggtgct gatggccatg 4560 gtgaagaaga agaacagcta aaggatccga taaaataaaa gattttattt agtctccaga 4620 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat 4680 tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac 4740 agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc 4800 tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac 4860 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta 4920 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag 4980 agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc 5040 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga 5100 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct 5160 tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat 5220 tagccactgt tttgaatcca catactccaa tactcctgaa atccatcgat ggagttcatt 5280 atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac 5340 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 5400 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 5460 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 5520 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5580 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5640 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5700 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5760 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5820 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5880 cgctttctca tagctcacgc ttaggttatc tcagttcggt gtaggtcgtt cgctccaagc 5940 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 6000 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 6060 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 6120 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 6180 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6240 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6300 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6360 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6420 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6480 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6540 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6600 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6660 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6720 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6780 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6840 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6900 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6960 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 7020 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 7080 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7140 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7200 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7260 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7320 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7380 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7440 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 7500 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 7560 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 7620 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 7680 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 7740 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 7800 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 7860 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccacgctct 7920 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 7980 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg 8040 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 8100 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 8160 ccggccacga tgcgtccggc gtagaggcga tttaaagaca ggatatcagt ggtccaggct 8220 ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa 8280 taaaagattt tatttagtct ccagaaaaag gggggaa 8317 <210> 36 <211> 9 <212> PRT <213> Homo sapiens <400> 36 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 <210> 37 <211> 119 <212> PRT <213> Homo sapiens <400> 37 Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly 1 5 10 15 Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr 20 25 30 Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala 35 40 45 Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys 50 55 60 Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr 65 70 75 80 Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala 85 90 95 Pro Gln Gly Tyr Gly Gly Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr 100 105 110 Arg Leu Thr Val Leu Glu Asp 115

Claims

1. A polynucleotide disposed in a vector, wherein: The polynucleotides encode the 3A1 VαT cell receptor polypeptide of SEQ ID NO:3 and the 3A1 VβT cell receptor polypeptide of SEQ ID NO:

4.

2. The polynucleotide of claim 1, wherein the carrier comprises a peptide that regulates the 3A1 VαT cell receptor polypeptide or the 3A1 VβT cell receptor polypeptide on CD8. + The polynucleotide sequence expressed within T cells.

3. The polynucleotide of claim 2, wherein the vector is a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, or a retrovirus vector.

4. The polynucleotide of claim 2, wherein the vector is a lentiviral vector.

5. The polynucleotide of claim 1, wherein the carrier comprises a polynucleotide encoding a Vα polypeptide or a polynucleotide encoding a Vβ polypeptide.

6. The polynucleotide of claim 1, wherein the vector comprises a combination of a polynucleotide encoding a Vα polypeptide and a polynucleotide encoding a Vβ polypeptide disposed therein, such that the Vα and Vβ T cell receptors (TCRs) are activated on CD8. + It is expressed on the surface of T cells.

7. A composition comprising a host cell transduced with a vector containing a polynucleotide as described in any one of claims 1-6.

8. The composition of claim 7, wherein the host cell is a human CD8+ cell. + T cells.

9. The composition of claim 8, wherein the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients selected from the group consisting of buffers, antimicrobial agents, tension modifiers, wetting agents, detergents, and pH adjusters.

10. The composition of claim 9, wherein: The CD8 + T cells were obtained from individuals diagnosed with cancer expressing the NY-ESO-1 antigen; and The CD8 + T cells were transduced using a vector containing a combination of polynucleotides encoding TCR Vα and TCR Vβ peptides, allowing heterologous TCRs to be transported to CD8 cells. + The heterologous TCR is expressed on the surface of T cells, wherein the heterologous TCR recognizes the NY-ESO-1 peptide associated with the human leukocyte antigen shown in SEQ ID NO.36, which is expressed on the surface of the cancer cells.

11. The composition of claim 10, wherein the vector is a retroviral vector.

12. Use of the polynucleotide of claim 1 or the composition of claim 7 in the preparation of a pharmaceutical agent, said pharmaceutical agent being used in a method for killing cancer cells expressing the NY-ESO-1 antigen, said method comprising reacting the cancer cells with CD8 as described in claim 8 under the following conditions. + T cell combination, thereby enabling the cancer cells to be recognized and killed, the conditions allowing heterologous TCRs on CD8 + The NY-ESO-1 peptide, associated with human leukocyte antigen (HLA) as shown in SEQ ID NO. 36, is expressed on the surface of the cancer cells, and the cancer cells are neuroblastoma cells, myeloma cells, metastatic melanoma cells, synovial sarcoma cells, bladder cancer cells, esophageal cancer cells, hepatocellular carcinoma cells, head and neck cancer cells, non-small cell lung cancer cells, ovarian cancer cells, prostate cancer cells, or breast cancer cells.

13. The use as described in claim 12, wherein the method is performed after infusing the CD8 + The T-cell therapy is performed in patients.

14. The use as claimed in claim 12, wherein the method comprises applying the first modified CD8 + T cells and second CD8 + The combination of T cells, the first modified CD8 + T cells target the NY-ESO-1 peptide associated with the first human leukocyte antigen (HLA) as shown in SEQ ID NO. 36, and the second CD8 peptide. + T cells target the NY-ESO-1 peptide, which is associated with the second human leukocyte antigen as shown in SEQ ID NO.

36. 15.CD8 + Use of T cells in the manufacture of agents for treating cancer, wherein the CD8 is transduced using a vector comprising the polynucleotide as described in any one of claims 1-6. + T cells, wherein the cancers mentioned are melanoma, neuroblastoma, myeloma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

16. The use as claimed in claim 15, wherein the melanoma is a metastatic melanoma.

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