Chimeric Antigen Receptors (CARs), Compositions, and Methods of Use Thereof

Abstract

The present invention relates to compositions and methods related to chimeric antigen receptor (CAR) polypeptides, and methods related thereto. In one embodiment, the present invention relates to engineered cells having chimeric antigen receptor polypeptides against at least two targets. In another embodiment, the present invention relates to engineered cells having chimeric antigen receptor polypeptides and enhancer moieties.

Description

[0001] This application is a divisional application of the patent application with the application date of June 24, 2016, application number 201680036459.4, and invention title "Chimeric Antigen Receptor (CAR), Compositions and Methods of Use Thereof".

[0002] Cross - reference to related applications

[0003] This application is an international PCT application, which claims the priority of U.S. Provisional Application No. 62 / 184,321 filed on June 25, 2015; No. 62 / 235,840 filed on October 1, 2015; and No. 62 / 244,435 filed on October 21, 2015, all of which are incorporated herein by reference in their entireties. Background of the Invention

[0004] T cells, a type of lymphocyte, play an important role in cell - mediated immunity. They are distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of T - cell receptors (TCRs) on their cell surface. Helper T cells, also known as CD4+ T or CD4 T cells, express the CD4 glycoprotein on their surface. Helper T cells are activated upon exposure to peptide antigens presented by major histocompatibility complex (MHC) class II molecules. Once activated, these cells rapidly proliferate and secrete cytokines that can regulate the immune response. Cytotoxic T cells, also known as CD8+ T cells or CD8 T cells, express the CD8 glycoprotein on the cell surface. CD8+ T cells are activated upon exposure to peptide antigens presented by MHC class I molecules. Memory T cells, a subset of T cells, persist for a long time and respond to their cognate antigens, thus providing the immune system with "memory" against past infections and / or tumor cells.

[0005] When genetically modified, T cells can produce a special receptor on their surface called a chimeric antigen receptor (CAR). A CAR is a protein that allows T cells to recognize a specific protein (antigen) on tumor cells. These engineered CAR T cells are then grown in the laboratory until their numbers reach billions. Then, the expanded population of CAR T cells is infused into a patient.

[0006] To date, clinical trials have confirmed the great promise of chimeric antigen receptor (CAR) T cells in hematologic malignancies resistant to standard chemotherapy. Most notably, specific CD19 CAR (CD19CAR) T cell therapies have shown remarkable efficacy, including long-term remissions in B cell malignancies (Kochenderfer, Wilson et al. 2010, Kalos, Levine et al. 2011, Porter, Levine et al. 2011, Davila, Riviere et al. 2013, Grupp, Frey et al. 2013, Grupp, Kalos et al. 2013, Kalos, Nazimuddin et al. 2013, Kochenderfer, Dudley et al. 2013, Kochenderfer, Dudley et al. 2013, Lee, Shah et al. 2013, Park, Riviere et al. 2013, Maude, Frey et al. 2014).

[0007] Despite the success of CAR therapies in B cell leukemia and lymphoma, no definitive CAR therapy has been applied to T cell malignancies. Given that therapies for T cell malignancies have significantly worse outcomes than those for B cell malignancies (Abramson, Feldman et al. 2014), CAR therapies have the potential to further address a large clinical need.

[0008] To date, current efforts have focused on CAR T cells that have shown efficacy in treating various B cell malignancies. Although an initial remission rate of approximately 90% is common in B-ALL using CD19CAR, most relapse within a year. At least part of the relapse is attributed to antigen escape. Therefore, there is an urgent need for more effective CAR T cell therapies to prevent relapse. Target exploration and selection are the initial steps because there are no general rules to ensure or guide effective CAR design.

[0009] There are currently some obstacles to the wider application of CAR therapeutic approaches. The most common challenges are: (1) antigen target and chimeric antigen receptor selection; (2) CAR design; (3) tumor heterogeneity, particularly differences in the surface expression of tumor antigens. Targeting a single antigen carries the risk of immune escape, which can be addressed by targeting multiple desired antigens.

[0010] Most chimeric antigen receptors (CARs) are single-chain variable fragments (scFvs) derived from monoclonal antibodies, and some of these monoclonal antibodies have been used in clinical trials or disease treatment. However, monoclonal antibodies have limited efficacy, indicating the need for alternative and more potent targeting methods such as CARs. ScFvs are the most commonly used chimeric antigen receptors for CARs. However, the CAR affinity binding and location of the epitopes recognized on the antigen can affect function. In addition, the surface CAR expression level of T cells or natural killer (NK) cells is affected by suitable leader sequences and promoters. In addition, overexpressed CAR proteins can be toxic to cells.

[0011] Accordingly, there remains a need for improved chimeric antigen receptor-based therapies that can account for more effective, safe, and efficient targeting of T cell-related malignancies. SUMMARY OF THE INVENTION

[0012] In one embodiment of the invention, the invention provides an engineered cell having a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain; wherein the first antigen recognition domain is different from the second antigen recognition domain.

[0013] In another embodiment, the invention provides an engineered polypeptide comprising a chimeric antigen receptor and an enhancer.

[0014] In another embodiment, the invention provides an engineered polypeptide comprising a chimeric antigen receptor polypeptide and an enhancer.

[0015] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising a signal peptide, a CD45 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. In another embodiment, the invention provides a polynucleotide encoding the foregoing polypeptide.

[0016] In another embodiment, the invention provides an engineered cell having the foregoing engineered polypeptide or polynucleotide.

[0017] In another embodiment, the present invention provides a method for reducing the number of target cells, which comprises the steps of (i.) contacting the target cells with an effective amount of engineered cells having at least one chimeric antigen receptor polypeptide, the engineered cells having a plurality of chimeric antigen receptor polypeptides, each chimeric antigen receptor polypeptide being independent; and (ii.) optionally analyzing the reduction in the number of such cells. The target cells include at least one cell surface antigen selected from the group consisting of interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD41, CD45, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1.

[0018] In another embodiment, the present invention provides a method for treating B-cell lymphoma, T-cell lymphoma, multiple myeloma, chronic myelogenous leukemia, B-cell acute lymphoblastic leukemia (B-ALL), and cell proliferative diseases by administering any of the above-mentioned engineered cells to a patient in need thereof.

[0019] Brief description of the drawings

[0020] Figure 1 . Schematic diagram of a cCAR construct (hereinafter, "multiple CARs or compound CARs"). The diverse or composite CAR targets multiple antigens (such as cell type 1 or cell type 2 or the same cell type). The diverse or cCAR T cell immunotherapy comprises individual component CARs, which comprise different or identical antigen recognition domains, hinge regions, transmembrane domains, multiple co-stimulatory domains, and intracellular signaling domains.

[0021] Figure 2A . Schematic diagram of a cCAR-T construct. The construct comprises an SFFV promoter that drives the expression of multiple modular units of CARs linked by P2A peptides. After linker cleavage, the cCARs are cleaved and engage when the target expresses CD33 and / or CD123. As a novel cCAR construct, the activation domains of the construct may include (but are not limited to) 4-1BB on the CD33 CAR segment and the CD28 region on the CD123CAR.

[0022] Figure 2B. Western blot depicting the expression of transduced CD33CD123 cCAR-T cells. The figure depicts the expression of two different CAR proteins, namely CD33 CAR and CD123 CAR. cCAR-T cells expressing CD33 and CD123 CAR upon linker cleavage produce two distinct and consistently strong protein bands. Green fluorescent protein (GFP) is included as a negative control.

[0023] Figure 2C . Flow cytometry of transduction efficiency. The upper figure shows the lentiviral titer of CD33CD123 cCAR (also known as CD33CD123-2G-CAR) tested on 293FT HEK (human embryonic kidney) cells to examine the maximum transduction efficiency before use on UCB (umbilical cord blood) and PB (peripheral blood) T cells. The lower figure shows CD33CD123 cCAR (also known as CD33CD123-2G-CAR) T cells transduced with a lentiviral vector containing the CD33CD123 cCAR construct and GFP-transduced cells as a control. The percentage indicated by the yellow ring is a surrogate for transduction efficiency.

[0024] Figure 3 . Schematic diagram showing a method for generating highly efficient complex CAR (cCAR).

[0025] Figure 4 . Co-culture analysis of CD33CD123-2G CAR-T cells (cCAR) co-cultured with the promyelocytic leukemia cell line HL60. cCAR-T cells (lower figure) are compared with control GFP-transduced T cells (upper figure). Cytotoxicity is measured by the CD33+ cell population remaining after approximately 24 hours of culture (enclosed in the yellow ring).

[0026] Figure 5 . Co-culture analysis of cCAR-T cells co-cultured with the myeloid leukemia cell line KG-1a, which expresses approximately 100% CD33 and approximately 50 - 80% CD123. cCAR-T cells (lower figure) are compared with control GFP-transduced T cells (upper figure). Cytotoxicity is measured by the CD33+ cell population remaining after approximately 24 hours of culture.

[0027] Figure 6. Co - culture analysis of cCAR - T cells with AML patient samples (referred to here as AML - 9). Patient cells include a mixed cell population, such as leukemia cells, monocytes, and other types of blasts. CD33 serves as a marker for CAR - T function and a marker for CD34 (a specific marker for leukemia cells). Compare the CAR - T graph (right) with control GFP - transduced T cells (middle). Cytotoxicity is measured by the CD33 + / CD34 + cell population remaining after culturing for at least 24 hours.

[0028] Figure 7 . Co - culture analysis of cCAR - T cells with B - ALL patient samples (referred to here as Sp - BM - B6). Patient cells include a mixed cell population, such as leukemia cells, monocytes, and other types of blasts. CD34 serves as a specific marker for leukemia cells. Compare the CAR - T graph (right) with control GFP - transduced T cells (middle). Cytotoxicity is measured by the CD34 + cell population remaining after culturing for at least 24 hours.

[0029] Figure 8 . CD33CD123 cCAR expression in NK - 92 cells. Detect CD33CD123 cCAR expression using goat anti - mouse antibody F(ab)2.

[0030] Figure 9 . Co - culture analysis of cCAR NK - 92 cells with HL - 60. Compare cCAR NK - 92 cells with GFP - transduced NK - 92 cells. Cytotoxicity is measured by the CD33 + cell population remaining after culturing for about 24 hours.

[0031] Figure 10 . Co - culture analysis of cCAR NK - 92 cells with KG1a. Compare the cCAR NK cell graph with GFP - transduced NK - 92 cells. Cytotoxicity is measured by the CD33 + cell population remaining after culturing for about 24 hours.

[0032] Figure 11 . Dose response of CD33CD123 cCAR (CAR - CD33 / 123) NK - 92 cells with HL - 60 or KG1a. Cytotoxicity is measured by the CD33 + cell population remaining after culturing for about 24 hours.

[0033] Figure 12. Comparison of CD33CD123 cCAR NK-92 cell killing with controls in two KG11 cell populations. Analyses were performed at different ratios of CAR-CD33 / 123 (CD33CD123 cCAR NK-92 cells) and target cells kG1a. Killing was measured by the CD33+CD123+ or CD33+CD123- cell population remaining after approximately 24 hours of incubation.

[0034] Figure 13 . Schematic of cCAR. The construct contains an SFFV promoter driving the expression of multiple modular units of a CAR linked by a linker. When the linker cleaves, the cCAR cleaves and engages when the target expresses a combination of multiple target antigens (CD19 and / or CD20, and / or CD22 and / or 138). Multiple cCARs use the same or different co-stimulatory domains such as (but not limited to) 4-1BB (also labeled 4-BB) and / or CD28.

[0035] Figure 14A - C.BCMA-CS1 cCAR construct combination (BC1cCAR). (A) The construct consists of an SFFV promoter driving the expression of two modular units of a CAR linked by a P2A peptide. When this P2A peptide cleaves, the cCAR cleaves and engages when the target expresses BCMA and / or CS1. The two-unit CAR uses the same co-stimulatory domain 4-1BB. (B) Flow cytometry analysis of BC1cCAR expression on the surface of T cells for the vector (left) and BC1cCAR showing 15.3% positive for F(ab)2 (right, highlighted by a square). Gating was performed against an isotype control. (C) Basic functional validation of BC1cCAR-T cells by co-culturing K562 cells transduced with BCMA cDNA (BCMA-K562) (obtained from Kochenderfer, NIH). The bar graph shows lysis of the BCMA-K562 cell line versus control T cells and lysis of wild-type K562 (wt-K562) versus control.

[0036] Figure 14D . BCMAC-S1-2G construct with two different co-stimulatory domains (4-1BB or CD28) for each unit. The construct includes an SFFV promoter driving the expression of two modular units of a CAR linked by a P2A peptide. When this P2A peptide cleaves, the cCAR cleaves and engages the target expressing BCMA and / or CS1. The two-unit CAR uses different co-stimulatory domains, namely 4-1BB or CD28. Flow cytometry analysis of BC1cCAR expression on the surface of T cells for the vector (left) and BC1cCAR showing rare positive cells for F(ab)2 (right, highlighted by a square). Gating was performed against an isotype control.

[0037] Figure 14E . Protein expression of BC1cCAR and BCMA-CS1-2G in HEK-293FT cells. HEK-293FT cells were transfected with lentiviral plasmids of GFP (lane 1), BC1cCAR (lane 2), and CD269-CS1-2G (lane 3) for 48 hours. After transfection, the supernatant was removed, and the cells were also removed. The cells were lysed with mouse anti-human CD3z antibody for Western blotting and investigation.

[0038] Figure 15A - Co-culture with the MM1S cell line. After co-culture for 24 hours, the cells were collected and analyzed by flow cytometry. Target MM1S cells (myeloma cells) were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. The population was gated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 15A : Flow cytometry plot of co-culture.

[0039] Figure 15B : Right: Overview plot of lysis control E:T ratio.

[0040] Figure 16A - Co-culture with the RPMI-8226 cell line. After co-culture for 24 hours, the cells were collected and analyzed by flow cytometry. Target RPMI-8226 cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. The population was gated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 16A : Flow cytometry plot of co-culture. Figure 16B : Overview plot of lysis control E:T ratio.

[0041] Figure 17A - Co-culture with the U266 cell line. After co-culture for 24 hours, the cells were collected and analyzed by flow cytometry. Target U266 cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. The population was gated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Left: Flow cytometry plot of co-culture, Right: Overview plot of lysis control E:T ratio.

[0042] Figure 18A- Co - culture of primary patient samples of - B.MM10 - G and specific lysis rate. Co - culture was carried out for 24 hours and samples were collected and analyzed by flow cytometry. Target MM10 - G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were gated by anti - BCMA (CD269) and anti - CS1 (CD319) antibodies. Notably, the gated display presented MM10 - G with different BCMA+ and CS1+ populations. Figure 18A : Flow cytometry plot of co - culture. Figure 18B : Overview plot of lysis vs E:T ratio.

[0043] Figure 19A - Co - culture of primary patient samples of - B.MM7 - G and specific lysis rate. Co - culture was carried out for 24 hours and samples were collected and analyzed by flow cytometry. Target MM7 - G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were gated by anti - BCMA (CD269) and anti - CS1 (CD319) antibodies. Figure 19A : Flow cytometry plot of co - culture. Figure 19B : Overview plot of lysis vs E:T ratio.

[0044] Figure 20A - Co - culture of primary patient samples of - B.MM11 - G and specific lysis rate. Co - culture was carried out for 24 hours and samples were collected and analyzed by flow cytometry. Target MM11 - G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were gated by anti - BCMA (CD269) and anti - CS1 (CD319) antibodies. Figure 20A : Flow cytometry plot of co - culture. Figure 20B : Overview plot of lysis vs E:T ratio.

[0045] Figure 21 . CD269 CS1 - BBCAR NK cells exhibit in vivo anti - leukemia effect. NSG mice were irradiated with a sublethal dose and on the next day were injected intravenously with luciferase - expressing MM.1S multiple myeloma cells to induce measurable tumor formation. After 3 days, 8×106 CD269 - CS1 - BBCAR NK cells or vector - control NK control cells were injected intravenously into the mice. On days 3, 6, and 8, RediJect D - luciferin was injected subcutaneously into the mice and the mice were subjected to IVIS imaging. The average light intensity of mice injected with CD269 - CS1 - BBCAR NK was compared with that of mice injected with vector - control NK.

[0046] Figure 22 . Measure the percentage of survival of the mice and based on data from Figure 21The study was compared between two groups.

[0047] Figure 23 . The CRISPR / Cas9 interference system. The expression of sgRNA and Cas9 puromycin was driven by the U6 and SFFV promoters, respectively. Cas9 was linked to the puromycin resistance gene by the E2A self-cleavage sequence.

[0048] Figure 24 . Provide a schematic diagram of an example of the steps for generating CAR T or NK cells targeting malignant blood diseases.

[0049] Figure 25 . Generation and sorting of NK-92 cells stably expressing the CD45 blocking gene using the CRISPR / Cas9 lentiviral system. Flow cytometry analysis indicated the amount of CD45 expression on the surface of NK-92 cells (left panel). After transduction of sgCD45B CRISPR into NK-92 cells, the transduced cells were cultured in medium containing puromycin for several weeks. CD45-negative NK-92 cells were determined using a CD45 antibody and sorted. The purity of the stable NK45i-92 (expressing the CD45 blocking gene) NK-92 cells was determined by flow cytometry analysis (right panel). This data confirmed the successful generation and acquisition of NK45i-92 cells.

[0050] Figure 26 . Cell growth curves of wild-type, GFP-transduced NK-92 or NK45i-92 NK cells. To evaluate the cell proliferation caused by CD45 blocking gene expression (KD) in NK-92 cells, the number of NK-92 (●), GFP-transduced -92 (■), and NK45i-92 (▲) cells was counted at the 48th and 96th hours after inoculation into a 24-well plate. IL-2 was added at the 48-hour time point (n = 3 independent experiments performed in duplicate). Data are mean ± S.D. These data indicate that the blocking gene expression of the CD45 receptor on NK-92 shows a similar cell growth curve compared to untransduced NK-92 or GFP-transduced NK-92 cells.

[0051] Figure 27A-B. Co-culture analysis was performed using CCRF-CEM (target: T) and GFP NK-92 or GFP NK45i-92 cells (effector: E) at a 5:1 (E:T) ratio. Incubation was for 16 hours. (A) Flow cytometry analysis of only CCRF-CEM (blue dots in the left panel), in co-culture with CCRF-CEM and control GFP-transduced NK-92 cells (middle panel) or GFP NK45i-92 cells (right panel). Blue dots in all panels indicate residual target CCRF-CEM cells and red dots show effector cells of the co-culture assay. The total incubation time was 16 hours and the effector T cell:target cell ratio was 5:1. All experiments were performed in duplicate. (B) Histogram indicating the percentage of lysis of GFP-transduced NK45i-92 cells compared to control GFP-transduced NK92 cells in the co-culture assay performed with CCRF-CEM. These data show that there is no significant difference in the killing activity against CCRF-CEM cells of the CD45-blocking gene expression in NK-92 cells compared to the in vitro co-culture assay with GFP control NK-92 cells. Blue dots are in the upper left portion.

[0052] Figure 28A-B. Co-culture assay using CCRF-CEM (target: T) and GFP NK-92, CD5CAR NK-92 or CD5CAR NK45i-92 cells (effector: E). 5:1 (E:T) ratio. 16-hour incubation (A) From right to left, flow cytometry analysis of only CCRF-CEM (left panel), in co-culture with CCRF-CEM and control GFP NK-92 cells (left middle panel), CD5CAR NK-92 cells (right middle panel), CD5CAR NK45i-92 cells (right panel). Blue dots in all panels indicate residual target CCRF-CEM cells and red dots show effector cells of the co-culture assay. The total incubation time was 16 hours and the ratio of effector T cells:target cells was 5:1. All experiments were performed in duplicate. (B) Bar graph indicating the percentage of lysis of CD5CAR NK-92 cells or CD5CAR NK45i-92 cells compared to control GFP NK92 cells in the co-culture assay using CCRF-CEM. Data are mean ± S.D.. Both CD5CAR NK cells and CD5CAR NK45i-92 cells showed approximately 100% cytotoxic activity against CD5-positive CCRF-CEM compared to control GFP NK-92 cells. These data demonstrate that compared to in vitro co-culture assay with GFP control NK-92 cells, CD5CAR NK cells and CD5CAR NK45i-92 cells can effectively lyse CD5-expressing CCRF-CEM cells and provide evidence that blockade of CD45 gene expression does not affect cell function regarding killing activity in NK-92 cells. Blue spots are located in the upper left portion of the first two panels starting from the left.

[0053] Figure 29A -B. Organization and expression of the CD45CAR construct. (A) Schematic diagram of the CD45CAR lentiviral vector. The CD45CAR construct is a modular signaling domain that contains: a leader sequence, anti-CD45 scFv, a hinge domain (H), a transmembrane domain (TM), two co-stimulatory domains (CD28 and 4-1BB) that define the construct as a 3rd generation CAR, and the intracellular signaling domain CD3ζ. (B) HEK-293FT cells were transfected with lentiviral plasmids of GFP (lane 1) and CD45CAR (lane 2). At 48 hours after transfection, the supernatant was removed and the cells were also removed. The cells were lysed with mouse anti-human CD3z antibody for Western blotting and investigation.

[0054] Figure 30A-B. Transduce CD45CAR into NK45i-92 cells and perform cell sorting on the CD45CAR-transduced cells. (A) After CD45CAR lentiviral transduction into NK45i-92 cells, the expression level of CD45CAR on NK45i-92 measured by flow cytometry analysis (blue circle in the middle panel) was compared with that of NK45i-92 cells (left panel). Sort the CD45CAR-expressing NK45i-92 cells and measure the CD45 expression level on the cell surface by flow cytometry analysis (right panel). (B) Approximately 87% CD45CAR expression on the cell surface was detected by flow cytometry analysis.

[0055] Figure 31A-31B . Perform co-culture analysis using CCRF-CEM (target: T) and GFP NK-92 or CD45CAR NK45i-92 cells (effector: E). 5:1 (E:T) ratio. 16-hour incubation. (A) Flow cytometry analysis of co-culture with CCRF-CEM and control GFP-transduced NK-92 cells (left panel) or CD45CAR NK45i-92 cells (right panel). The blue dots in all panels indicate the remaining target CCRF-CEM cells and the red dots show the effector NK-92 cells of the co-culture assay. The total incubation time was 16 hours and the ratio of effector T cells:target cells was 5:1. All experiments were performed in duplicate. (B) The bar graph indicates the percentage of cell lysis of CD45CAR NK45i-92 cells compared with control GFP NK92 cells in the co-culture assay using CCRF-CEM. Data are mean ± S.D.. CD45CAR NK45i-92 cells showed approximately 70% cell lysis against CCRF-CEM cells compared with control GFP NK-92 cells. These data indicate that CD45CAR NK45i-92 cells effectively lysed CCRF-CEM cells expressing CD45 compared with the in vitro co-culture assay with GFP control NK-92 cells.

[0056] Figure 32A-C. Co-culture analysis with Jurkat cells (target: T) and GFP control or CD45CAR NK45i-92 cells (effector: E). 5:1 or 2:1 (E:T) ratios. 6-hour incubation. (A) Flow cytometry analysis was performed after staining Jurkat cells with the CMTMR cell tracker dye. These data confirmed that Jurkat cells were CD45 positive (left panel) and mostly CD56 negative cells (right panel). (B) Flow cytometry analysis of co-cultures with Jurkat cells (target: T) and control or CD45CAR NK45i-92 cells (effector: E). Co-culture analysis was performed at 5:1 or 2:1 (E:T) ratios. The left panel shows co-cultures with control GFP or CD45CAR / CD45KD NK-92 cells at a 5:1 (E:T) ratio and the right panel indicates co-cultures with control GFP or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. The blue dots in the figures indicate residual target Jurkat cells and the red dots represent effector cells of the co-culture assay. The total incubation time was 6 hours. All experiments were performed in duplicate. (C) Bar graph shows the percentage of lysis of CD45CAR NK45i-92 cells compared to control GFP NK92 cells at 5:1 or 2:1 (E:T) ratios. Data are mean ± S.D.. At both conditions, CD45CAR NK45i-92 cells showed approximately 60% lysis of Jurkat cells compared to control GFP NK-92 cells. This data indicates that CD45CAR NK45i-92 cells efficiently lysed Jurkat cells expressing CD45 on the cell surface compared to in vitro co-culture assay with GFP control NK-92 cells.

[0057] Figure 33A-C. Co-culture assay using GFP-NK-92 cells (target: T) and untransduced NK-92 cells or CD45CAR NK45i-92 cells (effector: E). 5:1 or 2:1 (E:T) ratio. 6-hour incubation (A) Flow cytometry analysis using GFP control NK-92 cells. These data confirm that GFP control NK-92 cells are approximately 99% GFP-positive cells (green dots). (B) Flow cytometry analysis of co-culture assay with GFP control NK-92 cells (target: T) and untransduced or CD45CAR NK45i-92 cells (effector: E). Co-culture assay was performed at a ratio of 5:1 or 2:1 (E:T). The left panel shows co-culture with untransduced or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio and the right panel indicates co-culture with untransduced or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Green dots in the figures indicate residual target GFP NK-92 cells and red dots represent effector cells of the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (C) Bar graph shows the percentage of cell lysis of GFP NK-92 cells by CD45CAR NK45i-92 cells compared to untransduced NK-92 cells at 5:1 or 2:1 (E:T) ratios. Data are mean ± S.D. CD45CAR NK45i-92 cells show approximately 20% cell lysis at a 2:1 (E:T) ratio and approximately 55% cell lysis at a 5:1 (E:T) ratio for GFP NK-92 cells compared to untransduced NK-92 cells. This data indicates that CD45CAR NK45i-92 cells effectively lyse GFP NK-92 cells expressing CD45 on the cell surface compared to in vitro co-culture assay with untransduced NK-92 cells. Green dots are located in the upper right portion of each figure.

[0058] Figure 33D-E. Transduce CD45b-BB or CD45b-28 into NK45i-92 cells and perform cell sorting of NK45i-92 cells transduced with CD45b-BB or CD45b-28. (D) After lentiviral transduction of CD45b-BB or CD45b-28 into NK45i-92 cells, the surface expression levels of CD45b-BB CAR or CD45b-28 CAR on NK45i-92 were determined by flow cytometry analysis compared to NK45i-92 cells (left panel) (blue circles in the middle panel). (E) Sort NK45i-92 cells expressing CD45b-BB or CD45b-28 CAR by flow cytometry analysis. Approximately 74% CD45b-BB CAR or 82% CD45b-28 CAR expression on the cell surface was detected by flow cytometry analysis.

[0059] Figure 33F -G. Co-culture assay with REH cells (target: T) and GFP NK-92 cells or CD45CAR NK45i-92 cells or CD45b-BB NK45i-92 cells or CD45b-28 NK45i-92 cells (effector: E). 5:1 (E:T) ratio. 20-hour incubation. (F) Flow cytometry analysis of co-cultures of only REH cells (left panel), REH cells and control GFP-transduced NK-92 cells (second panel from the left), CD45CAR NK45i-92 cells (middle panel), CD45b-BB NK45i-92 cells (fourth panel from the left) or CD45b-28 NK45i-92 cells (right panel). The blue dots in all panels indicate residual target REH cells and the red dots show effector GFP or CAR-NK-92 cells of the co-culture assay. REH is a B acute lymphoblastic leukemia cell line. The total incubation time was 20 hours and the ratio of effector NK-cells: target cells was 5:1. All experiments were performed in duplicate. (G) Bar graph indicating the percentage of cell lysis of CD45CAR NK45i-92 cells, CD45b-BB NK45i-92 cells or CD45b-28 NK45i-92 cells compared to control GFP NK92 cells in the co-culture assay with REH cells. Data are mean ± S.D.. Compared to control GFP NK-92 cells, for REH cells, CD45CAR NK45i-92 cells showed approximately 76% cell lysis, CD45b-BB NK45i-92 cells showed approximately 79% cell lysis and CD45b-28 NK45i-92 showed 100% cell lysis. These data indicate that all three CD45CARs are effective in lysing REH cells.

[0060] Figure 34A-B. Schematic diagram of the construct and its expression in T or NK cells. (A) A combination of CAR (third generation) and sushi / IL-15 is assembled on an expression vector and driven to express by the SFFV promoter. The CAR with sushi / IL-15 is linked with the P2A cleavage sequence. The sushi / IL-15 part consists of an IL-2 signal peptide fused to the sushi domain and is linked to IL-5 via a 26-amino acid polyproline linker. (B) CAR and sushi / IL15 are present on T or NK cells.

[0061] Figure 35A -B. CD4IL15RA-CAR expression. (a) HEK-293FT cells were transfected with lentiviral plasmids of GFP (lane 1), CD4IL15RA CAR (lane 2), and the positive control CD4CAR (lane 3). At 48 hours after transfection, the supernatant was removed, and the cells were also removed and subjected to Western blotting with a mouse anti-human CD3z antibody. (b) HEK-293 cells were transduced with the GFP (left) or CD4IL15RA CAR (right) viral supernatant from transfected HEK-293FT cells. After 3 days of culture, the cells were collected, stained with goat anti-mouse F(Ab')2, and analyzed by flow cytometry.

[0062] Figure 36 . Transduction of NK cells with CD4IL15RA CAR. NK-92 cells were transduced with the GFP (left) or CD4IL15RA CAR (right) viral supernatant from transfected HEK-293FT cells. A second transduction was performed 24 hours after the first transduction. At 24 hours after the second transduction, the cells were collected, washed, and transferred to a tissue culture dish with fresh medium and IL-2. After 3 days of culture, the cells were collected and stained with a goat anti-mouse F(Ab')2 antibody or goat IgG (control) at 1:250 for 30 minutes. The cells were washed, stained with streptavidin-PE conjugate at 1:500, washed, suspended in 2% formalin, and analyzed by flow cytometry.

[0063] Figure 37. Transduce T cells with CD4IL15RA CAR. Western blot is shown on the left. HEK-293FT cells were transfected with lentiviral plasmids of GFP (lane 1) and CD4IL15RA-CAR (lane 2). At 48 hours after transfection, the supernatant was removed, and the cells were also collected and subjected to Western blot with mouse anti-human CD3ζ antibody. CD4IL15RA CAR expression is shown on the right. Activated T cells from umbilical cord blood buffy coat were transduced with viral supernatants of GFP (left) or CD4IL15RA CAR (right) from transfected HEK-293FT cells. The second transduction was performed 24 hours after the first transduction. At 24 hours after the second transduction, the cells were collected, washed and transferred to tissue culture plates with fresh medium and IL-2. After 3 days of culture, the cells were collected and stained with goat anti-mouse F(Ab')2 or isotype control for 30 minutes. Transduced with GFP (left) or CD4IL15RA (right). The cells were washed and stained with streptavidin-PE conjugate at 1:250, washed, suspended in 2% formalin and analyzed by flow cytometry.

[0064] Figure 38A -B. CD4CAR NK-92 cells and CD4IL15RA CAR NK-92 cells eliminate KARPAS299T leukemia cells in co-culture. NK-92 cells transduced with GFP control (upper right), CD4CAR (lower left) or CD4IL15RA (lower right) lentiviral supernatants were cultured with KARPAS 299 cells at a ratio of 5:1. After 4 hours of co-culture, the cells were stained with mouse anti-human CD4 (APC) and CD3 (PerCp) antibodies and analyzed by flow cytometry (N = 2). The upper left panel shows the labeled Karpas 299 cells alone. The percentage of lysed target cells is shown in the figure.

[0065] Figure 39 . CD4CAR NK-92 cells and CD4IL15RA CAR NK-92 cells eliminate MOLT4T leukemia cells expressing CD4 in co-culture. NK-92 cells transduced with GFP control (left), CD4CAR (middle) or CD4IL15RA (second from the right) lentiviral supernatants were cultured with MOLT4 cells at an effector:target ratio of 1:1 or 2:1. After overnight co-culture, the cells were stained with mouse anti-human CD4 (APC) and CD56 (PerCp) antibodies and analyzed by flow cytometry (N = 2). The upper right panel shows the labeled MOLT4 cells alone. The percentage of lysed target cells is shown in the figure.

[0066] Figure 40.CD4IL15RA CAR T cells exhibited more potent anti-leukemia effects in vivo than CD4CAR. NSG mice were irradiated at a sublethal dose and on the next day were injected intravenously (tail vein) with MOLM13 cells expressing luciferase to induce measurable tumor formation. After 3 days, a course of 8×106 CD4CAR, or CD4IL15RA CAR T cells or vector control T control cells were injected intravenously into the mice. On days 3, 6, 9, and 11, RediJect D-luciferin was injected subcutaneously into the mice and the mice were subjected to IVIS imaging.

[0067] Figure 41 . The percentage of tumor reduction in the mice was measured and comparisons were made between the three groups based on the study from Figure 40 . The mean light intensity measured in the mice injected with CD4CAR and CD4IL15RA CAR T was compared with that in the mice injected with vector control T, and was related to the remaining tumor burden. In each group consisting of two injection cases, CD4CAR T was on the left and CD4IL15RA CAR T was on the right.

[0068] Figure 42 . In 6-well tissue culture plates, in DMEM with 10% FBS, using the indicated volumes, HEK 293 cells were transduced with EF1-GFP or SFFV-GFP virus supernatants. The medium was changed the next morning. Forty-eight hours later, the transduced cells were observed on an EVOS fluorescence microscope using GFP at 10×.

[0069] Figure 43 . Using the volumes from the previous figure, the HEK293 cells transduced with EF1-GFP or SFFV-GFP virus supernatants were trypsinized, suspended in formalin and analyzed by flow cytometry, using the FITC channel to determine the percentage of GFP+ cells.

[0070] Figure 44A-44B . On days 7, 14, 21, and 28 after transduction, in the case of a small or large amount of virus supernatant, the activated cord blood leukocyte layer T cells transduced with EF1-GFP or SFFV-GFP virus supernatants were trypsinized, suspended in formalin and analyzed by flow cytometry, using the FITC channel to determine the percentage of GFP+ cells.

[0071] (A) Percentage of GFP+ T cells in the transduced cells in the case of a small or large supernatant.

[0072] (B) The percentage of GFP+ T cells transduced in the presence of a large amount of EF1-GFP supernatant relative to the percentage of GFP+ cells in T cells transduced in the presence of a small amount of SFFV-GFP supernatant. (Using 50 μL of SFFV-GFP and 1 mL of EF1-GFP supernatant). (N = 2).

[0073] Figure 45 . Ligand-receptor interactions in malignant plasma cells. The APRIL ligand binds to TAC1 or BCMA. The BAFF ligand binds to TAC1, BCMA, or BAFF-R. Embodiments

[0074] The present invention provides chimeric antigen receptor (CAR) compositions, methods for their manufacture and use.

[0075] Chimeric antigen receptor (CAR) polypeptides include a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0076] First-generation CARs include CD3z as the intracellular signaling domain, while second-generation CARs include at least one single co-stimulatory domain derived from a variety of proteins. Examples of co-stimulatory domains include (but are not limited to) CD28, CD2, 4-1BB (CD137, also known as "4-BB"), and OX-40 (CD124). Third-generation CARs include two co-stimulatory domains, such as (but not limited to) CD28, 4-1BB, CD134 (OX-40), CD2, and / or CD137 (4-1BB).

[0077] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise the protein or peptide sequence. Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds. The terms used herein refer to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, of which there are many types). "Polypeptide" includes, in particular, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0078] "Signal peptide" includes a peptide sequence that directs the trafficking and localization of an intracellular peptide and any linked polypeptides, for example, trafficking and localization to an organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0079] A signal peptide is a peptide of any secreted or transmembrane protein that directs the polypeptide disclosed herein to the cell membrane and cell surface and provides correct localization of the polypeptide of the present invention. Specifically, the signal peptide of the present invention directs the polypeptide of the present invention to the cell membrane, where the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is located in the cytoplasmic portion or inside the cell.

[0080] In one embodiment, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. In one embodiment, the signal peptide is a type I, II, III, or IV human protein. In one embodiment, the signal peptide includes an immunoglobulin heavy chain signal peptide.

[0081] "Antigen recognition domain" includes a polypeptide that is selective for or targets any of the following: an antigen of a target, a receptor, a peptide ligand, or a protein ligand; or a polypeptide of a target.

[0082] The antigen recognition domain can be obtained from any of a wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. The antigen recognition domain can include a portion of an Ig heavy chain linked to a portion of an Ig light chain, which constitutes a variable single-chain fragment (scFv) that specifically binds to a target antigen. The antibody can be a monoclonal or polyclonal antibody or can be of any type that specifically binds to a target antigen. In another embodiment, the antigen recognition domain can be a receptor or a ligand. In a particular embodiment, the target antigen is specific for a particular disease condition and the disease condition can be of any type, provided that it has a cell surface antigen that can be recognized by at least one of the chimeric receptor constructs present in the compound CAR architecture. In a particular embodiment, the chimeric receptor can be used in any cancer in which there is a specific monoclonal or polyclonal antibody or the ability to generate a specific monoclonal or polyclonal antibody. Specifically, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia have antigens to which the chimeric receptor binds.

[0083] The target-specific antigen recognition domain preferably includes an antigen-binding domain derived from an antibody against the antigen of the target, or a peptide that binds the antigen of the target, or a peptide or protein that binds an antibody (which binds the antigen of the target), or a peptide or protein ligand (including, but not limited to, growth factors, cytokines, or hormones) that binds a receptor on the target, or a domain derived from a receptor (including, but not limited to, growth factor receptors, cytokine receptors, or hormone receptors) that binds a peptide or protein ligand on the target.

[0084] In one embodiment, the antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody that is selective for the target.

[0085] In another embodiment, the antigen recognition domain comprises a Camelid single domain antibody, or a portion thereof. In one embodiment, the Camelid single domain antibody comprises a heavy chain antibody found in camels, or a VHH antibody. The VHH antibody of Camelids (e.g., camels, dromedaries, llamas and alpacas) refers to the variable fragment of the camel single chain antibody (see Nguyen et al., 2001; Muyldermans, 2001), and also includes isolated camel VHH antibodies, recombinant camel VHH antibodies or synthetic camel VHH antibodies.

[0086] In another embodiment, the antigen recognition domain comprises a ligand that binds to its cognate receptor. As an example, APRIL is a ligand that binds to the TAC1 receptor or the BCMA receptor. According to the invention disclosed herein, the antigen recognition domain comprises APRIL, or a fragment thereof. As another example, BAFF is a ligand that binds to the BAFF-R receptor or the BCMA receptor. According to the invention disclosed herein, the antigen recognition domain comprises BAFF, or a fragment thereof. In another embodiment, the antigen recognition domain is humanized.

[0087] The antigen recognition domain may include some variability within its sequence and still be selective for the targets disclosed herein. Thus, it is contemplated that polypeptides of the antigen recognition domain may have at least 95%, at least 90%, at least 80% or at least 70% identity with the antigen recognition domain polypeptides disclosed herein, and still be selective for the targets described herein and within the scope of the present invention.

[0088] Targets include interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15 CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, ROR1, PSMA, MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2 and CD138.

[0089] In another embodiment, the targets include any portion of the interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, TACI, ROR1, PSMA, MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0090] In one embodiment, the targets include the surface-exposed portions of any of the following: interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, TACI, ROR1, PSMA, MAGE A3, glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138 polypeptide.

[0091] In another embodiment, the target antigens include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV), or EBV (Epstein Barr virus) antigens; portions thereof; or surface-exposed regions thereof.

[0092] In one embodiment, the TACI antigen recognition domain comprises SEQ ID NO:24.

[0093] In one embodiment, the BCMA antigen recognition domain comprises SEQ ID NO:25.

[0094] In one embodiment, the CS1 antigen recognition domain comprises SEQ ID NO:26.

[0095] In one embodiment, the BAFF-R antigen recognition domain comprises SEQ ID NO:27.

[0096] In one embodiment, the CD33 antigen recognition domain comprises SEQ ID NO:28.

[0097] In one embodiment, the CD123 antigen recognition domain comprises SEQ ID NO:29.

[0098] In one embodiment, the CD19 antigen recognition domain comprises SEQ ID NO:30.

[0099] In one embodiment, the CD20 antigen recognition domain comprises SEQ ID NO:31. In another

[0100] embodiment, the CD20 antigen recognition domain comprises SEQ ID NO:32.

[0101] In one embodiment, the CD22 antigen recognition domain comprises SEQ ID NO:33.

[0102] In one embodiment, the CD45 antigen recognition domain comprises SEQ ID NO:34.

[0103] The hinge region is a sequence disposed, for example, between a chimeric antigen receptor and at least one co-stimulatory domain and a signaling domain. Hinge sequences can be obtained, including, for example, any suitable sequence from any genus, including human or a portion thereof. Such hinge regions are known in the art. In one embodiment, the hinge region comprises a hinge region of a human protein, including CD-8α, CD28, 4-1BB, OX40, CD3-ζ, T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0104] In one embodiment, the hinge region comprises the CD8a hinge region.

[0105] In some embodiments, the hinge region comprises one selected from (but not limited to) immunoglobulins such as IgG1, IgG2, IgG3, IgG4, and IgD.

[0106] The transmembrane domain comprises a hydrophobic polypeptide that spans the cell membrane. Specifically, the transmembrane domain spans from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0107] The transmembrane domain can be in the form of an α-helix or a β-barrel or a combination thereof. The transmembrane domain can include heterologous proteins that have multiple transmembrane segments, each in the form of an α-helix, a β-sheet, or a combination thereof.

[0108] In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain is selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0109] For example, the transmembrane domain includes the transmembrane domains of the following: the α or β chain of the T cell receptor, the CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD68, CD134, CD137, ICOS, CD41, CD154, their functional derivatives, and combinations thereof.

[0110] In one embodiment, the transmembrane domain is artificially designed such that more than 25%, more than 50%, or more than 75% of the amino acid residues in the domain are hydrophobic residues such as leucine and valine. In one embodiment, a triad of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0111] In one embodiment, the transmembrane domain is the CD8 transmembrane domain. In another embodiment, the transmembrane domain is the CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0112] The signaling domain and the co-stimulatory domain include polypeptides that can activate immune cells to stimulate or activate at least some aspects of the immune cell signaling pathway.

[0113] In one embodiment, the signaling domain includes polypeptides of the functional signaling domains of the following: CD3ζ, common FcRγ (FCER1G), FcγRlla, FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DNAX activating protein 10 (DAP10), DNAX activating protein 12 (DAP12), their active fragments, their functional derivatives, and combinations thereof. Such signaling domains are known in the art.

[0114] In one embodiment, the CAR polypeptide further comprises one or more co-stimulatory domains. In one embodiment, the co-stimulatory domain is a functional signaling domain from a protein including the following: OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; Natural Killer group 2 member C (NKG2C); Natural Killer group 2 member D (NKG2D), B7-H3; a ligand that binds to at least one of CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137); CD8; ICAM-1; LFA-1 (CD11a / CD18); CD40; CD27; CD7; B7-H3; NKG2C; PD-1; ICOS; an active fragment thereof; a functional derivative thereof; and combinations thereof.

[0115] As used herein, at least one co-stimulatory domain and a signaling domain may be collectively referred to as an intracellular domain. As used herein, a hinge region and antigen recognition may be collectively referred to as an extracellular domain.

[0116] The present invention also provides a polynucleotide encoding the above-mentioned chimeric antigen receptor polypeptide.

[0117] The term "polynucleotide" as used herein is defined as a nucleotide chain. Polynucleotides include DNA and RNA. In addition, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the common knowledge that nucleic acid is a polynucleotide, and a polynucleotide can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include (but are not limited to) all nucleic acid sequences obtained by any means available in the art, including (but not limited to) recombinant means, that is, using general cloning techniques to clone nucleic acid sequences from recombinant libraries or cellular genomes, and polymerase chain reaction (PCR), and similar means, and by synthesis in segments.

[0118] The polynucleotide encoding the CAR can be readily prepared from the amino acid sequence specifying the CAR by any conventional method. Regarding the amino acid sequence of each domain, the base sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or GenBank accession number, and the nucleic acid of the present invention can be prepared using standard molecular biological and / or chemical procedures. For example, based on the base sequence, a polynucleotide can be synthesized, and the polynucleotide of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0119] In one embodiment, the polynucleotide disclosed herein is part of a gene, or an expression or cloning cassette.

[0120] The above polynucleotides can be cloned into a vector. A "vector" is a composition of matter that includes an isolated polynucleotide and can be used to deliver the isolated polynucleotide into the interior of a cell. A wide variety of vectors known in the art include (but are not limited to) linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, phagemids, cosmids, and viruses. Viruses include bacteriophages, bacteriophage derivatives. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be considered to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include (but are not limited to) adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like. In one embodiment, the vector includes a cloning vector, an expression vector, a replication vector, a probe generating vector, an integration vector, and a sequencing vector.

[0121] In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a retroviral vector or a lentiviral vector. In one embodiment, the engineered cell is transduced virally to express a polynucleotide sequence.

[0122] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. The selected gene can be inserted into a vector and encapsulated into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or ex vivo to the cells of a patient. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0123] Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses suitable as vectors include (but are not limited to) retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, appropriate restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0124] The ability of lentiviral vectors to efficiently transfer genes into human T cells is well known, but the expression of the vector-encoded genes depends on internal promoters that drive their expression. For third- or fourth-generation CARs with additional costimulatory domains or genes encoding proliferative cytokines, strong promoters are particularly important because the increased CAR body size does not ensure equivalent expression levels. There are a variety of promoters with different strengths and cell type specificities. Gene therapy using CAR T cells depends on the ability of T cells to express sufficient amounts of the CAR body and maintain expression over a long period of time. The EF-1α promoter is commonly selected for CAR expression.

[0125] The present invention relates to expression vectors containing strong promoters for high gene expression levels in T cells or NK cells. In other embodiments, the inventors disclose strong promoters suitable for high CAR expression levels in T cells or NK cells. In certain embodiments, the strong promoter is related to the SFFV promoter, which is selectively introduced into the expression vector to obtain high expression levels and maintain expression over a long period of time in T cells or NK cells. The genes expressed preferably include CARs, T cell costimulatory factors, and cytokines for immunotherapy.

[0126] An example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high expression levels of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1a (EF-1a). However, other constitutive promoter sequences can also be used, including (but not limited to) the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as (but not limited to) actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters and also encompasses inducible promoters as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked thereto when such expression is needed, or turn off the expression when it is not needed. Examples of inducible promoters include (but not limited to) metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0127] Expression of the chimeric antigen receptor polynucleotide can be achieved using, for example, expression vectors including (but not limited to) SFFV (spleen focus-forming virus) (e.g., SEQ ID NO:23) or the human elongation factor 1α (EF) promoter, CAG (chicken β-actin promoter with CMV enhancer) promoter, or at least one of the human elongation factor 1α (EF) promoter. Examples of weaker / lower-expressing promoters that can be used include (but are not limited to) the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate early promoter, the ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter or portions thereof. Inducible expression of the chimeric antigen receptor can be achieved using, for example, a tetracycline-responsive promoter, including (but not limited to) TRE3GV (Tet-responsive element, including all generations and preferably the 3rd generation), the inducible promoter (Clontech Laboratories, Mountain View, CA), or portions or combinations thereof.

[0128] In a preferred embodiment, the promoter is the SFFV promoter or a derivative thereof. And it was unexpectedly found that the SFFV promoter provides stronger expression and a greater degree of persistence in the transduced cells according to the present invention.

[0129] "Expression vector" refers to a vector including a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide. The expression vector can be a bicistronic or polycistronic expression vector. The bicistronic or polycistronic expression vector can include (1) multiple promoters fused to each open reading frame; (2) splicing signals inserted between genes; fusions expressing genes driven by a single promoter; (3) proteolytic cleavage sites inserted between genes (self-cleaving peptides); and (iv) internal ribosome entry sites (IRES) inserted between genes.

[0130] In one embodiment, the present invention provides an engineered cell having at least one chimeric antigen receptor polypeptide or polynucleotide.

[0131] "Engineered cell" means any cell of any organism that has been modified, transformed or otherwise manipulated by the addition or modification of a gene, DNA or RNA sequence or a protein or polypeptide. The isolated cells, host cells and genetically engineered cells of the present invention include isolated immune cells, such as NK cells and T cells that contain a DNA or RNA sequence encoding a chimeric antigen receptor or a chimeric antigen receptor complex and express the chimeric receptor on the cell surface. The isolated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, treat cancer and treat infectious diseases.

[0132] In one embodiment, the engineered cells include immunomodulatory cells. Immunomodulatory cells include T cells, such as CD4 T cells (helper T cells), CD8 T cells (cytotoxic T cells, CTLs) and memory T cells or memory stem cell T cells. In another embodiment, the T cells include natural killer T cells (NK T cells).

[0133] In one embodiment, the engineered cells include natural killer cells. Natural killer cells are well known in the art. In one embodiment, the natural killer cells include cell lines, such as NK-92 cells. Other examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells and NKL cells.

[0134] NK cells mediate anti-tumor effects without the risk of GvHD and have a relatively short lifespan compared to T cells. Thus, NK cells will be depleted soon after destroying cancer cells, reducing the need for an inducible suicide gene on the CAR construct that would ablate the modified cells.

[0135] According to the present invention, we unexpectedly found that NK cells provide readily available cells engineered to contain and express the chimeric antigen receptor polypeptides disclosed herein.

[0136] Allogeneic or autologous NK cells induce a rapid immune response but disappear relatively quickly from the circulation due to their limited lifespan. Thus, the applicant unexpectedly found that using CAR cell-based therapies can reduce the problem of persistent side effects.

[0137] The present invention includes methods of generating cCAR. In some embodiments, T cells are used to generate cCAR. In other embodiments, cCAR is generated using native NK cells isolated from peripheral blood or cord blood and NK-92 cells such that it can be "off-the-shelf" administered to any mammal suffering from a disease or cancer.

[0138] According to one aspect of the present invention, NK cells can be amplified and transfected with the CAR polynucleotides according to the present invention, and the NK cells can be derived from umbilical cord blood, peripheral blood, iPS cells, and embryonic stem cells. According to one aspect of the present invention, NK-92 cells are amplified and transfected with CAR. NK-92 is a continuously growing cell line that has the characteristics and features of natural killer (NK) cells (Arai, Meagher et al. 2008). The NK-92 cell line is IL-2 dependent and has been demonstrated to be safe (Arai, Meagher et al. 2008) and viable. The CAR-expressing NK-92 cells can be amplified in serum-free medium with or without co-culture with feeder cells. A pure population of NK-92 carrying the relevant CAR can be obtained by sorting.

[0139] In one embodiment, the engineered cells include allogeneic T cells obtained from a donor and modified to inactivate components of the TCR (T cell receptor) involved in MHC recognition. Thus, TCR-deficient T cells will not cause graft-versus-host disease (GVHD).

[0140] In some embodiments, the engineered cells can be modified to prevent the expression of cell surface antigens. For example, the engineered cells can be genetically modified to delete the native CD45 gene to prevent its expression and cell surface presentation.

[0141] In some embodiments, the engineered cells include an inducible suicide gene ("safety switch") or a combination of safety switches that can be assembled on a vector, such as (but not limited to) a retroviral vector, a lentiviral vector, an adenoviral vector, or a plasmid. The introduction of the "safety switch" can greatly increase the safety profile and limit the on-target or off-tumor toxicity of the compound CAR. The "safety switch" can be an inducible suicide gene, such as (but not limited to) caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450. Other safety switches for eliminating unwanted modified T cells involve the expression of CD20 or CD19 or truncated epidermal growth factor receptor in T cells. All possible safety switches are covered and implemented in the present invention.

[0142] In some embodiments, the suicide gene is integrated into the genome of the engineered cells.

[0143] In one embodiment, the present invention provides engineered cells having a CD45 chimeric antigen receptor polynucleotide.

[0144] In one embodiment, the CD45 CAR polypeptide comprises SEQ ID NO:13 and the corresponding polynucleotide sequence SEQ ID NO:14. In another embodiment, the CD45 CAR polypeptide comprises SEQ ID NO:15 and the corresponding polynucleotide sequence SEQ ID NO:16. In another embodiment, the CD45 CAR polypeptide comprises SEQ ID NO:17 and the corresponding polynucleotide sequence SEQ ID NO:18.

[0145] Multiple CAR units

[0146] The present invention provides engineered cells having at least two different CAR polypeptides.

[0147] As used herein, a compound CAR (cCAR) or multiple CARs refers to an engineered cell having at least two different chimeric antigen receptor polypeptides. As used herein, a "different chimeric antigen receptor polypeptide" has a unique antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. Thus, two distinct chimeric antigen receptor polypeptides will have different antigen recognition domains. Between two different chimeric antigen receptor polypeptides, the signal peptide, hinge region, transmembrane domain, at least one co-stimulatory domain, and signaling domain may be the same or different. As used herein, a chimeric antigen receptor (CAR) unit refers to a different chimeric antigen receptor polypeptide or a polynucleotide encoding a different chimeric antigen receptor polypeptide.

[0148] As used herein, a unique antigen recognition domain is an antigen recognition domain that is specific for or targets a single target or a single antigenic determinant of a target.

[0149] In some embodiments, the compound CAR targets the same antigen. For example, the cCAR targets different antigenic determinants or portions of a single antigen. In some embodiments, each of the CAR units in the compound CAR targets a different antigen specific for the same or different disease conditions or side effects caused by a disease condition.

[0150] In some embodiments, the compound CAR targets two different antigens.

[0151] Generating compound CARs carrying different CAR units can be extremely challenging: (1) Interactions between CARs can have adverse effects and suitable CAR designs are key to counteracting such effects; (2) Compound CARs in a single construct can increase the length of the expression cassette, which can lead to reduced viral titers and protein expression levels; (3) Suitable designs are needed to incorporate multiple CAR body elements, especially selection strategies for expressing multiple CARs in a single vector; (4) Strong promoters are particularly important for compound CARs carrying additional CAR units; (5) The hinge regions in CARs need to be designed to preferably avoid interactions between the hinge regions of each CAR unit; (6) Two or more CAR units expressed in a cell can cause toxic effects (CAR-CAR interactions). The applicants herein provide novel and unexpected CAR compositions and methods that can address these obstacles.

[0152] In one embodiment, the present invention provides engineered cells having multiple CAR units. This enables a single engineered cell to target multiple antigens. Simultaneous targeting of multiple surface markers or antigens by multiple CAR units can prevent the selection of resistant clones and reduce tumor recurrence. Multiple CAR T cell immunotherapies for any malignancy have not been developed, where each individual component CAR contains multiple domains and activation sites.

[0153] In one aspect of the present invention, cCARs include multiple CAR units. In some embodiments, cCARs include at least two CAR units. In another embodiment, cCARs include at least three CAR units. In another embodiment, cCARs include at least four units.

[0154] In one embodiment, the present invention provides engineered cells having at least two different chimeric antigen receptor polypeptides, each having a different antigen recognition domain.

[0155] In a preferred embodiment, the engineered cells having at least two different chimeric antigen receptor polypeptides are native NK cells isolated from peripheral blood or cord blood and NK-92 cells, such that they are "off-the-shelf" for administration to any mammal suffering from a disease or cancer.

[0156] In one embodiment, the engineered cells include (i.) a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and (ii.) a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain. The first antigen recognition domain is different from the second antigen recognition domain.

[0157] In a preferred embodiment, each engineered CAR unit polynucleotide has a different nucleotide sequence to avoid homologous recombination.

[0158] In one embodiment, the targets of the first antigen recognition domain are selected from the group consisting of interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1; and the targets of the second recognition domain are selected from the group consisting of interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1.

[0159] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD20 recognition domain. In one embodiment, this engineered cell includes the polypeptide of SEQ ID NO:3 and the corresponding polynucleotide of SEQ ID NO:4.

[0160] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD22 recognition domain. In one embodiment, this engineered cell includes the polypeptide of SEQ ID NO:5 and the corresponding polynucleotide of SEQ ID NO:6.

[0161] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 recognition domain. In one embodiment, this engineered cell includes the polypeptide of SEQ ID NO:7 and the corresponding polynucleotide of SEQ ID NO:8.

[0162] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In one embodiment, this engineered cell comprises the polypeptide of SEQ ID NO:9 and the corresponding polynucleotide of SEQ ID NO:10. In another embodiment, this engineered cell comprises the polypeptide of SEQ ID NO:11 and the corresponding polynucleotide of SEQ ID NO:12.

[0163] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a BAFF-R antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS1 antigen recognition domain.

[0164] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD269 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS1 recognition domain. In one embodiment, the engineered cell comprises the polypeptide of SEQ ID NO:19 and the corresponding polynucleotide SEQ ID NO:20. In one embodiment, the engineered cell comprises the polypeptide of SEQ ID NO:21 and the corresponding polynucleotide SEQ ID NO:22.

[0165] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 recognition domain.

[0166] In one embodiment, each CAR unit comprises the same or different hinge regions. In another embodiment, each CAR unit comprises the same or different transmembrane regions. In another embodiment, each CAR unit comprises the same or different intracellular domains.

[0167] In one embodiment, each CAR unit comprises a CD3ζ chain signaling domain.

[0168] In one embodiment, each different CAR unit comprises a different co-stimulatory domain to avoid cross-interaction. For example, the first chimeric antigen receptor polypeptide comprises a 4-1BB co-stimulatory domain; and the second chimeric antigen receptor polypeptide comprises a CD28 co-stimulatory domain.

[0169] In another embodiment, the hinge region is designed to exclude amino acids that may cause unwanted intra- or intermolecular interactions. For example, the hinge region can be designed to exclude or minimize cysteine residues to prevent the formation of disulfide bonds. In another embodiment, the hinge region can be designed to exclude or minimize hydrophobic residues to prevent unwanted hydrophobic interactions.

[0170] The compound CAR can kill cells independently or in combination. The multiple or compound CARs comprise the same or different hinge regions, the same or different transmembrane regions, the same or different co-stimulatory regions, and the same or different intracellular domains. Preferably, the hinge regions are selected to avoid interaction sites.

[0171] The compound CARs of the present invention can target the same or different tumor populations in T or NK cells. For example, the first CAR can target a large tumor population and subsequently or the second CAR can, for example, eradicate cancer or leukemia stem cells to avoid cancer recurrence.

[0172] According to the present invention, it was unexpectedly found that compound CARs targeting different or the same tumor populations in T or NK cells counter tumor factors of cancer cells that can cause resistance to the killing activity of CARs, thereby resulting in downregulation of the target antigen from the cancer cell surface. It was also unexpectedly found that this can cause cancer cells to "evade" CAR therapy, called "antigen escape", and tumor heterogeneity, whereby different tumor cells can present different surface antigen expression profiles.

[0173] Engineered cells having a CAR polypeptide and an enhancer

[0174] In another embodiment, the present invention provides engineered cells having at least one chimeric antigen receptor polypeptide and an enhancer.

[0175] In one embodiment, the present invention provides engineered cells having at least two different chimeric antigen receptor polypeptides and an enhancer.

[0176] As used herein, an enhancer includes a biomolecule that promotes or enhances the activity of an engineered cell having a chimeric antigen receptor polypeptide. Enhancers include cytokines. In another embodiment, the enhancer includes IL-2, IL-7, IL-12, IL-15, IL-21, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFRβ, their receptors, and functional fragments thereof.

[0177] The enhancer can be expressed by the engineered cells described herein and presented on the surface of the engineered cells, or the enhancer can be secreted by the engineered cells into the surrounding extracellular space. Methods of surface presentation and secretion are well known in the art. For example, the enhancer can be a fusion protein having a peptide that provides surface presentation or secretion into the extracellular space.

[0178] The action of the enhancer can be supplemented by other factors, such as enhancer receptors and functional fragments thereof. The other factors can be co-expressed with the enhancer in the form of a fusion protein or expressed as separate peptides and secreted into the extracellular space.

[0179] In one embodiment, the enhancer is IL-15. In this case, the other factors are the IL-15 receptor, and its functional fragments. The functional fragments include the IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA. Examples of suitable domains include SEQ ID NO:35. According to the present invention, any chimeric antigen receptor polypeptide disclosed herein includes human interleukin 15 having the human interleukin 2 signal peptide SEQ ID NO:36.

[0180] Interleukin (IL)-15 and its specific receptor chain IL-15Rα (IL-15-RA) play key functional roles in a variety of effector cells, including NK and CD8 T cells. CD8+ T cells can be modified to express autocrine growth factors, including (but not limited to) IL-2, IL-7, IL21, or IL-15, to maintain survival after transfer in vivo. Without wishing to be bound by theory, IL-15 can address CD4 deficiency to induce naive and recall memory CD8 T cells. Overexpression of IL-15-RA or an IL-15 / IL-RA fusion on CD8 T cells significantly enhances their survival as well as in vitro and in vivo proliferation. In some embodiments, the CD4CAR or any CAR can include expression of any one or more or a portion or combination of IL-15, IL15RA, and IL-15 / IL-15R or IL-15-RA / IL-15 to enhance the survival or proliferation of CAR T or NK, and improve the expansion of memory CAR CD8+ T cells.

[0181] The present invention is directed to engineered cells having a CAR and any one or more or a portion or combination of IL-15, IL15RA, and IL-15 / IL-15R or IL15-RA / IL-15 as described herein to enhance the survival, persistence, or proliferation of CAR T or NK for the treatment of cancer in a patient.

[0182] In one embodiment, the engineered cell includes a CD4 chimeric antigen receptor polypeptide and IL to 15RA (SEQ ID NO:1), and the corresponding polynucleotide (SEQ ID NO:2).

[0183] Methods of producing engineered cells

[0184] Any of the polynucleotides disclosed herein can be introduced into an engineered cell by any method known in the art.

[0185] In one embodiment, the CAR polynucleotide is delivered into an engineered cell by any viral vector as disclosed herein.

[0186] In one embodiment, to obtain an enhanced safety profile or therapeutic index, any of the engineered cells disclosed herein is engineered into a transiently RNA-modified "biodegradable" version or derivative, or a combination thereof. The RNA-modified CARs of the invention can be electroporated into T cells or NK cells. Expression of the CAR compound can gradually decline within a few days.

[0187] In some embodiments of the invention, any of the engineered cells disclosed herein can be engineered in a transposon system (also known as "Sleeping Beauty") that integrates CAR DNA into the host genome without a viral vector.

[0188] Method for producing engineered cells having multiple CAR units

[0189] In another embodiment, the invention provides a method for producing engineered cells having at least two CAR units.

[0190] In some embodiments, multiple CAR units are expressed in T or NK cells using a bicistronic or polycistronic expression vector. There are several strategies that can be used to engineer a bicistronic or polycistronic vector, including (but not limited to) (1) fusing multiple promoters to the open reading frame of the CAR; (2) inserting splicing signals between CAR units; expressing fusions of CARs driven by a single promoter; (3) inserting proteolytic cleavage sites between CAR units (self-cleaving peptides); and (iv) inserting an internal ribosome entry site (IRES).

[0191] In a preferred embodiment, multiple CAR units are expressed in a single open reading frame (ORF), thereby producing a single polypeptide having multiple CAR units. In this embodiment, an amino acid sequence or linker containing a highly efficient cleavage site is placed between each CAR unit.

[0192] As used herein, high cleavage efficacy is defined as more than 50%, more than 70%, more than 80% or more than 90% cleavage of the translated protein. Cleavage efficacy can be measured by Western blot analysis, as described by Kim 2011.

[0193] In addition, in a preferred embodiment, there are equal amounts of cleavage products, as shown by Western blot analysis.

[0194] Examples of highly efficient cleavage sites include porcine teschovirus-1 2A (P2A), FMDV 2A (referred to herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thosea asigna virus 2A (T2A), Bombyx mori cytoplasmic polyhedrosis virus 2A (BmCPV2A), and Bombyx mori infectious flacherie virus 2A (BmIFV2A), or combinations thereof. In a preferred embodiment, the highly efficient cleavage site is P2A. The highly efficient cleavage site is described in Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, et al. (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice. PLoS ONE 6(4):e18556, the content of which is incorporated herein by reference.

[0195] In embodiments where multiple CAR units are expressed in a single open reading frame (ORF), expression is controlled by a strong promoter. Examples of strong promoters include the SFFV promoter, and derivatives thereof.

[0196] Engineered cells having a CAR polypeptide and an enhancer

[0197] In another embodiment, the present invention provides a method for producing engineered cells that express at least one CAR unit and an enhancer.

[0198] In some embodiments, a bicistronic or polycistronic expression vector is used to express at least one CAR unit and an enhancer in T or NK cells. There are several strategies that can be used to construct bicistronic or polycistronic vectors, including (but not limited to) (1) fusing multiple promoters to the open reading frame of the CAR; (2) inserting splicing signals between CAR units; expressing fusions of CARs driven by a single promoter; (3) inserting proteolytic cleavage sites between CAR units (self-cleaving peptides); and (iv) inserting an internal ribosome entry site (IRES).

[0199] In a preferred embodiment, at least one CAR unit and an enhancer are expressed in a single open reading frame (ORF), thereby generating a single polypeptide having at least one CAR unit and an enhancer. In this embodiment, an amino acid sequence or linker containing a highly efficient cleavage site is placed between each CAR unit and between the CAR unit and the enhancer. In this embodiment, the ORF is controlled by a strong promoter. Examples of strong promoters include the SFFV promoter and its derivatives.

[0200] In addition, in a preferred embodiment, there are equal amounts of cleavage products, as shown by Western blot analysis.

[0201] A method of treatment using the compositions disclosed herein

[0202] In another embodiment, the present invention provides a method for targeting CD45 for modulation prior to allotransplantation in cancer treatment. CD45, also known as leukocyte common antigen (LCA), is a tyrosine phosphatase expressed on almost all hematopoietic-derived cells except red blood cells and platelets. Most hematological malignancies express CD45. For example, 85% to 90% of acute lymphoid and myeloid leukemias express CD45. CD45 is not found in non-hematopoietic sources. In addition, CD45 is expressed at a high density of approximately 200,000 molecules per cell on malignant cells and white blood cells. CD45 represents an ideal target for a variety of hematological malignancies. However, CAR T and NK cells also express CD45. Without inactivation of endogenous CD45, CAR T or NK cells with a CAR targeting CD45 can cause suicide.

[0203] The binding of CD45 to the TCR complex is essential for regulating T cell activation in response to an antigen. The inability of CD45-deficient T cells to present an antigen is attributed to reduced signal transduction via the T cell receptor (TCR). The TCR is a cell surface receptor that plays an important role in T cell activation in response to antigen presentation. The TCR is typically made up of two chains, namely α and β, which are associated with the transducer unit CD3 to form the T cell receptor complex presented on the cell surface.

[0204] It has been unexpectedly found that multiple CARs (Compound CAR, cCAR) of the present invention counter an important mechanism of cancer cells for resisting CAR activity, namely the downregulation or non-uniform expression of the target antigen from the cancer cell surface. This mechanism enables cancer cells to "evade" CAR therapy, a phenomenon called 'antigen escape'. The present invention preempts cancer antigen escape by identifying a combination of two or more antigens that can rapidly eliminate tumors.

[0205] The present invention provides methods of using cCARs to simultaneously target multiple antigens, which result in improved tumor control by minimizing the likelihood of tumor selection based on target antigen loss or downregulation.

[0206] The present invention as disclosed includes a compound (multiplicity or compound) cCAR in T or NK cells that targets different or identical surface antigens presented in tumor cells. The compound chimeric antigen receptors of the present invention each comprise at least a multiplicity of chimeric receptor constructs linked by a linker and targeting the same or different antigens. For example, each CAR construct in a compound CAR (cCAR) construct includes an antigen recognition domain, an extracellular domain, a transmembrane domain, and / or a cytoplasmic domain. The extracellular domain and the transmembrane domain can be derived from any desired source of such domains. The multiplicity of CAR constructs are linked by a linker. The expression of the compound CAR constructs is driven by a promoter. The linker can be a part of a peptide or a protein that results in post-cleavage of the protein or peptide (also referred to as a self-cleaving peptide).

[0207] In one embodiment, the compound CARs of the present invention target myelodysplastic syndromes and acute myeloid leukemia (AML) populations. Myelodysplastic syndromes (MDS) remain incurable hematopoietic stem cell malignancies that most commonly occur in the elderly, with approximately 14,000 new cases arising in the United States each year. Approximately 30 - 40% of MDS cases progress to AML. The incidence of MDS continues to increase as the population ages. Despite extensive research on MDS and AML, no satisfactory treatment has been developed.

[0208] The compositions and methods of the present invention can be used to generate populations of T lymphocytes or NK cells that deliver primary and co-stimulatory signals for immunotherapy used in the treatment of cancer (particularly the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, brain cancer, sarcoma, leukemia, and lymphoma).

[0209] Immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The effector can be a lymphocyte carrying surface molecules that directly or indirectly interact with tumor cell targets. A variety of effector cells include cytotoxic T cells, NK cells, and NK-92 cells. The compositions and methods described in the present invention can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, and the like. The compositions and methods described in the present invention can be used for other disease conditions that rely on an immune response, such as inflammation, immune diseases, and infectious diseases.

[0210] In some embodiments, the compound CARs of the present invention can act as a bridge to bone marrow transplantation by achieving complete remission in patients with minimal residual disease and no longer responsive to chemotherapy. In other embodiments, the compound CARs eliminate leukemia cells and then rescue bone marrow stem cells to support leukopenia.

[0211] In some embodiments, the compound CAR of the present invention can counter cancer cells by downregulating target antigens, which is an important mechanism for resisting CAR activity. In another embodiment, the compound CAR of the present invention can also counter the heterogeneity of cancer cells, which poses significant challenges in conventional CAR T / NK cell therapies. In another embodiment, the disclosed compound CAR is designed such that the first CAR targets a large tumor population and the other eradicates cancer or leukemia stem cells to avoid cancer recurrence.

[0212] In one embodiment, the present invention provides a method of destroying cells having a CD33 antigen or a CD123 antigen, or by contacting such cells with engineered cells containing at least one of a chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. The engineered cells can be T or NK cells.

[0213] Cells having at least one of a CD33 antigen and a CD123 antigen include acute myeloid leukemia, precursor acute lymphoblastic leukemia, chronic myeloproliferative neoplasm, chronic myeloid leukemia, myelodysplastic syndrome, blastic plasmacytoid dendritic neoplasm (BPDCN), Hodgkin's lymphoma, mastocytosis, and hairy cell leukemia cells.

[0214] In another embodiment, the present invention provides a method of providing a myelosuppressive conditioning regimen for hematopoietic stem cell transplantation. In this embodiment, T or NK engineered cells having a CD33 unit and a CD123 unit are administered to a patient in need thereof.

[0215] In other embodiments, the present invention provides a method of eradicating or killing leukemia stem cells (LSCs) or primary leukemia cells expressing CD123 or CD33 or both. In this embodiment, T or NK engineered cells having a CD33 unit and a CD123 unit are administered to a patient in need thereof.

[0216] In other embodiments, the compound CAR in T or NK cells can be used to eradicate or kill CD34+CD38- leukemia stem cells or primary leukemia cells expressing CD123 or CD33 or both.

[0217] In some embodiments, the compound CAR targets cells expressing the CD19 or CD20 antigen or both. In another embodiment, the compound CAR targets cells expressing the CD19 or CD22 antigen or both. The targeted cells can be cancer cells, such as (but not limited to) B cell lymphoma or leukemia. In other embodiments, the target antigen can include (but not limited to) at least one of this group: ROR1, PSMA, MAGE A3, glycolipid, phosphatidylinositol proteoglycan 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2 and CD138. The target antigen can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0218] In some embodiments, the compound CAR targets cells expressing the CD19 or CD123 antigen or both. The targeted cells are cancer cells, such as (but not limited to) B cell lymphoma or leukemia.

[0219] In other embodiments, the compound CAR targets cells expressing CS1 and / or B cell maturation antigen (BCMA) or both. In another embodiment, the target cells are malignant plasma cells, such as (but not limited to) multiple myeloma.

[0220] In some embodiments, the compound CAR targets cells expressing multiple antigens, including (but not limited to) CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, CD20, interleukin 6 receptor and NY-ESO-1 antigen. In another embodiment, the target cells are malignant plasma cells, such as (but not limited to) multiple myeloma.

[0221] In some embodiments, the compound CAR targets cells expressing multiple antigens, including (but not limited to) alpha-fetoprotein (AFP) and phosphatidylinositol proteoglycan-3 (GPC3). In another embodiment, the target cells are hepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal hamartoma of the liver, squamous cell carcinoma of the lung, testicular non-seminomatous germ cell tumor, liposarcoma, ovarian and extragonadal yolk sac tumor, ovarian choriocarcinoma, teratoma, ovarian clear cell carcinoma and placental site trophoblastic tumor.

[0222] According to the present invention, T or NK cells comprising a compound CAR targeting different or the same antigen can counteract tumor escape and at the same time be capable of targeting tumor cells.

[0223] T or NK host cells comprising the compound CAR disclosed herein are practiced in the present invention. The nucleotide and polypeptide constructs, sequences, host cells, vectors of the compound CAR are considered a part of the present invention and are practiced herein.

[0224] In some embodiments, the compound CAR is administered in combination with any chemotherapeutic reagent currently under investigation or available on the market. In some embodiments, the compound CAR is administered as a first-line treatment for diseases, including (but not limited to) hematological malignancies, cancers, non-hematological carcinomas, inflammatory diseases, infectious diseases, such as HIV and HTLV, etc. In one embodiment, T cells expressing the compound CAR are co-administered with NK cells expressing the same or different compound CARs as an adoptive immunotherapy. The compound CAR NK cells provide rapid, innate activity against targeted cells, while the compound T cells provide relatively long-lasting adoptive immune activity.

[0225] In one embodiment, cells expressing the compound CAR are administered as a bridge for bone marrow stem cell transplantation in mammals, for example, patients who are resistant to chemotherapy and not suitable for bone marrow stem cell transplantation.

[0226] In some embodiments, the compound CAR co-expresses a transgene in the targeted tumor lesion and releases a transgene product (such as IL-12) and further modulates the tumor microenvironment.

[0227] In one embodiment, a part of the treatment of the disease is to eliminate the bone marrow of a mammal with cells expressing the compound CAR.

[0228] In a specific embodiment, cells expressing the compound CAR (which can be T cells or NK cells) are administered to a mammal (such as a human). The present invention includes a method for treating a mammal suffering from a disorder or disease by administering the compound CAR. The target cells can be, for example, cancer cells, or cells affected by any other disease condition (such as infectious diseases, inflammation, and autoimmune disorders).

[0229] The present invention is intended to include the use of fragments, mutants, or variants (such as modified forms) of the compound CAR or antigen that retain the ability to induce the stimulation and proliferation of T / NK cells. "Form of a protein" is intended to mean a protein that shares significant homology with at least one CAR or antigen and is capable of achieving the stimulation and proliferation of T / NK cells. As used herein, the term "biologically active" or "biologically active form of a protein" is intended to mean a form of a protein or variant that is capable of achieving the anti-tumor activity of cells.

[0230] The compositions and methods of the present invention can be used to generate populations of T / NK cells that can deliver primary and co-stimulatory signals for immunotherapy for the treatment of cancer, particularly for the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia, and lymphoma. The compositions and methods described in the present invention can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, and the like.

[0231] In some embodiments, the present invention discloses methods for depleting B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells in patients with autoimmune diseases by administering CAR or compound CAR T cells or NK cells to the patient. The cells targeted by CAR are B or plasma cells that express one or both of the antigens, BCMA, TACI, and BAFF-R. Autoimmune diseases include systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjorgen's syndrome, polymyositis, granulomatosis, and vasculitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane disease.

[0232] The value of a variety of extracellular cell markers as tumor-associated antigens and thus as potential targets for CAR T / NK cell therapy is currently being investigated. However, in addition to off-target toxicity, the expression of such antigens on healthy tissues that cause on-target, off-tumor adverse events remains a major safety concern. In addition, a major limitation of CAR T / NK cell therapy is the potential selection of antigen escape variants when targeting molecules that are not essential for tumorigenesis. Thus, malignant cells that maintain minimal or no expression of the target antigen can evade CAR T / NK cells, even if they have high-affinity interactions.

[0233] According to the present invention, natural killer (NK) cells represent an alternative cytotoxic effector for CAR-driven killing. Unlike T cells, NK cells do not require pre-activation and constitutively exhibit lytic function. Further expression of cCAR in NK cells enables NK cells to effectively kill cancer, particularly cancer cells that are resistant to NK cell therapy.

[0234] In addition, NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD).

[0235] Studies have shown abnormal overexpression of CD123 on CD34+CD38-AML cells, while normal bone marrow counterparts CD34+CD38- do not express CD123 (Jordan, Upchurch et al. 2000). This CD123+, CD34+CD38- population is considered LSC because these cells are capable of initiating and maintaining the leukemic process in immunodeficient mice.

[0236] The number of CD34+ / CD38- / CD123+ LSCs can be used to predict the clinical outcome of AML patients.

[0237] CD34+ / CD38- / CD123+ cells (greater than 15% in AML patients) are associated with incomplete remission and adverse cytogenetic profiles. In addition, the presence of more than 1% CD34+ / CD38- / CD123+ cells can also have an adverse effect on disease-free survival and overall survival.

[0238] Currently, the therapies for MDS and AML focus on leukemic blasts because they very well and clearly represent the majority of the problems faced by the patient. Importantly, leukemia stem cells (LSCs) are significantly different from other leukemic cells ("blast" cells), and they constitute a rare subset. Although killing blasts can provide short-term remission, if not destroyed, LSCs will always regrow, causing relapse in the patient. LSCs must be destroyed to achieve a durable cure of MDS disease. Unfortunately, standard drug regimens are ineffective against MDS or AML LSCs. Therefore, it is important to develop new therapies that can specifically target the leukemia stem cell population and the large leukemic population. The compound CAR disclosed in the present invention targets these populations and is implemented herein.

[0239] According to the present invention, it has been unexpectedly found that NK cells provide a ready-made product that can be used as an allogeneic product for treatment. Therefore, according to the present invention, as required by the current state of the art, cCAR cell therapy based on the patient-specificity is needed. The applicant of the present invention has discovered a novel immunotherapy in which an effective CAR cell-based therapy can be achieved without isolating the patient's lymphocytes or tumor-infiltrating lymphocytes.

[0240] Allogeneic or autologous NK cells are expected to induce a rapid immune response, but disappear relatively rapidly from circulation due to their limited lifespan. Therefore, the applicant has unexpectedly found that using cCAR cell-based therapy can reduce the problem of persistent side effects.

[0241] According to one aspect of the present invention, NK cells can be amplified and transfected with cCAR according to the present invention. NK cells can be derived from umbilical cord blood, peripheral blood, iPS cells, and embryonic stem cells. According to one aspect of the present invention, NK-92 cells are amplified and transfected with cCAR. NK-92 is a continuously growing cell line that has the characteristics and features of natural killer (NK) cells. The NK-92 cell line is IL-2 dependent and has been proven to be safe and viable. NK-92 cells expressing cCAR can be amplified in serum-free medium with or without co-culture with feeder cells. A pure population of NK-92 carrying the relevant cCAR can be obtained by sorting.

[0242] The identification of suitable surface target antigens is a prerequisite for generating CART / NK cells in adoptive immunotherapy.

[0243] In one aspect of the present invention, the CD123 antigen is one of the targets for cCAR therapy. CD123 (the alpha chain of the interleukin-3 receptor) is overexpressed in a variety of hematological malignancies, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, and blastic plasmacytoid dendritic cell neoplasm. CD123 is absent or minimally expressed on normal hematopoietic stem cells. More importantly, CD123 is expressed on a subset of leukemia cells associated with leukemia stem cells (LSCs), and eliminating these leukemia stem cells is necessary to prevent disease refractoriness and recurrence.

[0244] In one aspect of the present invention, the CD33 antigen is one of the targets for cCAR therapy. CD33 is a transmembrane receptor expressed on 90% of malignant cells in acute myeloid leukemia. Therefore, according to the present invention, the CD123 and CD33 target antigens are particularly attractive from a safety perspective.

[0245] According to the present invention, the compound CD33CD123CAR can be highly effective in the therapeutic treatment of a chronic myeloid leukemia (CML) population. In chronic myeloid leukemia (CML), there is a rare subset of cells that are CD34+CD38-. It is believed that this population contains LSCs. An increased number of LSCs is associated with the progression of the disease. It has been demonstrated that small molecule Bcr-Abl tyrosine kinase inhibitors (TKIs) can significantly improve the overall survival of patients with CP-CML. However, it is believed that LSCs are resistant to TKI therapy. There is an urgent need for novel therapies targeting CML-resistant LSCs for the treatment of CML, and the novel therapy is implemented in the form of the compound CD33CD123CAR disclosed in the present invention. The expression of CD123 is higher in the CD34+CD38- population. According to the present invention, the compound CD33CD123CAR is highly effective in the therapeutic treatment of this population.

[0246] In one embodiment of the present invention, leukemic cells expressing CD123 and CD33 in cCAR are used for therapeutic treatment. CD33 is expressed on myeloid cells, myeloid leukemic blasts, and mature monocytes but not on normal pluripotent hematopoietic stem cells (Griffin, Linch et al. 1984). CD33 is widely expressed in leukemic cells in CML, myeloproliferative neoplasms, and MDS.

[0247] Since a large number of acute myeloid leukemia (AML) patients are difficult to treat with standard chemotherapy regimens or experience disease relapse after treatment (Burnett 2012), the study of CART cell immunotherapy for AML has the potential to address a huge clinical need. In most of these patients, leukemic cells express CD123 and CD33, providing broad clinical applicability for the compound CD33CD123CAR disclosed herein. Accordingly, the present invention discloses novel multiplex cCART / NK cell constructs that comprise multiplex CARs targeting multiple leukemia-associated antigens, thereby counteracting antigen escape mechanisms, targeting leukemic cells, including leukemia stem cells, by the synergistic effects of co-stimulatory domains activation, and thereby providing a more potent, safe, and effective therapy.

[0248] The present invention also discloses compound CAR constructs that have enhanced anti-tumor activity efficacy against cells co-expressing target antigens and still maintain sensitivity to tumor cells expressing only one antigen. In addition, each CAR in the compound CAR includes one or two co-stimulatory domains and potent killing in the presence of a specific target.

[0249] In preclinical studies of dual-specific, reverse-signaling CARs targeting solid tumors, including breast cancer and epithelial ovarian cancer, the CD3ζ intracellular signaling domain was separated from the co-stimulatory domain of the second-generation CAR. In other words, one CAR contains the first-generation CAR without any co-stimulatory domain, and the other does not have the CD3ζ intracellular domain. Therefore, the presence of two target antigens is required for T cell activation and potent killing. Accordingly, it is proposed as a way to reduce off-tumor toxicity caused by the expression of one of the two target antigens in healthy tissues, increasing target specificity but at the cost of sensitivity. In one embodiment, the compound CAR is the compound CD123CD19CAR. It has been demonstrated that over 90% of the population subset of B-ALL expresses CD123. Similar to AML and MDS, a rare LSC population is thought to exist in B-ALL. Accordingly, according to the present invention, targeting leukemia stem cells and large leukemic populations can be applied to B-ALL. According to the present invention, the CD123 and CD19 surface antigens expressed in B-ALL can be targets because CD19 is highly expressed at different stages of the B cell lymphoid population.

[0250] Multiple myeloma (MM) is the second most common hematologic malignancy in the United States and is caused by monoclonal plasma cells that accumulate in the bone marrow or extramedullary sites. MM is an incurable disease with a median survival of approximately 4.5 years (Kumar, Rajkumar et al. 2008). Anti-myeloma CARs in preclinical studies have been investigated and CAR targets include CD38, CS1, B cell maturation antigen (BCMA), and CD38. However, there is usually heterogeneity in surface antigen expression in malignant plasma cells (Ruiz-Arguelles and San Miguel 1994), making it difficult to target them with CARs. Malignant plasma cells also express low levels of CD19. It has previously been demonstrated that myeloma stem cells also express some B cell markers, including CD19. Targeting this population in combination with standard and other myeloma CAR therapies can be effective in the treatment of myeloma.

[0251] Multiple myeloma (MM) is a hematologic malignancy with monoclonal expansion of plasma cells. Despite important developments in treatment, myeloma remains an incurable disease; thus, there is an urgent need for novel treatment methods.

[0252] CS1 (also known as CD319 or SLAMF7) is a protein encoded by the SLAMF7 gene. The surface antigen CS1 is a stable marker of normal and myeloma cells (malignant plasma cells).

[0253] Tumor necrosis factor receptor superfamily, member 17 (TNFRSF17), also known as B cell maturation antigen (BCMA) or CD269 is almost completely expressed at the terminal stage of plasma cells and malignant plasma cells. Its expression is not present in other tissues, indicating potential as a target for CART or NK cells.

[0254] Malignant plasma cells show variable degrees of antigen heterogeneity of CD269 and CS1. A single CAR unit product targeting CD269 or CS1 can target most cells in the main tumor, causing an initial stable anti-tumor response. Subsequently, a very small number of remaining untargeted cells expand and cause disease recurrence. Although multiple myeloma is particularly heterogeneous, this phenomenon necessarily applies to other leukemias or tumors. Current clinical trials using BCMACART cells at the NIH show promising results, with complete responses in some multiple myeloma patients. However, these patients relapse after 17 weeks, which can be attributed to antigen escape. Antigen escape has also been found in CD19CAR and NY-ESO1CART cell therapies. Therefore, there is an urgent need for more effective CART cell therapies to prevent recurrence.

[0255] In one aspect of the present invention, BCMA and CS1 are targets of the BCMACS1CAR therapy.

[0256] In some embodiments, the compound CAR targets cells expressing BCMA or CS1 antigen or both. The target cells can be cancer cells, such as (but not limited to) lymphoma, or leukemia or plasmacytoma. In other embodiments, the plasmacytoma is selected from plasmacytic leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenstrom's macroglobulinema, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0257] BAFF (B-cell activating factor) and APRIL (a proliferation-inducing ligand) are two TNF homologs that specifically bind to TACI (also known as TNFRSF13B or CD267) and BCMA with high affinity. BAFF (also known as BLyS) binds to BAFF-R and is functionally involved in promoting the survival and late proliferation of B cells. BAFF has been shown to be associated with some autoimmune disorders. APRIL plays an important role in promoting antibody class switch. BAFF and APRIL have been shown to be growth and survival factors for malignant plasma cells.

[0258] The ligand-receptor interactions in malignant plasma cells are described below:

[0259] Ligand-receptor interactions in malignant plasma cells

[0260]

[0261] In some embodiments, the compound CAR targets cells expressing TACI or CS1 antigen or both. In another embodiment, the compound CAR targets cells expressing TACI or CS1 antigen or both. The target cells can be cancer cells, such as (but not limited to) lymphoma, or leukemia or plasmacytoma. In other embodiments, the plasmacytoma is selected from plasmacytic leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenstrom's macroglobulinema, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma. The target cells can also be B cells, immature B cells, naive B cells, centroblasts, centrocytes, memory B cells, plasmablasts, long-lived plasma cells, plasma cells of one or two or more different cell types. These cells are associated with autoimmune diseases, including systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjogren's syndrome, polymyositis, granulomatosis with polyangiitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane disease.

[0262] In some embodiments, the compound CAR targets cells expressing BAFF-R or CS1 antigen or both. In another embodiment, the compound CAR targets cells expressing BAFF-R or CS1 antigen or both. The target cells can be cancer cells, such as (but not limited to) lymphoma, or leukemia or plasmacytoma. In other embodiments, the plasmacytoma is selected from plasmacytic leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldenström's macroglobulinemia, heavy chain disease, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma.

[0263] In some embodiments, the compound CAR (cCAR) targets cells expressing one or both or all of BAFF-R, BCMA, TACI, and CS1 antigens.

[0264] In some embodiments, the units of CAR in cCAR can include: 1) scFv against BAFF-R, BCMA, TACI, and CS1; 2) a hinge region; 3) a co-stimulatory domain and an intracellular signaling domain.

[0265] In some embodiments, the units of CAR in cCAR can include: 1) a BCMA or TACI or BAFF-R binding domain, or an APRIL binding domain; 2) a hinge region; 3) a co-stimulatory domain and an intracellular signaling domain.

[0266] In another embodiment, the BCMA or TAC1 or BAFF-R binding domain can be the entire APRIL and BAFF molecules or a part thereof.

[0267] In some embodiments, the units of CAR in cCAR can include: 1) scFv against BCMA or CS1; 2) a hinge region; 3) a co-stimulatory domain and an intracellular signaling domain.

[0268] In other embodiments, cCAR can include one or two or more units of CAR. Each unit CAR can have the same or different hinge regions and co-stimulatory domains.

[0269] In other embodiments, the target antigen can include (but is not limited to) at least one of the following group: ROR1, PSMA, MAGEA3, glycolipid, phosphatidylinositol proteoglycan 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. The target antigen can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0270] In some embodiments, the cCAR targets cells expressing the CD19 or CD20 antigen or both. In another embodiment, the cCAR targets cells expressing the CD19 or CD22 antigen or both. The target cells are cancer cells, such as B cell lymphoma or leukemia.

[0271] Acute graft-versus-host disease (GVHD) remains the most important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. In the effector phase of GVHD, the T cell receptor (TCR), a heterodimer of the alpha and beta chains, is expressed on the surface of T cells, the TCR recognizes some antigens on HLA molecules on host cells, enhances T cell proliferation, and releases cytotoxic agents that cause damage to host cells. The TCR gene is effective in preventing potential graft-versus-host reactions. Inactivation of the TCR can lead to prevention of TCR recognition of allogeneic antigens and thus GVHD.

[0272] The role of CD45 in NK cells is significantly different from that in T cells. NK cells from CD45-deficient mice have normal cytotoxic activity against the prototype tumor cell line Yac-1. In addition, CD45-deficient NK cells proliferate normally and respond to IL15 and IL-21. Therefore, disruption or deletion of CD45 will not affect the killing and proliferative properties of NK cells.

[0273] The present invention includes a method of permanently deleting CD45 in T or NK cells followed by stable introduction of a CD45-specific CAR. Thus, the engineered T cells display the desired properties of redirecting specificity against CD45 without causing suicide and response to antigen presentation. In another embodiment, the engineered T cells can have the efficacy as an off-the-shelf therapy for treating malignancies or other diseases.

[0274] The present invention relates to a method in which T cells are engineered to allow proliferation even when TCR signaling is reduced or lost via inactivation or deletion of endogenous CD45. The result of reducing or losing TCR signaling can prevent GVHD.

[0275] In another embodiment, T cells in which TCR signaling is reduced or lost by inactivation of CD45 can be used as an "off-the-shelf" therapeutic product.

[0276] The present invention includes methods of modified T or NK cells, comprising: (a) modifying T or NK cells by inactivating CD45; (b) expanding these modified cells; (c) sorting modified T or NK cells that do not express CD45; (d) introducing CD45CAR.

[0277] In embodiments, the CD45CAR gene encodes a chimeric antigen receptor (CAR), where the CAR comprises at least one of an antigen recognition domain, a hinge region, a transmembrane domain, and a T cell activation domain, and the antigen recognition domain redirects against the CD45 surface antigen presented on the cell. The antigen recognition domain includes a monoclonal or polyclonal antibody against the CD45 antigen. The antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody.

[0278] In some embodiments, modified T cells are obtained from allogeneic donors and used as "off-the-shelf products".

[0279] Targeting CD45 using CAR T or NK cells can cause suicide because T and NK cells express this surface antigen. To overcome this drawback, the inventors propose using engineered CRISPR / Cas9 systems, zinc finger nucleases (ZFNs), and TALE nucleases (TALENs) and meganucleases to inactivate the CD45 gene. Loss of CD45 in T or NK cells is further transduced by a CAR targeting tumors that express CD45.

[0280] The present invention includes methods for eliminating or reducing abnormal or malignant cells in bone marrow, blood, and organs. In some embodiments, malignant cells expressing CD45 are present in patients with the following diseases: acute leukemia, chronic leukemia, B and T cell lymphomas, myeloid leukemia, acute lymphoblastic lymphoma or leukemia, primary effusion lymphoma, reticulohistiocytosis, transient myeloproliferative disorder of Down's syndrome, lymphocyte-predominant Hodgkin's lymphoma, myeloid leukemia or sarcoma, dendritic cell tumors, histiocytic sarcoma, giant cell tumor of the tendon sheath, interdigitating dendritic cell sarcoma, post-transplant lymphoproliferative disorder, etc.

[0281] In some embodiments, CD45CAR cells can be used to create space in the bone marrow for bone marrow stem cell transplantation by removing hematopoietic cells, while removing leukemia / lymphoma cells or immune cells capable of transplant rejection.

[0282] In another embodiment, CD45CAR cells can be used to pre-treat a patient before the patient undergoes a bone marrow transplant to receive stem cells. In another embodiment, CD45CAR can be used as a myeloablative conditioning regimen for hematopoietic stem cell transplantation.

[0283] In some embodiments, CD45CAR cells are used to treat or prevent residual disease after stem cell transplantation and / or chemotherapy.

[0284] In some embodiments, CD45CAR is part of an expression gene or cassette. In a preferred embodiment, in addition to CD45CAR, the expression gene or cassette can include accessory genes or tags or portions thereof. The accessory gene can be an inducible suicide gene or a portion thereof, including (but not limited to) caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450. The "suicide gene" elimination method can improve the safety of gene therapy and kill cells only when initiated by a specific compound or molecule. In some embodiments, the suicide gene is inducible and activated using a specific dimerization chemical inducer (CID).

[0285] In some embodiments, the safety switch can include an accessory tag, which is a c-myc tag, CD20, CD52 (Campath), a truncated EGFR gene (EGFRt), or a portion or combination thereof. The accessory tag can be used as a non-immune gene selection tool or as a tracking marker.

[0286] In some embodiments, the safety switch can include a 24-residue peptide corresponding to residues 254-277 of the RSVF glycoprotein A2 strain (NSELLSLINDMPITNDQKKLMSNN). In some embodiments, the safety switch can include the amino acid sequence of TNFα bound by a monoclonal anti-TNFα drug.

[0287] The use of any of the engineered cells described herein can be applied together with a CAR enhancer. Examples of CAR enhancers include immunomodulatory drugs that enhance CAR activity, such as (but not limited to) agents that target immune checkpoint pathways, inhibitors of colony-stimulating factor-1 receptor (CSF1R) for better treatment outcomes. Agents that target immune checkpoint pathways include small molecules, proteins, or antibodies that bind and inhibit inhibitory immune receptors CTLA-4, PD-1, and PD-L1 and cause CTLA-4 and PD-1 / PD-L1 blockade. As used herein, enhancer includes booster as described above.

[0288] As used herein, "patient" includes mammals. The mammals referred to herein can be any mammal. As used herein, the term "mammal" refers to any mammal, including (but not limited to) mammals of the order Rodentia (such as mice and hamsters) and mammals of the order Lagomorpha (such as rabbits). Mammals can be from the order Carnivora, including felines (cats) and canines (dogs). Mammals can be from the order Artiodactyla, including bovines (cows) and swines (pigs), or the order Perissodactyla, including equines (horses). Mammals can be primates, ceboids or simoids (monkeys) or anthropoids (humans and apes). The mammal is preferably a human. A patient includes an individual.

[0289] In certain embodiments, the patient is a human who is 0 to 6 months, 6 to 12 months, 1 to 5 years, 5 to 10 years, 5 to 12 years, 10 to 15 years, 15 to 20 years, 13 to 19 years, 20 to 25 years, 25 to 30 years, 20 to 65 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years, 60 to 65 years, 65 to 70 years, 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years, or 95 to 100 years old.

[0290] As used herein, the "effective amount" and "therapeutically effective amount" of an engineered cell mean an amount of the engineered cell sufficient to provide the desired therapeutic or physiological effect or result. Such effects or results include a reduction or improvement in the symptoms of a cellular disease. Undesirable effects, such as side effects, sometimes occur in conjunction with the desired therapeutic effect; thus, a physician balances the potential benefits against the potential risks in determining what is a suitable "effective amount". The exact amount required will vary between patients depending on the species, age and general health of the patient, the mode of administration, and similar factors. Thus, it may not be possible to specify an exact "effective amount". However, the suitable "effective amount" in any individual case can be determined by one of ordinary skill in the art using only routine experimentation. Generally, the engineered cells are administered in an amount and under conditions sufficient to reduce the proliferation of the target cells.

[0291] After administration of a delivery system for treating, inhibiting or preventing cancer, the efficacy of the therapeutically engineered cells can be evaluated by a variety of methods well known to those skilled in the art. For example, one of ordinary skill in the art should understand that therapeutically engineered cells delivered in combination with a chemical adjuvant can effectively treat or inhibit cancer in a patient by observing that the therapeutically engineered cells reduce the cancer cell burden or prevent further increase in the cancer cell burden. The cancer cell burden can be measured by methods known in the art, such as using polymerase chain reaction assays to detect the presence of certain cancer cell nucleic acids or to identify certain cancer cell markers in the blood, using antibody assays, for example, to detect the presence of markers in a sample from an individual or patient (such as, but not limited to, blood), or by the content of circulating cancer cell antibodies in a patient.

[0292] Throughout this specification, quantities are defined by a range and the lower and upper limits of the range. Each lower limit can be combined with each upper limit to define a range. The lower and upper limits should each be considered a separate element. Throughout this specification, reference to "an embodiment" or "an example" means that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment of the invention. Thus, the phrase "in one embodiment" or "an example" appearing multiple times throughout this specification need not necessarily refer to the same embodiment or example. Further, in one or more embodiments or examples, the particular features, structures, or characteristics may be combined in any suitable combination and / or sub-combination. Additionally, it should be understood that the figures provided with it are for illustrative purposes for those of ordinary skill in the art and the figures are not necessarily drawn to scale.

[0293] As used herein, the terms "comprises / comprising", "includes / including", "has / having" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.

[0294] Further, unless expressly stated to the contrary, "or" refers to the inclusive "or" and not the exclusive "or". For example, condition A or condition B is satisfied by any one of the following: A is true (or present) and B is false (or absent); A is false (or absent) and B is true (or present); and both A and B are true (or present).

[0295] In addition, any examples or illustrations provided herein should not be construed as in any way binding, limiting, or defining any of the terms used therein. The fact is that such examples or illustrations are considered to describe a particular embodiment and are for illustrative purposes only. Those of ordinary skill in the art will understand that any terms used in conjunction with such examples or illustrations will cover other embodiments that may or may not be provided in conjunction therewith or elsewhere in the specification, and all such embodiments are intended to be included within the scope of that or those terms. Language designating such non-limiting examples and illustrations includes, but is not limited to: "for example," "such as," and "in one embodiment."

[0296] In this specification, groups of various parameters containing multiple members are described. Within a parameter group, each member can be combined with any one or more other members to form other subgroups. For example, if the group members are a, b, c, d, and e, then other specifically covered subgroups include any one, two, three, or four of these members, such as a and c; a, d, and e; b, c, d, and e, etc.

[0297] As used herein, the XXXX antigen recognition domain is a polypeptide that is selective for XXXX. Thus, XXXX is the target. For example, the CD38 antigen recognition domain is a polypeptide that is specific for CD38.

[0298] As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.

[0299] The present invention can be better understood with reference to the examples set forth below. The following examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be illustrative only and not intended to limit the present invention.

[0300] Examples

[0301] Generate the compound CAR (cCAR)

[0302] The construction of CD33CD123cCAR follows Figure 2ASchematic diagram therein. It includes an SFFV (spleen focus-forming virus) promoter that drives the expression of a functional compound CAR (cCAR) with two different CAR units. Antigen receptor orientation, nucleotide sequences of scFv (single-chain variable fragment) against CD33 and CD123. The P2A peptide derived from picornavirus is used due to its efficient mechanism for the self-cleavage kinetics of the dicistronic gene construct. The self-cleaving P2A peptide is used to link together two independent units among CAR, CD33CAR, and CD123CAR during expression. The advantages of this method compared to the internal ribosome entry site (IRES) commonly used in the literature include its smaller size and high cleavage efficacy between the two upstream and downstream unit proteins of the 2A peptide. In addition, when using IRES, the use of the self-cleaving P2A peptide can avoid the problem of expression level differences between genes before and after IRES.

[0303] Module unit, CD33CAR includes a CD33scFv domain, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and an intracellular domain of the CD3ζ chain. The second module CAR, CD123CAR, has the same hinge, transmembrane, and intracellular signaling domains as CD33CAR but has different scFv and co-stimulatory domains. CD33CAR recognizes its corresponding antigen and CD123CAR binds to its corresponding antigen. The hinge region is designed to avoid sequences with disulfide interactions therein. Different co-stimulatory domains 4-1BB and CD28 are used. The CD33CD123 compound CAR is subcloned into a lentiviral plasmid.

[0304] Generate an efficient compound CAR (cCAR)

[0305] The compound CAR lentivirus is generated by transfecting HEK-293FT cells with Lipofectamine 2000 according to the manufacturer's instructions, but 2-fold vector DNA is used to increase the titer due to the large size of the insert, as Figure 2B shown. After culturing for about 12 - 16 hours, the medium containing Lipofectamine is removed and replaced with DMEM containing 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, and 1 mM sodium butyrate. After about 24 hours, the supernatant is collected and refrigerated, and the medium is replaced with fresh medium. After about 24 hours, the supernatant is collected and combined with the previous supernatant and filtered through a 0.45 μM filter. The supernatant is divided into aliquots, rapidly frozen with liquid nitrogen, and stored at -80 °C. HEK-293FT cells are collected, cryopreserved, and lysed for subsequent electrophoresis and Western blotting.

[0306] PB (peripheral blood) or CB (human cord blood) buffy coat cells were activated with anti-CD3 antibody and IL-2 for 2 days. The cCAR lentiviral supernatant was spin-coated onto a multi-well culture dish coated with recombinant human fibrin fragment. The activated T cells were transduced at a low concentration of approximately 0.3×10 6 cells / ml with the lentiviral supernatant in multiple wells to increase the transduction efficiency ( Figure 2C ).

[0307] After the first overnight transduction, unless the cells appeared unhealthy, the cells were directly added to a second virus-coated dish without washing for the second transduction. After the second overnight transduction, the cells were washed, pooled and cultured in a tissue culture-treated dish. The CART cells were expanded for up to approximately 5 days, followed by co-culture killing assay. After culturing for approximately 3 days, the cells were cultured with biotin-conjugated goat anti-mouse F(ab')2 or goat IgG (isotype) antibody, washed and then cultured with streptavidin-PE and conjugated anti-human CD3. After washing and suspending in 2% formalin, the cells were then analyzed by flow cytometry to determine the percentage of transduction efficiency.

[0308] Characterization of CD33CD123cCAR

[0309] The transfected CD33CD123cCAR HEK293T cells underwent Western blot analysis to confirm the compound construct. Immunoblotting with anti-CD3ζ monoclonal antibody demonstrated bands of the predicted size of the compound CARCD3ζ fusion protein ( Figure 2B ). Importantly, as expected, two different bands of similar intensity were observed at the band of successful high cleavage of the signal transduction P2A peptide. As expected, no CD3ζ expression of the GFP control vector was found. The surface expression of the scFv was also tested on HEK293 cells ( Figure 2C ) and primary T cells ( Figure 2C ).

[0310] The transduction efficiency of the compound CD33CD23CAR lentivirus was tested in the HEK293 cell line and analyzed by flow cytometry (Beckman Coulter) ( Figure 2C ). Flow cytometry indicated that approximately 67% of the HEK cells expressed CD33CD123CAR. Human peripheral blood (PB) is commonly used for autologous T cell therapy. Human PB buffy coat cells were activated with anti-CD3 antibody and IL-2, and transduced with CD4CAR or control (GFP) lentivirus. After transduction, flow cytometry analysis indicated that approximately 22% of the T cells expressed CD33CD123CAR ( Figure 2C ).

[0311] Results

[0312] CD33CD123c CART cells derived from umbilical cord blood (UCB) and peripheral blood (PB) specifically kill tumor cells expressing CD33

[0313] CD33CD123c CART cells or GFP T cells (controls) were cultured for approximately 24 hours in approximately 1 - 2 mL of T cell medium without IL-2 at ratios in the range of 0.5:1 to 50:1, preferably at ratios of approximately 2:1, 5:1, 10:1, 20:1, 50:1, with approximately 100,000, 200,000, 500,000, approximately 1 million or 2 million effector cells to approximately 50,000, 100,000, 200,000 target cells. The target cells were leukemia cell lines and leukemia cells from leukemia patients. After approximately 24 hours of co-culture, the cells were stained with mouse anti-human CD33, CD123, CD34, and CD3 antibodies.

[0314] Generate CD33CD123c CART cells expressing CD33 CAR and CD123 CAR and test the anti-leukemia function using HL60 and KG-1a cell lines. The HL60 cell line is a promyelocytic leukemia cell line highly enriched for CD33. Approximately 100% of its cell population is CD33+, with a small subset (<10%) being dim CD123+. In culture, this cell line was tested to determine the efficacy of CD33CD123 CAR, with an emphasis on targeting leukemia cells expressing CD33. Additionally, due to the strong CD33 expression in HL60, the CD33CD123c CAR effect can be significant. Indeed, during 24-hour co-culture conditions with various effector:target cell ratios, the CD33CD123c CAR exhibited significant leukemia cell killing properties ( Figure 3 ). First, the ability of CB-derived CD33CD123 CART cells to kill HL60 cells was tested. At approximately 24 hours of culture and at low effector:target (E:T) ratios in the range of approximately 0.5:1 to 50:1, preferably 1:1 to approximately 5:1, more preferably approximately 2:1 to 4:1, CD33CD123 CAR cells eliminated approximately 55% of CD33-expressing HL60 cells when compared to the GFP control. At a ratio of approximately 5:1, the killing effect increased to approximately 82%.

[0315] CD33CD123 CAR derived from peripheral blood mononuclear cells (PBMC) was co-cultured with the myeloid leukemia cell line KG1a, which also moderately expressed approximately 100% CD33 as compared to HL60 and 50 - 80% CD123. Thus, KG1a was a relatively dual-target cell population that was doubly positive for the antigens targeted by CD33CD123 CAR. A cultivation of approximately 24 hours and a low effector:target (E:T) ratio in the range of approximately 0.5:1 to 50:1 were used. Although at a low E:T ratio of approximately 2:1, CD33CD123 CAR still exhibited a moderate anti-leukemia activity of approximately 26%, increasing the E:T ratio to 10:1 caused killing of approximately 62% of KG1a compared to the GFP control ( Figure 4 ), indicating that the intensity of the CD33 marker could be an indicator of killing power, where HL60 was strongly presented and more CAR action was utilized compared to KG1a. These experiments provided evidence for the functionality of the fully CD33CD123 CAR against its relevant antigen-presenting cell population.

[0316] Another compound CAR, CD33CD123 - BBcCAR, has been generated. This compound CAR contains two independent CAR units, CD33 and CD123. The first CAR contains an scFv that binds to CD33 and the second CAR has a different scFv that recognizes CD123. Both CARs contain the same hinge region, transmembrane, co-stimulatory, and intracellular domains. A CD33CD123 - BBcCAR lentivirus was generated and its killing power was tested in KG-1a cells. As Figure 5 shown, substantial killing was present at a ratio of approximately 10:1, but it was weaker in intensity compared to CD33CD123cCAR.

[0317] CD33CD123cCAR has activity against patient samples expressing CD33 and / or CD123

[0318] In addition to cell line experiments, patient samples were also studied to test the functionality of each individual CAR unit. The efficacy of CD33CD123cCAR was tested using invasive acute myeloid leukemia (AML), AML-9. Due to the heterogeneity of the patient cell population, which included multiple cell types in the AML-9 sample, leukemic blasts were gated with CD34 and CD33 as they were positive for these two markers. At a certain ratio of CAR T cells:target cells, depletion of this CD33+CD34+ population of leukemic cells was observed to be 48% higher than the GFP control ( Figure 6 ).

[0319] Leukemia cells that are also CD123 positive and CD33 negative were also tested. For this purpose, a human B cell acute lymphoblastic leukemia (B-ALL) sample, Sp-BM-B6, was selected. All leukemic blasts in this sample were CD34+CD33−, and more than about 50% were positive for CD123. Compared to the GFP control, the depletion rate of the CD34+ leukemic cell population by CD33CD123cCART cells was approximately 86%( Figure 7 ). Based on cell line and human sample studies, our data clearly show that the compound CD33CD123CAR can target leukemic cells expressing CD33 or CD123 or both.

[0320] CD33CD123cCAR NK cells target leukemic cells expressing CD33 or CD23 or both

[0321] Natural killer (NK) cells are CD56+CD3− and can effectively kill infected and tumor cells like CD8+ T cells. Different from CD8+ T cells, NK cells initiate cytotoxicity against tumors without activation to kill cells. NK cells are safer than effector cells because they can avoid potentially lethal complications of cytokine storms. However, the use of CD33 or CD123 or both CAR NK cells to kill leukemia has not been explored at all.

[0322] Generation of CD33CD123cCAR NK cells

[0323] NK-92 cells were transduced with CD33CD123CAR lentiviral supernatant for two consecutive overnights, with the difference between the transductions being culture dishes coated with recombinant human fibrin fragments and virus-coated culture dishes. The transduced cells were expanded for 3 or 4 days and then analyzed for CAR expression by flow cytometry. Cells were collected and incubated with goat anti-mouse F(Ab')2 at about 1:250 for about 30 minutes. The cells were washed, suspended and stained with streptavidin-PE for about 30 minutes. The cells were washed and suspended in 2% formalin and analyzed by flow cytometry. Then, NK-92 cells expressing CD33CD123cCAR were labeled as described above and sorted on a FACSAria, where the top 0.2% of cells expressing F(Ab')2 were collected and cultured. Subsequent labeling of the sorted and expanded cells showed that approximately 89% of NK-92 cells were positive for anti-mouse F(ab')2( Figure 8 ).

[0324] CD33CD123cCAR NK cells effectively lyse or eliminate leukemic cells

[0325] First, we tested the function of CD33CD123cCAR NK-92 cells by evaluating their ability to kill the HL-60 cancer cell line in co-culture. Almost all HL-60 cells highly express CD33, but CD123 expression in this cell line is only less than 10% (weak). Therefore, the killing ability of CD33CD123cCAR may depend on the ability of the cCAR to correctly target CD33.

[0326] CD33CD123cCAR NK-92 cells were co-cultured with HL-60 cells in NK cell medium without IL-2 for approximately 24 hours. After incubation, CD33CD123cCAR NK-92 cells were labeled and compared with non-CAR control GFP NK-92 cells. Compared with the control GFP NK-92 cells, CD33CD123cCAR NK-92 cells were observed to significantly kill HL-60 cells. In addition, the killing ability of CD33CD123cCAR NK-92 cells was dose-dependent, where at a ratio of approximately 10:1 compared to the control, it was approximately 100% ( Figure 9 and 11 ).

[0327] A second co-culture experiment was performed using KG1a, a myeloid leukemia cell line. KG1a expresses CD33 in all cells, but at a moderate level compared to HL-60. CD123 antigen is expressed in approximately 50 - 80% of KG1a cells. The experimental design was similar to the first experiment of the HL-60 killing assay above, with the same incubation time, effector:cancer cell ratio, and GFP NK-92 cell control. The results showed that CD33CD123cCAR NK-92 cells significantly killed KG1a cells in a dose-dependent manner compared to the GFP NK-92 cell control. At an effector:target ratio of 10:1, CD33CD123cCAR NK-92 cells killed approximately 85% of KG1a cells compared to the GFP control ( Figure 10 and 11 ).

[0328] Analysis of KG1a cells showed two different populations, CD33+CD123- and CD33+CD123-. Figure 11 A dose-dependent increase in cell killing was shown to be found in both populations. Surprisingly, the double-positive population showed higher effective killing at each increasing ratio, indicating a possible synergistic effect of the two-module CAR of CD33 and CD123 ( Figure 12 ).

[0329] Generate CD19CD20, CD19CD22, CD19CD138cCAR

[0330] Three cCARs ( Figure 13 ) have been generated using a strategy similar to the above CD33CD123cCAR.

[0331] cCARs for the treatment of multiple myeloma have been generated, including BCMACS1cCAR and BCMACD19cCAR

[0332] Preclinical studies of cCARs have been conducted to target surface antigens, including CD38, CS1, CD138, B cell maturation antigen (BCMA), and CD38. CD19CAR has also shown some efficacy in the treatment of multiple myeloma in phase I clinical trials. However, given the heterogeneity of surface antigen expression that is commonly present in malignant plasma cells (Ruiz-Arguelles and San Miguel 1994), a single target is unlikely to be sufficient to eliminate this disease. BCMACS1cCAR, BCMACD19cCAR, BCMACD38cCAR, and BCMACD138cCAR have been generated and the experimental design is similar to that of the CD33CD123cCAR described above.

[0333] cCARs for the treatment of multiple myeloma have been generated, including BCMACS1cCAR (BC1cCAR)

[0334] Generation and characterization of the BCMA-CS1cCAR (BC1cCAR) construct

[0335] The modular design of BC1cCAR consists of an anti-CD269 (BCMA, B cell maturation antigen) single-chain variable fragment (scFv) region fused to anti-CD319 (CS1) by a self-cleaving P2A peptide, a CD8-derived hinge (H) and transmembrane (TM) region, and a tandem 4-1BB co-activation domain linked to the CD3ζ signaling domain ( Figure 14A ). A strong spleen focus-forming virus promoter (SFFV) and a CD8 leader sequence are used for the efficient expression of the BC1cCAR)CAR molecule on the T cell surface. The two-unit CAR uses the same co-stimulatory domain 4-1BB. Transfected BC1cCAR HEK293T cells were subjected to western blot analysis to confirm the compound construct. Immunoblotting with an anti-CD3ζ monoclonal antibody demonstrated bands of the predicted size for the compound CARCD3ζ fusion protein ( Figure 14E ). Importantly, as expected, two distinct bands of similar intensity were observed at the spot of successful high cleavage of the signaling P2A peptide. As expected, no CD3ζ expression was found for the GFP control vector.

[0336] Generation of BC1cCAR (cCAR) T cells

[0337] After 2 days of activation, T cells isolated from the cord blood (UCB) buffy coat were transduced with BC1cCAR lentivirus. Two units of CAR used the same co-stimulatory domain 4-1BB. The transduction efficiency of BC1cCAR was determined to be approximately 15%, as determined by flow cytometry ( Figure 14B ). BC1cCAR T cells were first tested on a CML (chronic myeloid leukemia) cell line negative for the myeloma markers BCMA and CS1. As expected, no lysis was induced by control T cells or BC1cCAR T cells against wild-type K562 ( Figure 14C ). BCMA-K562 (Kochenderfer, NIH) is a K562 cell transduced with cDNA encoding BCMA to express BCMA in >80% of the cell population. BC1cCAR T cells were co-cultured with this cell line at E:T ratios of 2:1 and 5:1 and showed >30% lysis (undetectable) compared to the control ( Figure 14C ). These results are consistent with other cultures performed on cell lines transduced with other CARs, such as CS1 CAR T cells.

[0338] However, when BCMA-CS1-2G (cCAR) used different co-stimulatory domains, 4-BB or CD28 for each unit, rare surface CAR expression was detected, indicating that appropriate co-stimulatory domain selection may be important for ensuring surface CAR expression on T cells ( Figure 14D ).

[0339] Although protein was detected by Western blotting in HEK cells ( Figure 14E ), we could not detect surface expression in activated T cells transduced with CD269-CS1-2G lentiviral supernatant. This may be attributed to the inability to export the expressed protein to the cell membrane. In the future, we may need to optimize the sequence of this construct to allow greater cell surface expression.

[0340] BC1cCAR T cells specifically lyse BCMA+ and CS1+ cell lines

[0341] To evaluate the cytotoxic ability of BC1cCART cells, we performed co-culture assays with myeloma cell lines: MM1S (BCMA+CS1+), RPMI-8226 (BCMA+CS1-), and U266 (BCMA+CS1dim). The ability of BC1cCART cells to lyse target cells was quantified by flow cytometry analysis, and the target cells were stained with the Cytotracker dye (CMTMR). In 24-hour co-cultures, BC1cCAR showed near-complete lysis of MM1S cells, consuming over 90% of the target cells at an E:T ratio of 2:1 and over 95% at an E:T of 5:1 (Figure 15). In RPMI-8226 cells, BC1cCAR lysed over 70% of BCMA+ target cells at an E:T ratio of 2:1 and over 75% at an E:T of 5:1 (Figure 16). In 24-hour co-cultures with U266 target cells, BC1cCAR lysed 80% of BCMA+ U266 cells at an E:T ratio of 2:1, reaching saturation (Figure 17).

[0342] BC1cCART cells specifically target BCMA+ and CS1+ populations in primary patient myeloma samples

[0343] Flow cytometry analysis of MM10-G patient samples showed distinct and invariant subsets of BCMA+ and CS1+ populations (Figure 18). The MM7-G sample exhibited a complete BCMA+CS1+ phenotype, while MM11-G presented a contaminated BCMAdimCS1dim phenotype, possibly attributable to its nature as a bone marrow aspirate. After 24 hours, BC1cCART cells showed stable elimination of the MM7-G primary patient sample, lysing over 75% at an E:T ratio of 5:1 and increasing to over 85% at 10:1 (Figure 19). For MM11-G (Figure 20), BC1cCART cells were able to lyse over 45% of the BCMA+CS1+ population at an E:T of 10:1.

[0344] BC1cCAR demonstrated targeting and specific lysis rates by significantly eliminating subsets of BCMA+CS1+ and BCMA-CS1+ populations in MM10-G co-cultures within 24 hours. At an E:T ratio of 2:1, BC1cCART cells eliminated over 60% of the BCMA+CS1+ population and 70% of the CS1+ population alone. At an E:T ratio of 5:1, elimination of the CS1+ population alone increased to 80% (Figure 18).

[0345] BC1cCART cells showed significant control and reduction of tumors in vivo

[0346] To evaluate the in vivo anti-tumor activity of BC1c CART cells, we used a xenograft mouse model of NSG mice that were sub-lethally irradiated and intravenously injected with luciferase-expressing MM.1S cells (a multiple myeloma cell line) to induce measurable leukemia formation. On the third day after tumor cell injection, 8 × 106 BC1c CART cells or vector control cells were intravenously injected into the mice as a single dose. On days 3, 6, and 8, RediJect D-Luciferin (PerkinElmer) was subcutaneously injected into the mice and the mice were subjected to IVIS imaging to measure the tumor burden ( Figure 21 ). The average light intensity of the mice injected with BC1c CART cells was compared with the average light intensity of the mice injected with the vector control to determine the percentage of tumor cells in the treated mice compared to the control mice ( Figure 21 and 22 ). Unpaired T-test analysis showed a highly significant difference between the two groups on day 8 (P = 0.0001), where the group injected with BC1c CART cells had a lower light intensity and thus a lower tumor burden compared to the control group (p < 0.0001). On day 1 and then every other day, the tumor size area was measured and the average tumor size between the two groups was compared ( Figure 21 ). In summary, these in vivo data indicate that CD269-CS1-BB CART cells significantly reduce the tumor burden in NSG mice injected with MM.1S compared to vector control NK control cells.

[0347] CD45 CAR Therapy

[0348] Three pairs of sgRNAs were designed using CHOPCHOP to target the relevant genes. The gene-specific sgRNAs were then cloned into a lentiviral vector (LentiU6-sgRNA-SFFV-Cas9-puro-wpre) expressing human Cas9 and puromycin resistance gene, which was linked to an E2A self-cleaving linker. The U6-sgRNA cassette was located before the Cas9 element. The expression of sgRNA and Cas9puro was driven by the U6 promoter and SFFV, respectively ( Figure 23 ).

[0349] The following gene-specific sgRNA sequences were used and constructed.

[0350] In a non-limiting example of the present invention, exemplary gene-specific sgRNAs were designed and constructed as set forth below:

[0351] CD45 sgRNA construct:

[0352] Lenti-U6-sgCD45a-SFFV-Cas9-puroGTGGTGTGAGTAGGTAA

[0353] Lenti-U6-sgCD45b-SFFV-Cas9-puroGAGTTTTGCATTGGCGG

[0354] Lenti-U6-sgCD45c-SFFV-Cas9-puroGAGGGTGGTTGTCAATG

[0355] Figure 24 Show the steps of generating CD45 CAR T or NK cells targeting hematological malignancies.

[0356] CRISPR / Cas nuclease targets CD45 on NK cells

[0357] Transduce NK-92 cells with lentivirus carrying gene-specific sgRNA. Determine the reduction of CD45 expression on NK-92 cells by flow cytometry analysis. Sort and expand the CD45-negative population of NK-92 cells ( Figure 25 ). Generate CD45 CAR NK cells using the sorted and expanded CD45-negative NK-92 cells. Test the ability of the resulting CD45 CAR NK cells to kill CD45+ cells.

[0358] Functional characterization of CD45-inactivated NK-92 cells (NK45i-92) after CRISPR / Cas nuclease targeting. We demonstrated that after CRISPR / Cas nuclease inactivation of CD45, the growth of NK45i-92 cells was similar to that of wild-type NK-92 cells ( Figure 26 ).

[0359] Inactivation of CD45 did not significantly affect the cell proliferation of NK-92. In addition, we confirmed that when the cells were co-cultured with leukemia cells CCRF, the lysis ability of NK45i-92 cells was compatible with that of wild-type NK-92 (Figure 27).

[0360] To demonstrate the compatibility of CD45-inactivated NK-92 with CAR lysis, NK45i-92 cells and their wild-type NK-92 were transduced with lentiviruses expressing CD5 CAR or GFP. CD5 CAR NK45i-92 cells and GFP NK45i-92 obtained by FACS sorting were used to compare their ability to kill target cells. CD5 CAR NK45i-92 cells showed a stable ability to kill CD5+ target leukemia cells at ratios of 2:1 and 5:1 (E:T) when co-cultured with CCRF-CEM cells. There was a similar in vitro CCRF-CEM cell elimination efficacy between our CD5 CAR NK45i-92 and CD5 CAR NK-92 cells (Figure 28). This indicates that the reduction in CD45 expression does not reduce the anti-tumor activity of CAR NK cells.

[0361] Generate the CD45 CAR construct

[0362] We then investigated the response of CD45 CAR in NK45i-92 cells to the CD45 antigen in leukemia cells. We generated CD45 CAR. CD45 CAR consists of an anti-CD45 single-chain variable fragment (scFv) region, a CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28 and 4-1BB co-activation domains linked to the CD3ζ signaling domain ( Figure 29A ). The strong spleen focus-forming virus promoter (SFFV) and the CD8 leader sequence were used. Western blotting of HEK293-FT cells transfected with the CD45 CAR lentiviral plasmid was used to characterize the CD45 CAR protein using a suitable vector control. In addition, immunoblotting with an anti-CD3ζ monoclonal antibody showed a band of the predicted size of the CD45 CAR protein, where no band was observed in the vector control ( Figure 29B ).

[0363] CD45 CAR NK45i-92 NK cells

[0364] After fluorescence-activated cell sorting (FACS) to enrich NK45i-92 cells, the transduction efficacy of CD45 CAR NK-92 was determined to be 87%, as measured by flow cytometry after sorting (Figure 30). After FACS collection of NK45i-92 cells, the CD45 CAR expression level remained consistently stable in at least 10 passages.

[0365] CD45 CAR NK45i-92 cells specifically lyse CD45+ leukemia cells

[0366] To evaluate the anti-leukemia activity of CD45CAR NK45i-92, we performed co-culture assays using T-ALL cell lines, CCRF-CEM and Jurkat, and the NK cell line, NK-92, as they all express CD45 (Figs. 31, 32). We demonstrated that CD45CAR NK45i-92 cells consistently showed stable lysis of leukemia cells. After 6 hours of incubation at low to medium effector-to-target (E:T) ratios (5:1) against target cells, CD45CAR NK45i-92 cells efficiently lysed more than 60% of CCRF-CEM cells (Fig. 31). After 6 hours of co-culture, CD45CAR NK45i-92 cells were also able to eliminate approximately 60% of Jurkat cells at E:T ratios between 2:1 and 5:1 (Fig. 32). After 6 hours of co-culture, CD45CAR NK45i-92 cells efficiently lysed 20% of CD45-positive NK-92 cells at an E:T ratio of 2:1, and nearly 60% at an E:T ratio of 5:1 ( Figure 33A-33C ).

[0367] To further analyze the CD45 targeting in hematological malignancies, we also generated two additional CARs: CD45-28 and CD45-BB, and transduced NK45i-92 cells with lentivirus expressing either CD45-28 or CD45-BB CAR. CD45-28 and CD45-BB CAR contain novel anti-CD45 scFv that are different from the above CD45CAR. CD45-28CAR uses the CD28 co-stimulatory domain, while CD45-BB has the 4-1BB co-stimulatory domain. The CARs use a CD8-derived hinge (H), transmembrane (TM) region, and CD3ζ signaling domain. CD45CAR showed stable lysis of the B acute lymphoblastic cell line, REH. CD45CAR NK45i-92 cells lysed approximately 76% of REH cells compared to control GFP NK-92 cells. CD45b-BB CAR NK45i-92 cells and CD45b-28CAR NK45i-92 cells showed approximately 79% and 100% lysis of REH cells, respectively ( Figure 33D -G). CD45b-28CAR NK45i-92 cells exhibited the highest REH cell lysis capacity.

[0368] IL15 and its receptor enhance CAR T and NK cell function

[0369] Recent studies have shown that T cell persistence is well correlated with the efficacy of CAR T cell therapy. Current trials have shown that potent and persistent anti-tumor activity can be generated by infusing a small number of CAR T cells, indicating that the quality rather than the quantity of the infused product is more important for promoting anti-tumor activity. Interleukin (IL)-15 is a cytokine that promotes the development and homeostasis of lymphocytes. Increased levels of IL-15 can promote T cell proliferation and enhance T cell effector responses. Data from recent studies have confirmed that IL-15 is important for the generation and maintenance of memory CD8 T cells, an important factor associated with anti-tumor activity. IL-15 binds to the IL-15 receptor alpha chain (also known as IL15RA or RA), promoting IL-15-mediated effects such as T cell survival, proliferation, and the generation of memory T cells.

[0370] The IL-15Rα binds to the βγ complex in the surface of T cells and IL15 signals by binding to this IL-15Rα / βγ complex on the cell surface of T cells and other types of cells.

[0371] Current data indicate that IL-15 transfection alone does not show an impact on T cell function, but IL-15 / 1IL-15RA enables T cells to survive and proliferate spontaneously.

[0372] The function of all administered IL-15 alone can be limited by the availability of free IL-15RA and its short half-life. Administering soluble IL-15 / RA complexes greatly enhances the half-life and bioavailability of IL-15 in vivo. Therefore, treating mice with this complex rather than IL-15 alone can cause stable proliferation and maintenance of memory CD8 T cells and NK cells. Recent studies have shown that a portion of the extracellular region of IL-15RA (called the sushi domain) is required for its binding to IL15 (WEI et al., J. Immunol., Vol. 167 (1), pp. 277-282, 2001). IL-15 / RA fusion proteins or IL-15 / sushi fusion proteins containing linkers are more potent than IL-15 alone and soluble IL-15RA. The combination of IL-15 / RA or IL-15 / sushi can maximize IL-15 activity. However, it is not clear whether the design of incorporating CAR and IL-15 / RA or IL15 / sushi in the same construct can maintain their desired biological properties in T or NK cells, as the insert sequence length can affect transfection function and gene expression levels.

[0373] The present invention provides engineered cells that have CAR and IL15 / RA or IL15 / sushi in a single construct. In some embodiments, the present invention includes methods of generating higher viral titers and using stronger promoters to drive CAR and IL15 / RA or IL-15 / sushi therein.

[0374] In some embodiments, the present invention provides engineered cells that have: (1) a CAR that targets an antigen, which includes (but is not limited to) CD4, CD2, CD3, CD7, CD5, CD45, CD20, CD19, CD33, CD123, CS1, and B cell maturation antigen (BCMA); and (2) 15; (3) IL15RA (RA) or sushi. In other embodiments, the CAR comprises a chimeric antigen receptor, one or more of the co-stimulatory intracellular domains such as CD28, CD2, 4-1BB, and OX40, and CD3ζ. In other embodiments, the strong promoter can be (but is not limited to) SFFV, CAR, IL-15 / RA, or sushi, and an inducible suicide gene (“safety switch”), or can be assembled on a vector such as a lentiviral vector, an adenoviral vector, a retroviral vector, or a plastid. The introduction of the “safety switch” can show an increased safety profile and limit the on-target or off-tumor toxicity of the CAR.

[0375] Characterize CD4IL15RA-CAR

[0376] CD4IL15RA-CAR has been produced, which contains a third-generation CD4 CAR linked to IL15RA (Figure 34). Assembled on an expression vector (third generation), the combination of sushi / IL-15 is driven by the SFFV promoter to express the CAR with sushi / IL-15 linked by a P2A cleavage sequence. The sushi / IL-15 moiety consists of an IL-2 signal peptide fused to the sushi domain and is linked to IL-5 via a 26-amino acid polyproline linker (Figure 34).

[0377] To verify the CD4IL15RA construct, HEK293FT cells were transfected with GFP (control) or viral plasmids prior to CD4IL15RA. At approximately 60 hours post-transfection, HEK-293FT cells and supernatants were collected. Cells were lysed in RIPA buffer containing protease inhibitor mixture and subjected to electrophoresis. Gels were transferred to ImmobilonFL blot membranes, blocked and probed with mouse anti-human CD3z antibody at 1:500. After washing, the membranes were probed with goat anti-mouse HRP conjugate, washed, and exposed to film after treatment with HyGloHRP substrate. CD4IL15RA-CAR was successfully expressed in HEK293 cells (lane 2, Figure 35a, as shown by recombinant IL-15 protein in lane 3 (arrow)). The CD4IL15RA-CAR lentiviral supernatant was further tested by transduction of fresh HEK-293 cells (Figure 35a). HEK-293 cells sourced from transfected HEK-293FT cells were transduced with GFP or CD4IL15RA-CAR viral supernatant adding polybrene to 4 μL / mL. After 16 hours, the medium was changed and replaced with medium without viral supernatant or polybrene. Three days after transduction, cells were collected and stained with goat anti-mouse F(ab')2 antibody at 1:250 for 30 minutes, washed, stained with streptavidin-PE conjugate at 1:500, washed, suspended in 2% formalin and analyzed by flow cytometry. Figure 34b shows that HEK-293 cells transduced with CD4IL15RA-CAR lentivirus were 80% positive for F(ab)2-PE (circles, Figure 35b), while cells transduced with GFP control lentivirus were minimal for F(ab)2-PE ( Figure 35B , left).

[0378] Generation of CD4IL15RA-CAR NK cells

[0379] NK-92 cells were transduced with CD4IL15RA-CAR lentiviral supernatant. After culturing for 5 days, cells were collected and incubated with goat anti-mouse F(ab')2 at 1:250 for 30 minutes. Cells were washed, suspended and stained with streptavidin-PE for 30 minutes. Cells were washed and suspended in 2% formalin, and analyzed by flow cytometry, resulting in approximately 70% of the transduced cells expressing CD4IL15RA-CAR (circles, Figure 36 ). Other experimental tests of CD4IL15RA-CAR will include in vitro and in vivo leukemia / lymphoma killing assays, and comparison of target killing and proliferation rates of cells transduced with CD4CAR. The inventors also used the same strategy as described above to generate CD19IL15RA-CAR.

[0380] Generation of CD4IL15RA-CAR T cells

[0381] Human umbilical cord leukocyte layer cells were transduced with CD4IL15RA-CAR lentiviral supernatant. After culturing for 5 days, the cells were collected and incubated with goat anti-mouse F(Ab')2 at 1:250 for 30 minutes. The cells were washed, suspended and stained with streptavidin-PE for 30 minutes. The cells were washed and suspended in 2% formalin, and analyzed by flow cytometry, resulting in 63% of the transduced cells expressing CD4IL15RA-CAR (circles, Figure 37 ). Other experimental tests of CD4IL15RA-CAR will include in vitro and in vivo leukemia / lymphoma killing assays, and comparison of the target killing and proliferation rates of CD4CAR-transduced cells.

[0382] The in vitro anti-leukemia activity of CD4IL15RA-CAR NK cells relative to CD4CAR NK cells was tested by co-culturing them with the following CD4-positive cell lines: Karpas299 and MOLT4

[0383] The Karpas299 cell line was derived from a patient with polymorphic large T cell lymphoma. The MOLT4 cell line expressing CD4 was generated from the peripheral blood of a 19-year-old patient with acute lymphoblastic leukemia (T-ALL). During the 4-hour co-culture experiment, CD4IL15RA-CAR NK cells showed significant killing (95%) of Karpas299 cells at an effector:target ratio of 5:1, even at a higher rate than CD4CAR NK cells (82%, Figure 38). Similarly, when co-cultured with MOLT4 cells at 1:1, in the overnight assay, CD4IL15RA-CAR NK cells lysed target cells at a higher rate (84% to 65%) than CD4CAR NK cells ( Figure 39 ). These results confirm that CD4IL15CAR NK cells can eliminate tumor cells at least as well as CD4CAR NK cells.

[0384] CD4CAR and CD4IL15RA-CAR T cells exhibited more potent in vivo anti-tumor activity than CD4CAR

[0385] To evaluate the in vivo anti-tumor activity of CD4CAR and CD4IL15RACAR T cells, and to determine the possible increase in the persistence of CD4IL15RACAR T cells relative to CD4CAR T cells, we studied a xenograft mouse model of NSG mice that were irradiated with a sublethal dose and intravenously injected with luciferase-expressing MOLM13 cells (an acute myeloid leukemia cell line (M5) that is 100% CD4) to induce measurable tumor formation. Three days after tumor cell injection, a course of 8 × 106 CD4CAR, CD4IL15RACAR T cells, or vector control T cells was intravenously injected into each of 6 mice. On days 3, 6, 9, and 11, RediJect D-Luciferin (PerkinElmer) was injected subcutaneously into the mice and the mice were subjected to IVIS imaging to measure tumor burden ( Figure 40 ). On day 6, mice treated with CD4CAR T cells had a 52% reduction in tumor burden relative to the control, while mice treated with CD4IL15RACAR T cells had a 74% reduction in tumor burden ( Figure 41 ). On day 11, almost all tumor cells lysed in both of these groups. On day 9, unpaired t-test analysis showed a highly significant difference (P = 0.0045) between the control and the two groups, with lower light intensity and thus lower tumor burden in the groups treated with CD4CAR and CD4IL15RACAR T cells compared to the control.

[0386] Promoter testing using a GFP reporter

[0387] HEK293FT cells were transfected with lentiviral plasmids expressing GFP under the SFFV, EF1, or CAG promoter. At approximately 60 hours after transfection, supernatants were collected from each sample. Relative viral titers were determined by first transducing HEK293 cells with supernatants from each of the 3 promoters. HEK-293 cells were transduced with GFP viral supernatants from each of the 3 transfected HEK-293FT cells. Polybrene was added to a final concentration of 4 μL / mL. The medium was changed after 16 hours and replaced with medium without viral supernatant or polybrene. Three days after transduction, cells were collected, washed, resuspended in 2% formalin, and GFP expression (FITC) was analyzed by flow cytometry. GFP expression was detected in each sample, but was highest in cells transduced with virus prepared using the SFFV promoter.

[0388] The activated human umbilical cord leukocyte layer cells were transduced with GFP lentiviral supernatants from each promoter (amount based on the results of HEK293 transduction efficacy). After culturing for 5 days, the cells were collected, washed and suspended in 2% formalin, and GFP expression was analyzed by flow cytometry. 43% of the cells expressed GFP in large amounts (>103), while GFP expression in the cells transduced with the viruses using the promoters EF1 (15%) and CAG (3%) was significantly lower. After five days, the cells analyzed in the same manner showed almost the same respective percentages (46%, 15% and 3% respectively; Figure 23 ). These results indicate that the SFFV promoter causes stronger expression than the EF1 or CAG promoters, and this expression remains high for at least 10 days after transduction. Other experimental tests will include longer culturing times of the transduced cells outside the 10-day window.

[0389] There is provided a method for generating a CAR gene comprising a T antigen recognition moiety (at least one or a part or combination of CD4, CD8, CD3, CD5, CD7 and CD2), a hinge region and at least one of a T cell activation domain.

[0390] There is provided a method for generating multiple CAR (cCAR) units targeting an antigen, the antigen(s) comprising at least one of CD33, CD123, CD19, CD20, CD22, CD269, CS1, CD38, CD52, ROR1, PSMA, CD138 and GPC3, or a part or combination of a hinge region and a T cell activation domain. All references cited and / or disclosed herein are incorporated herein by reference in their entirety.

[0391] The methods provided also include: 1) generating CART or NK cells targeting leukemias and lymphomas expressing CD45 and avoiding suicide; 2) generating "armored" CART or NK cells, which are designed to overcome the inhibitory tumor microenvironment and exhibit enhanced anti-tumor activity and long-term persistence. The present invention is not limited to the embodiments described and illustrated above and can be varied and modified within the scope of the appended claims. Throughout this specification, various publications are cited, including patent cases, published applications, technical papers and academic papers. Each of the cited publications is incorporated herein by reference in its entirety and used for all purposes. Throughout this specification and the claims, various terms related to aspects of the present invention are used. Unless otherwise indicated, such terms will have their ordinary meaning in the art. Other specifically defined terms are interpreted in a manner consistent with the definitions provided herein.

[0392] The functional titer of viral vector particles in the supernatant (GFP cell % as measured by flow cytometry allows surrogate virus titer regulation because higher titer viruses infiltrate more cells, causing a higher % of GFP cell population).

[0393] To determine the functional titer of viral vector particles in each of our supernatants, in 12-well tissue culture-treated plates, HEK293 cells were transduced with EF1-GFP or SFFV-GFP virus supernatants at 30 μL (low), 125 μL (medium), or 500 μL (high) / well. The medium was changed to DMEM plus 10% FBS the next morning.

[0394] Subsequently, the transduced cells were trypsinized, washed, and suspended in formalin and subjected to flow analysis. The percentage of GFP+ cells under each condition was determined by flow cytometry using the FITC channel ( Figure 43 ). In all cases, the percentage of GFP+ in cells transduced with SFFV-GFP was higher than that in cells transduced with the corresponding volume of EF1-GFP virus supernatant (50% vs. 18% (low), 80% vs. 40% (medium), and 82% vs. 70% (high)). Thus, we determined that in terms of titer, using the highest volume of EF1 promoter virus was similar to using the lowest volume of SFFV promoter virus, and the relative promoter concentrations of the following transduction experiments will be compared.

[0395] Also, under the same exposure conditions for each well, the transduced cells were observed on an EVOS fluorescence microscope at 20× using GFP ( Figure 42 ). Cells transduced with SFFV-GFP virus supernatant were significantly brighter than those transduced with EF1-GFP. In addition, comparing the images of the EF1 promoter at high viral volume load with the images of the SFFV promoter at low viral volume load showed similar fluorescence intensities. This indicates that the SFFV promoter is a stronger gene expression driver.

[0396] Comparison of surface expression and persistence of different promoters in primary T cells (The % of GFP cells in T cell transduction measured by flow cytometry showed the expected differences in the GFP cell population as expected from the previous experiments with HEK293 cells)

[0397] To determine the promoter transduction efficacy and surface expression persistence in primary T cells, activated cord blood leukocyte layer T cells were transduced with 50 μL SFFV-GFP or 1 mL EF1-GFP EF1-GFP viral supernatant in a 12-well tissue culture-treated plate pre-coated with recombinant human fibrin fragment (Clontech). After two overnight transductions, the cells were cultured with 300 IU / mL IL-2 (Peprotech) in T cell medium and maintained at a concentration of 1.0 - 4.0 × 106 cells / mL. At 7, 14, 21, and 28 days after transduction, the cells were washed, suspended in formalin, and subjected to flow cytometry analysis using the FITC channel to determine the percentage of GFP+ cells. The percentage of GFP+ cells in T cells transduced with SFFV-GFP was consistently higher ( Figure 44A ) than that in T cells transduced with EF1-GFP, even though the total percentage of GFP+ cells decreased during this period. Another comparison confirmed that between day 7 and day 28, the percentage of T cells transduced with a higher (1 mL) amount of EF1-GFP supernatant actually decreased, from over 60% to below 40% ( Figure 44B ), relative to the percentage of GFP+ cells transduced with a lower amount (50 μL, or 20-fold lower) of SFFV-GFP. This indicates that transduction using the SFFV promoter results in greater persistence of the transduced cells.

[0398] BCMA or TACI or BAFF-R CAR NK cells or T cells targeting at least one of the BCMA or TACI or BAFF-RCAR antigens

[0399] To evaluate the cytotoxic ability of a CAR targeting at least one of BCMA or TACI or BAFF-R NK cells or T cells, co-culture assays were performed with cell lines or primary human cells expressing at least one of BCMA or TACI or BAFF-R. The ability of the aforementioned CAR NK cells or T cells to lyse target cells was quantified by flow cytometry analysis, and the target cells were stained with Cytotracker dye (CMTMR). Lysis was observed over a 24-hour culture.

[0400] BAFF or APRIL CAR NK or T cells targeting cells expressing at least one of the BCMA or TACI or BAFF-R antigens.

[0401] The chimeric antigen receptor in the CAR is a ligand for BCMA or TACI or BAFF-R.

[0402] To evaluate the cytotoxic ability of CARs after targeting at least one of BCMA, TACI, BAFF-R or T cells, co-culture assays were performed with cell lines or primary human cells expressing at least one of BCMA, TACI or BAFF-R. The ability of the aforementioned CAR NK or T cells to lyse target cells was quantified by flow cytometry analysis, and the target cells were stained with the Cytotracker dye (CMTMR). Lysis was observed over a 24-hour culture period.

[0403] References

[0404] Arai, S., R. Meagher, M. Swearingen, H. Myint, E. Rich, J. Martinson and H. Klingemann (2008). "Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial." Cytotherapy 10(6):625-632.

[0405] Boissel, L., M. Betancur-Boissel, W. Lu, D. S. Krause, R. A. Van Etten, W. S. Wels and H. Klingemann (2013). "Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity."

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[0407] Burnett, A. K. (2012). "Treatment of acute myeloid leukemia: are we making progress?" Hematology-American Society Hematology Education Program:1-6.

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[0409] Corbin, A. S., A. Agarwal, M. Loriaux, J. Cortes, M. W. Deininger and B. J. Druker (2011). "Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity." J Clin Invest 121(1):396-409.

[0410] Dinndorf, P. A., R. G. Andrews, D. Benjamin, D. Ridgway, L. Wolff and I. D. Bernstein (1986). "Expression of normal myeloid-associated antigens by acute leukemia cells." Blood 67(4):1048-1053.

[0411] Djokic, M., E. Bjorklund, E. Blennow, J. Mazur, S. Soderhall and A. Porwit (2009). "Overexpression of CD123 correlates with the hyperdiploid genotype in acute lymphoblastic leukemia." Haematologica 94(7):1016-1019.

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[0413] Firor, A. E., A. Jares and Y. Ma (2015). "From humble beginnings to success in the clinic: Chimeric antigen receptor - modified T - cells and implications for immunotherapy." Exp Biol Med (Maywood).

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[0415] Ghosh, N. and W. Matsui (2009). "Cancer stem cells in multiple myeloma." Cancer Lett 277(1): 1 - 7.

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[0417] Jilani, I., E. Estey, Y. Huh, Y. Joe, T. Manshouri, M. Yared, F. Giles, H. Kantarjian, J. Cortes, D. Thomas, M. Keating, E. Freireich and M. Albitar (2002). "Differences in CD33 intensity between various myeloid neoplasms." Am J Clin Pathol 118(4):560 - 566.

[0418] Jordan, C.T., D. Upchurch, S.J. Szilvassy, M.L. Guzman, D.S. Howard, A.L. Pettigrew, T. Meyerrose, R. Rossi, B. Grimes, D.A. Rizzieri, S.M. Luger and G.L. Phillips (2000). "The interleukin - 3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells." Leukemia 14(10):1777 - 1784.

[0419] Klingemann, H. (2014). "Are natural killer cells superior CAR drivers?" Oncoimmunology 3:e28147.

[0420] Kumar, S.K., S.V. Rajkumar, A. Dispenzieri, M.Q. Lacy, S.R. Hayman, F.K. Buadi, S.R. Zeldenrust, D. Dingli, S.J. Russell, J.A. Lust, P.R. Greipp, R.A. Kyle and M.A. Gertz (2008). "Improved survival in multiple myeloma and the impact of novel therapies." Blood 111(5):2516 - 2520.

[0421] Lang, S., N.L. Vujanovic, B. Wollenberg and T.L. Whiteside (1998). "Absence of B7.1 - CD28 / CTLA - 4 - mediated co - stimulation in human NK cells." Eur J Immunol 28(3):780 - 786.

[0422] Lanitis, E., M. Poussin, A.W. Klattenhoff, D. Song, R. Sandaltzopoulos, C.H. June and D.J. Powell, Jr. (2013). "Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo." Cancer Immunol Res 1(1):43 - 53.

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[0424] Maus, M.V., J.A. Fraietta, B.L. Levine, M. Kalos, Y. Zhao and C.H. June (2014). "Adoptive immunotherapy for cancer or viruses." Annu Rev Immunol 32:189 - 225.

[0425] Olson, J.A., D.B. Leveson - Gower, S. Gill, J. Baker, A. Beilhack and R.S. Negrin (2010). "NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects." Blood 115(21):4293 - 4301.

[0426] Ruiz - Arguelles, G.J. and J.F. San Miguel (1994). "Cell surface markers in multiple myeloma." Mayo Clin Proc 69(7):684 - 690.

[0427] Testa, U., E. Pelosi and A. Frankel (2014). "CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies." Biomark Res 2(1):4.

[0428] Vergez, F., A. S. Green, J. Tamburini, J. E. Sarry, B. Gaillard, P. Cornillet-Lefebvre, M. Pannetier, A. Neyret, N. Chapuis, N. Ifrah, F. Dreyfus, S. Manenti, C. Demur, E. Delabesse, C. Lacombe, P. Mayeux, D. Bouscary, C. Recher and V. Bardet (2011). "High levels of CD34+CD38low / -CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study." Haematologica 96(12):1792 - 1798.

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[0430]

Symbol Explanation

[0431] None Sequence Listing <110> ICELL GENE THERAPEUTICS LLC MA, Yupo PINZ, Kevin JIANG, Xun WADA, Masayuki CHEN, Kevin <120> Chimeric Antigen Receptor (CAR), Composition and Method of Use Thereof <150> 62 / 184,321 <151> 2015-06-25 <160> 36 <170> SIPOSequenceListing 1.0 <210> 1 <211> 830 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Ile Val Met Thr Gly Ser Pro Ala Ser Leu 20 25 30 Ala Val Ser Leu Gly Gly Ala Val Thr Met Ala Cys Leu Ser Ser Gly 35 40 45 Ser Leu Leu Thr Ser Thr Ala Gly Leu Ala Thr Leu Ala Thr Thr Gly 50 55 60 Gly Leu Pro Gly Gly Ser Pro Leu Leu Leu Ile Thr Thr Ala Ser Thr 65 70 75 80 Ala Gly Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr 85 90 95 Ala Pro Thr Leu Thr Ile Ser Ser Val Gly Ala Gly Ala Val Ala Val 100 105 110 Thr Thr Cys Gly Gly Thr Thr Ser Thr Ala Thr Pro Gly Gly Gly Thr 115 120 125 Leu Leu Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Val Gly Leu Gly Gly Ser Gly Pro Gly Val Val 145 150 155 160 Leu Pro Gly Ala Ser Val Leu Met Ser Cys Leu Ala Ser Gly Thr Thr 165 170 175 Pro Thr Ser Thr Val Ile His Thr Val Ala Gly Leu Pro Gly Gly Gly 180 185 190 Leu Ala Thr Ile Gly Thr Ile Ala Pro Thr Ala Ala Gly Thr Ala Thr 195 200 205 Ala Gly Leu Pro Leu Gly Leu Ala Thr Leu Thr Ser Ala Thr Ser Thr 210 215 220 Ser Thr Ala Thr Met Gly Leu Ser Ser Leu Ala Ser Gly Ala Thr Ala 225 230 235 240 Val Thr Thr Cys Ala Ala Gly Leu Ala Ala Thr Ala Thr Gly Ala Thr 245 250 255 Pro Ala Thr Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser Thr Thr 260 265 270 Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly 275 280 285 Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Thr Cys Ala Ser Leu Ala Ser Ala Leu Leu His Ser Ala Thr Met 340 345 350 Ala Met Thr Pro Ala Ala Pro Gly Pro Thr Ala Leu His Thr Gly Pro 355 360 365 Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr Ala Ser Leu Ala Gly Ala 370 375 380 Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly 385 390 395 400 Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly 405 410 415 Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala 420 425 430 Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu 435 440 445 Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala 450 455 460 Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly 465 470 475 480 Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly 485 490 495 Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu 500 505 510 Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His 515 520 525 Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu 530 535 540 Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro Met Thr Ala 545 550 555 560 Met Gly Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu Val Thr Ala 565 570 575 Ser Gly Ile His Val Pro Ile Leu Gly Cys Pro Ser Ala Gly Leu Pro 580 585 590 Leu Thr Gly Ala Ala Thr Val Ala Val Ile Ser Ala Leu Leu Leu Ile 595 600 605 Gly Ala Leu Ile Gly Ser Met His Ile Ala Ala Thr Leu Thr Thr Gly 610 615 620 Ser Ala Val His Pro Ser Cys Leu Val Thr Ala Met Leu Cys Pro Leu 625 630 635 640 Leu Gly Leu Gly Val Ile Ser Leu Gly Ser Gly Ala Ala Ser Ile His 645 650 655 Ala Thr Val Gly Ala Leu Ile Ile Leu Ala Ala Ala Ser Leu Ser Ser 660 665 670 Ala Gly Ala Val Thr Gly Ser Gly Cys Leu Gly Cys Gly Gly Leu Gly 675 680 685 Gly Leu Ala Ile Leu Gly Pro Leu Gly Ser Pro Val His Ile Val Gly 690 695 700 Met Pro Ile Ala Thr Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 705 710 715 720 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu Gly 725 730 735 Ala Pro Ala Ala Ala Ala Gly Cys Ala Thr Leu Gly Leu Pro Ala Leu 740 745 750 Leu Leu Leu Leu Leu Leu Ala Pro Pro Ala Thr Ala Gly Ile Thr Cys 755 760 765 Pro Pro Pro Met Ser Val Gly His Ala Ala Ile Thr Val Leu Ser Thr 770 775 780 Ser Leu Thr Ser Ala Gly Ala Thr Ile Cys Ala Ser Gly Pro Leu Ala 785 790 795 800 Leu Ala Gly Thr Ser Ser Leu Thr Gly Cys Val Leu Ala Leu Ala Thr 805 810 815 Ala Val Ala His Thr Thr Thr Pro Ser Leu Leu Cys Ile Ala 820 825 830 <210> 2 <211> 2509 <212> DNA <213> Artificial Sequence <400> 2 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatcgtg atgacccaaa gccccgacag cctggccgtg agcctgggcg 120 agagggtgac catgaactgc aaaagcagcc agtccctgct gtactccacc aaccagaaga 180 actacctggc ttggtatcaa cagaagcccg gacagagccc caagctgctg atctattggg 240 ccagcactag ggaaagcggc gtgcccgata ggttcagcgg cagcgggagc ggcacagact 300 tcactctgac cattagcagc gtgcaggctg aggatgtggc cgtctactac tgccagcagt 360 actacagcta caggaccttt gggggcggaa ctaagctgga gatcaaggga ggggggggat 420 ccgggggagg aggctccggc ggaggcggaa gccaagtgca actgcagcag agcggcccag 480 aggtggtcaa acctggggca agcgtgaaga tgagctgcaa ggctagcggc tataccttca 540 ccagctatgt gatccactgg gtgaggcaga aaccaggaca gggcctggac tggatcggct 600 acatcaaccc ctacaatgac ggcaccgatt atgacgaaaa attcaagggg aaggccaccc 660 tgaccagcga caccagcaca agcaccgcct acatggagct gtccagcctg aggtccgagg 720 acaccgccgt gtattactgt gccagggaga aggacaatta cgccaccggc gcttggttcg 780 cctactgggg ccagggcaca ctggtgacag tgagcagcac cacgacgcca gcgccgcgac 840 caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc 900 ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt gatatctaca 960 tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt atcacccttt 1020 actgcaggag taagaggagc aggctcctgc acagtgacta catgaacatg actccccgcc 1080 gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac ttcgcagcct 1140 atcgctccaa acggggcaga aagaaactcc tgtatatatt caaacaacca tttatgagac 1200 cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa gaagaagaag 1260 gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg taccagcagg 1320 gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac gatgttttgg 1380 acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg aagaaccctc 1440 aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 1500 ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 1560 cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgcggaagcg 1620 gagccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cccggcccca 1680 tgtacagaat gcagctgctg agctgcatcg ccctgagcct ggccctggtg accaacagcg 1740 gcatccacgt gttcatcctg ggctgcttca gcgccggcct gcccaagacc gaggccaact 1800 gggtgaacgt gatcagcgac ctgaagaaga tcgaggacct gatccagagc atgcacatcg 1860 acgccaccct gtacaccgag agcgacgtgc accccagctg caaggtgacc gccatgaagt 1920 gcttcctgct ggagctgcag gtgatcagcc tggagagcgg cgacgccagc atccacgaca 1980 ccgtggagaa cctgatcatc ctggccaaca acagcctgag cagcaacggc aacgtgaccg 2040 agagcggctg caaggagtgc gaggagctgg aggagaagaa catcaaggag ttcctgcaga 2100 gcttcgtgca catcgtgcag atgttcatca acaccagctc cggcggcggc tccggcggcg 2160 gcggctccgg cggcggcggc tccggcggcg gcggctccgg cggcggctcc ctgcaggccc 2220 ccagaagagc cagaggctgc agaaccctgg gcctgcccgc cctgctgctg ctgctgctgc 2280 tgagaccccc cgccaccaga ggcatcacct gccccccccc catgagcgtg gagcacgccg 2340 acatctgggt gaagagctac agcctgtaca gcagagagag atacatctgc aacagcggct 2400 tcaagagaaa ggccggcacc agcagcctga ccgagtgcgt gctgaacaag gccaccaacg 2460 tggcccactg gaccaccccc agcctgaagt gcatcagata agtttaaac 2509 <210> 3 <211> 995 <212> PRT <213> Artificial Sequence <400> 3 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly 35 40 45 Ala Ile Ser Leu Thr Leu Ala Thr Thr Gly Gly Leu Pro Ala Gly Thr 50 55 60 Val Leu Leu Leu Ile Thr His Thr Ser Ala Leu His Ser Gly Val Pro 65 70 75 80 Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Thr Ser Leu Thr Ile 85 90 95 Ser Ala Leu Gly Gly Gly Ala Ile Ala Thr Thr Pro Cys Gly Gly Gly 100 105 110 Ala Thr Leu Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Val Leu Leu Gly Gly Ser Gly Pro Gly Leu Val Ala Pro Ser Gly Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ala Thr Gly 165 170 175 Val Ser Thr Ile Ala Gly Pro Pro Ala Leu Gly Leu Gly Thr Leu Gly 180 185 190 Val Ile Thr Gly Ser Gly Thr Thr Thr Thr Ala Ser Ala Leu Leu Ser 195 200 205 Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu Ser Gly Val Pro Leu Leu 210 215 220 Met Ala Ser Leu Gly Thr Ala Ala Thr Ala Ile Thr Thr Cys Ala Leu 225 230 235 240 His Thr Thr Thr Gly Gly Ser Thr Ala Met Ala Thr Thr Gly Gly Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly 275 280 285 Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala 290 295 300 Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu Ala Gly Ala 325 330 335 Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly 340 345 350 Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly 355 360 365 Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala 370 375 380 Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu 385 390 395 400 Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala 405 410 415 Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly 420 425 430 Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly 435 440 445 Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu 450 455 460 Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His 465 470 475 480 Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu 485 490 495 Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro Met Ala Leu 500 505 510 Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala 515 520 525 Ala Pro Gly Ile Val Leu Ser Gly Ser Pro Ala Ile Leu Ser Ala Ser 530 535 540 Pro Gly Gly Leu Val Thr Met Thr Cys Ala Ala Ser Ser Ser Val Ser 545 550 555 560 Thr Ile His Thr Pro Gly Gly Leu Pro Gly Ser Ser Pro Leu Pro Thr 565 570 575 Ile Thr Ala Thr Ser Ala Leu Ala Ser Gly Val Pro Val Ala Pro Ser 580 585 590 Gly Ser Gly Ser Gly Thr Ser Thr Ser Leu Thr Ile Ser Ala Val Gly 595 600 605 Ala Gly Ala Ala Ala Thr Thr Thr Cys Gly Gly Thr Thr Ser Ala Pro 610 615 620 Pro Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu Gly Gly Gly Gly 625 630 635 640 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val Gly Leu Gly 645 650 655 Gly Pro Gly Ala Gly Leu Val Leu Pro Gly Ala Ser Val Leu Met Ser 660 665 670 Cys Leu Ala Ser Gly Thr Thr Pro Thr Ser Thr Ala Met His Thr Val 675 680 685 Leu Gly Thr Pro Gly Ala Gly Leu Gly Thr Ile Gly Ala Ile Thr Pro 690 695 700 Gly Ala Gly Ala Thr Ser Thr Ala Gly Leu Pro Leu Gly Leu Ala Thr 705 710 715 720 Leu Thr Ala Ala Leu Ser Ser Ser Thr Ala Thr Met Gly Leu Ser Ser 725 730 735 Leu Thr Ser Gly Ala Ser Ala Val Thr Thr Cys Ala Ala Ser Thr Thr 740 745 750 Thr Gly Gly Ala Thr Thr Pro Ala Val Thr Gly Ala Gly Thr Thr Val 755 760 765 Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala 770 775 780 Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala 785 790 795 800 Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys 805 810 815 Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 820 825 830 Leu Ser Leu Val Ile Thr Leu Thr Cys Ala Ser Leu Ala Ser Ala Leu 835 840 845 Leu His Ser Ala Thr Met Ala Met Thr Pro Ala Ala Pro Gly Pro Thr 850 855 860 Ala Leu His Thr Gly Pro Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr 865 870 875 880 Ala Ser Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly 885 890 895 Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly 900 905 910 Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly 915 920 925 Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly 930 935 940 Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu 945 950 955 960 Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu 965 970 975 Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu 980 985 990 Pro Pro Ala 995 <210> 4 <211> 3004 <212> DNA <213> Artificial Sequence <400> 4 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatccag atgacacaga ctacatcctc cctgtctgcc tctctgggag 120 acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 180 agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga ttacactcag 240 gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc accattagca 300 acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg cttccgtaca 360 cgttcggagg ggggaccaag ctggagatca caggtggcgg tggctcgggc ggtggtgggt 420 cgggtggcgg cggatctgag gtgaaactgc aggagtcagg acctggcctg gtggcgccct 480 cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac tatggtgtaa 540 gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata tggggtagtg 600 aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag gacaactcca 660 agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc atttactact 720 gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc caaggaacct 780 cagtcaccgt ctcctcaacc acgacgccag cgccgcgacc accaacaccg gcgcccacca 840 tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag 900 tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc ttggccggga 960 cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg ggcagaaaga 1020 aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 1080 atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt 1140 tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc 1200 tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg 1260 agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac aatgaactgc 1320 agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag cgccggaggg 1380 gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac acctacgacg 1440 cccttcacat gcaggccctg ccccctcgcg gaagcggagc caccaacttc agcctgctga 1500 agcaggccgg cgacgtggag gagaaccccg gccccatggc cttaccagtg accgccttgc 1560 tcctgccgct ggccttgctg ctccacgccg ccaggccgca gatcgtgctg agccagagcc 1620 ctgccatcct gtccgcaagc ccaggcgaga aggtgaccat gacctgtagg gccagcagct 1680 ccgtgagcta catccactgg tttcagcaga agcctggaag cagccctaag ccctggatct 1740 acgccacaag caatctggct agcggcgtgc ccgtgaggtt cagcggcagc gggagcggga 1800 ccagctacag cctgactatc agcagggtgg aggccgagga cgccgccaca tactactgcc 1860 aacagtggac ctccaaccca cccacctttg gaggagggac aaaactggag atcaaagggg 1920 gcggagggtc cggaggcggc ggaagcgggg gagggggaag ccaggtccaa ctgcaacagc 1980 ccggagcaga actggtcaaa ccaggcgcca gcgtgaagat gagctgcaag gccagcgggt 2040 acaccttcac ttcctataac atgcactggg tgaagcagac cccaggaagg ggcctggagt 2100 ggatcggggc aatctatccc ggcaacggcg acacaagcta caaccagaag ttcaagggga 2160 aagccactct gaccgccgac aagtccagct ccaccgccta catgcagctg agctccctga 2220 ccagcgagga cagcgccgtg tactattgcg ccagaagcac ttattacgga ggggactggt 2280 ccagcgagga cagcgccgtg tactattgcg ccagaagcac ttattacgga ggggactggt 2280 acttcaacgt gtggggggca gggaccaccg tgaccgtgtc cgccaccacg acgccagcgc 2340 acttcaacgt gtggggggca gggaccaccg tgaccgtgtc cgccaccacg acgccagcgc 2340 cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg 2400 cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg 2400 cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gcctgtgata 2460 cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gcctgtgata 2460 tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 2520 tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 2520 ccctttactg caggagtaag aggagcaggc tcctgcacag tgactacatg aacatgactc 2580 ccctttactg caggagtaag aggagcaggc tcctgcacag tgactacatg aacatgactc 2580 cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca cgcgacttcg 2640 cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca cgcgacttcg 2640 cagcctatcg ctccagagtg aagttcagca ggagcgcaga cgcccccgcg taccagcagg 2700 cagcctatcg ctccagagtg aagttcagca ggagcgcaga cgcccccgcg taccagcagg 2700 gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac gatgttttgg 2760 gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac gatgttttgg 2760 acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg aagaaccctc 2820 acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg aagaaccctc 2820 aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 2880 aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 2880 ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 2940 ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 2940 cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgctaagttt 3000 aaac 3004 <210> 5 <211> 1001 <212> PRT <213> Artificial Sequence <400> 5 Ala Ala Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 Leu Leu His Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Ala Ala Val Thr Ile Ser Cys Ala Ala 35 40 45 Ser Gly Ala Ile Ser Leu Thr Leu Ala Thr Thr Gly Gly Leu Pro Ala 50 55 60 Gly Thr Val Leu Leu Leu Ile Thr His Thr Ser Ala Leu His Ser Gly 65 70 75 80 Val Pro Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Thr Ser Leu 85 90 95 Thr Ile Ser Ala Leu Gly Gly Gly Ala Ile Ala Thr Thr Pro Cys Gly 100 105 110 Gly Gly Ala Thr Leu Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly 115 120 125 Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Val Leu Leu Gly Gly Ser Gly Pro Gly Leu Val Ala Pro Ser 145 150 155 160 Gly Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ala 165 170 175 Thr Gly Val Ser Thr Ile Ala Gly Pro Pro Ala Leu Gly Leu Gly Thr 180 185 190 Leu Gly Val Ile Thr Gly Ser Gly Thr Thr Thr Thr Ala Ser Ala Leu 195 200 205 Leu Ser Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu Ser Gly Val Pro 210 215 220 Leu Leu Met Ala Ser Leu Gly Thr Ala Ala Thr Ala Ile Thr Thr Cys 225 230 235 240 Ala Leu His Thr Thr Thr Gly Gly Ser Thr Ala Met Ala Thr Thr Gly 245 250 255 Gly Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala 275 280 285 Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly 290 295 300 Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu Ala 325 330 335 Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro 340 345 350 Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly 355 360 365 Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala 370 375 380 Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu 385 390 395 400 Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly 405 410 415 Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu Ala Pro Gly Gly 420 425 430 Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser 435 440 445 Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly 450 455 460 Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu 465 470 475 480 His Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser 485 490 495 Leu Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro Met Ala 500 505 510 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala 515 520 525 Ala Ala Pro Ala Ile Gly Met Thr Gly Ser Pro Ser Ser Leu Ser Ala 530 535 540 Ser Val Gly Ala Ala Val Thr Ile Thr Cys Ala Ser Ser Gly Ser Ile 545 550 555 560 Val His Ser Val Gly Ala Thr Pro Leu Gly Thr Thr Gly Gly Leu Pro 565 570 575 Gly Leu Ala Pro Leu Leu Leu Ile Thr Leu Val Ser Ala Ala Pro Ser 580 585 590 Gly Val Pro Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr 595 600 605 Leu Thr Ile Ser Ser Leu Gly Pro Gly Ala Pro Ala Thr Thr Thr Cys 610 615 620 Pro Gly Gly Ser Gly Pro Pro Thr Thr Pro Gly Gly Gly Thr Leu Val 625 630 635 640 Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 645 650 655 Gly Ser Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro 660 665 670 Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly Thr Gly Pro Ser 675 680 685 Ala Ser Thr Met Ala Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly 690 695 700 Thr Val Gly Ala Ile Thr Pro Gly Ala Gly Ala Thr Ala Thr Ser Gly 705 710 715 720 Leu Pro Leu Gly Ala Pro Thr Ile Ser Ala Ala Thr Ser Leu Ala Thr 725 730 735 Ala Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr 740 745 750 Thr Cys Ala Ala Ala Gly Ser Ser Thr Ala Thr Thr Pro Ala Val Thr 755 760 765 Gly Gly Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro 770 775 780 Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu 785 790 795 800 Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala 805 810 815 Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly 820 825 830 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu 835 840 845 Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala 850 855 860 Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro 865 870 875 880 Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser 885 890 895 Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly 900 905 910 Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala 915 920 925 Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu Ala Pro Gly 930 935 940 Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr 945 950 955 960 Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala 965 970 975 Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala 980 985 990 Leu His Met Gly Ala Leu Pro Pro Ala 995 1000 <210> 6 <211> 3016 <212> DNA <213> Artificial Sequence <400> 6 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatccag atgacacaga ctacatcctc cctgtctgcc tctctgggag 120 acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 180 agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga ttacactcag 240 gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc accattagca 300 acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg cttccgtaca 360 cgttcggagg ggggaccaag ctggagatca caggtggcgg tggctcgggc ggtggtgggt 420 cgggtggcgg cggatctgag gtgaaactgc aggagtcagg acctggcctg gtggcgccct 480 cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac tatggtgtaa 540 gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata tggggtagtg 600 aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag gacaactcca 660 agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc atttactact 720 gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc caaggaacct 780 cagtcaccgt ctcctcaacc acgacgccag cgccgcgacc accaacaccg gcgcccacca 840 tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag 900 tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc ttggccggga 960 cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg ggcagaaaga 1020 aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 1080 atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt 1140 tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc 1200 tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg 1260 agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga 1320 aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca 1380 aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc 1440 ttcacatgca ggccctgccc cctcgcggaa gcggagccac caacttcagc ctgctgaagc 1500 aggccggcga cgtggaggag aaccccggcc ccatggcctt accagtgacc gccttgctcc 1560 tgccgctggc cttgctgctc cacgccgcca ggccggatat ccagatgacc cagagcccca 1620 gctccctgtc cgcatccgtg ggcgacagag tgacaattac ctgtagaagc agccaaagca 1680 tcgtgcatag cgtcggcaac acttttctgg agtggtatca acagaagccc gggaaggccc 1740 ccaaactgct gatctacaag gtgagcaaca gattcagcgg ggtcccaagc agattctccg 1800 gcagcggctc cgggactgac ttcaccctga ccattagcag cctgcagcca gaggacttcg 1860 ccacatacta ctgcttccaa gggagccagt tcccctacac cttcggccaa ggcactaagg 1920 tggagatcaa agggggggga ggaagcggcg gaggagggag cggaggcggg ggatccgaag 1980 tgcaactggt cgaatccgga ggggggctgg tccagcctgg agggtccctg agactgagct 2040 gcgccgcaag cggctacgag ttctccaggt cctggatgaa ctgggtgagg caggccccag 2100 gaaaagggct ggaatgggtg ggcaggatct accctggcga cggcgatacc aactactccg 2160 gaaagttcaa gggcaggttc actatcagcg ccgacactag caagaatacc gcctacctgc 2220 agatgaatag cctgagggcc gaggacaccg ccgtgtatta ctgcgctaga gacggcagca 2280 gctgggattg gtacttcgac gtgtggggcc agggcactct ggtgactgtg agcagcacca 2340 cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc 2400 tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg gggctggact 2460 tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc cttctcctgt 2520 cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat atattcaaac 2580 aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc tgccgatttc 2640 cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc gcagacgccc 2700 ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg 2760 agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga aagccgagaa 2820 ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct 2880 acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc 2940 agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag gccctgcccc 3000 ctcgctaagt ttaaac 3016 <210> 7 <211> 990 <212> PRT <213> Artificial Sequence <400> 7 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly 35 40 45 Ala Ile Ser Leu Thr Leu Ala Thr Thr Gly Gly Leu Pro Ala Gly Thr 50 55 60 Val Leu Leu Leu Ile Thr His Thr Ser Ala Leu His Ser Gly Val Pro 65 70 75 80 Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Thr Ser Leu Thr Ile 85 90 95 Ser Ala Leu Gly Gly Gly Ala Ile Ala Thr Thr Pro Cys Gly Gly Gly 100 105 110 Ala Thr Leu Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Val Leu Leu Gly Gly Ser Gly Pro Gly Leu Val Ala Pro Ser Gly Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ala Thr Gly 165 170 175 Val Ser Thr Ile Ala Gly Pro Pro Ala Leu Gly Leu Gly Thr Leu Gly 180 185 190 Val Ile Thr Gly Ser Gly Thr Thr Thr Thr Ala Ser Ala Leu Leu Ser 195 200 205 Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu Ser Gly Val Pro Leu Leu 210 215 220 Met Ala Ser Leu Gly Thr Ala Ala Thr Ala Ile Thr Thr Cys Ala Leu 225 230 235 240 His Thr Thr Thr Gly Gly Ser Thr Ala Met Ala Thr Thr Gly Gly Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly 275 280 285 Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala 290 295 300 Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu Ala Gly Ala 325 330 335 Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly 340 345 350 Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly 355 360 365 Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala 370 375 380 Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu 385 390 395 400 Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala 405 410 415 Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly 420 425 430 Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly 435 440 445 Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu 450 455 460 Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His 465 470 475 480 Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu 485 490 495 Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro Met Ala Leu 500 505 510 Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala 515 520 525 Ala Pro Ala Val Gly Ile Thr Gly Ser Pro Ser Thr Leu Ala Ala Ser 530 535 540 Pro Gly Gly Thr Ile Thr Ile Ala Cys Ala Ala Ser Leu Ser Ile Ser 545 550 555 560 Leu Ala Leu Ala Thr Thr Gly Gly Leu Pro Gly Leu Thr Ala Leu Leu 565 570 575 Leu Ile Thr Ser Gly Ser Thr Leu Gly Ser Gly Ile Pro Ser Ala Pro 580 585 590 Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile Ser Ser Leu 595 600 605 Gly Pro Gly Ala Pro Ala Met Thr Thr Cys Gly Gly His Ala Leu Thr 610 615 620 Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu Gly Gly Gly 625 630 635 640 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val Gly Leu 645 650 655 Gly Gly Pro Gly Ala Gly Leu Val Ala Pro Gly Ala Ser Val Leu Leu 660 665 670 Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ser Thr Thr Met Ala Thr 675 680 685 Val Leu Gly Ala Pro Ala Gly Gly Leu Gly Thr Ile Gly Ala Ile Ala 690 695 700 Pro Thr Ala Ser Gly Thr His Thr Ala Gly Leu Pro Leu Ala Leu Ala 705 710 715 720 Ile Leu Thr Val Ala Leu Ser Ser Ser Thr Ala Thr Met Gly Leu Ser 725 730 735 Ser Leu Thr Ser Gly Ala Ser Ala Val Thr Thr Cys Ala Ala Gly Ala 740 745 750 Thr Ala Ala Thr Thr Gly Gly Gly Thr Thr Leu Thr Val Ser Ser Thr 755 760 765 Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 770 775 780 Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly 785 790 795 800 Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr 805 810 815 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 820 825 830 Thr Leu Thr Cys Ala Ser Leu Ala Ser Ala Leu Leu His Ser Ala Thr 835 840 845 Met Ala Met Thr Pro Ala Ala Pro Gly Pro Thr Ala Leu His Thr Gly 850 855 860 Pro Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr Ala Ser Ala Val Leu 865 870 875 880 Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly 885 890 895 Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu 900 905 910 Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala 915 920 925 Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu 930 935 940 Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala 945 950 955 960 Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu 965 970 975 Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala 980 985 990 <210> 8 <211> 2989 <212> DNA <213> Artificial Sequence <400> 8 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatccag atgacacaga ctacatcctc cctgtctgcc tctctgggag 120 acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 180 agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga ttacactcag 240 gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttattctctc accattagca 300 acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg cttccgtaca 360 cgttcggagg ggggaccaag ctggagatca caggtggcgg tggctcgggc ggtggtgggt 420 cgggtggcgg cggatctgag gtgaaactgc aggagtcagg acctggcctg gtggcgccct 480 cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac tatggtgtaa 540 gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata tggggtagtg 600 aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag gacaactcca 660 agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc atttactact 720 gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc caaggaacct 780 cagtcaccgt ctcctcaacc acgacgccag cgccgcgacc accaacaccg gcgcccacca 840 tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag 900 tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc ttggccggga 960 cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg ggcagaaaga 1020 aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 1080 atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt 1140 tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc 1200 tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg 1260 agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac aatgaactgc 1320 agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag cgccggaggg 1380 gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac acctacgacg 1440 cccttcacat gcaggccctg ccccctcgcg gaagcggagc caccaacttc agcctgctga 1500 agcaggccgg cgacgtggag gagaaccccg gccccatggc cttaccagtg accgccttgc 1560 tcctgccgct ggccttgctg ctccacgccg ccaggccgga cgtgcagatc acccagagcc 1620 ccagctacct ggccgccagc cccggcgaga ccatcaccat caactgcaga gccagcaaga 1680 gcatcagcaa ggacctggcc tggtaccagg agaagcccgg caagaccaac aagctgctga 1740 tctacagcgg cagcaccctg cagagcggca tccccagcag attcagcggc agcggcagcg 1800 gcaccgactt caccctgacc atcagcagcc tggagcccga ggacttcgcc atgtactact 1860 gccagcagca caacaagtac ccctacacct tcggcggcgg caccaagctg gagatcaagg 1920 gagggggggg atccggggga ggaggctccg gcggaggcgg aagccaggtg cagctgcagc 1980 agcccggcgc cgagctggtg agacccggcg ccagcgtgaa gctgagctgc aaggccagcg 2040 gctacacctt caccagctac tggatgaact gggtgaagca gagacccgac cagggcctgg 2100 agtggatcgg cagaatcgac ccctacgaca gcgagaccca ctacaaccag aagttcaagg 2160 acaaggccat cctgaccgtg gacaagagca gcagcaccgc ctacatgcag ctgagcagcc 2220 tgaccagcga ggacagcgcc gtgtactact gcgccagagg caactgggac gactactggg 2280 gccagggcac caccctgacc gtgagcagca ccacgacgcc agcgccgcga ccaccaacac 2340 cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 2400 cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc 2460 ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcagga 2520 gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc 2580 ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca 2640 gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct 2700 ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 2760 gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca 2820 atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc 2880 gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca 2940 cctacgacgc ccttcacatg caggccctgc cccctcgcta agtttaaac 2989 <210> 9 <211> 1066 <212> PRT <213> Artificial Sequence <400> 9 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Gly Ile Val Leu Thr Gly Ser Pro Ala Ile Met 20 25 30 Ser Ala Ser Pro Gly Gly Leu Val Thr Ile Thr Cys Ser Ala Ser Ser 35 40 45 Ser Ile Ser Thr Met His Thr Pro Gly Gly Leu Pro Gly Thr Ser Pro 50 55 60 Leu Leu Thr Ile Thr Thr Thr Ser Ala Leu Ala Ser Gly Val Pro Ala 65 70 75 80 Ala Pro Ser Gly Ser Gly Ser Gly Thr Ser Thr Ser Leu Thr Ile Ser 85 90 95 Ala Met Gly Ala Gly Ala Ala Ala Thr Thr Thr Cys His Gly Ala Ser 100 105 110 Thr Thr Pro Leu Thr Pro Gly Ser Gly Thr Leu Leu Gly Leu Leu Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val 130 135 140 Gly Leu Gly Gly Ser Gly Ala Gly Leu Ala Leu Pro Gly Ala Ser Val 145 150 155 160 Leu Met Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ser Thr Ala Met 165 170 175 His Thr Val Leu Gly Ala Pro Gly Gly Gly Leu Gly Thr Ile Gly Thr 180 185 190 Ile Ala Pro Ser Thr Gly Thr Thr Gly Thr Ala Gly Leu Pro Leu Ala 195 200 205 Leu Ala Thr Leu Thr Ala Ala Leu Ser Ser Ser Thr Ala Thr Met Gly 210 215 220 Leu Ser Ser Leu Thr Pro Gly Ala Ser Ala Val Thr Thr Cys Ala Ala 225 230 235 240 Gly Gly Gly Val Pro Ala Thr Thr Gly Gly Gly Thr Thr Leu Thr Val 245 250 255 Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr 260 265 270 Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala 275 280 285 Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile 290 295 300 Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 305 310 315 320 Leu Val Ile Thr Leu Thr Cys Ala Ser Leu Ala Ser Ala Leu Leu His 325 330 335 Ser Ala Thr Met Ala Met Thr Pro Ala Ala Pro Gly Pro Thr Ala Leu 340 345 350 His Thr Gly Pro Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr Ala Ser 355 360 365 Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met 370 375 380 Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro 385 390 395 400 Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala 405 410 415 Ser Ala Ala Ala Pro Ala Thr Leu Gly Gly Gly Ala Gly Leu Thr Ala 420 425 430 Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala 435 440 445 Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu Ala Pro 450 455 460 Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala 465 470 475 480 Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His 485 490 495 Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala 500 505 510 Ala Leu His Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala 515 520 525 Pro Ser Leu Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro 530 535 540 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 545 550 555 560 His Ala Ala Ala Pro Ala Val Gly Ile Thr Gly Ser Pro Ser Thr Leu 565 570 575 Ala Ala Ser Pro Gly Gly Thr Ile Thr Ile Ala Cys Ala Ala Ser Leu 580 585 590 Ser Ile Ser Leu Ala Leu Ala Thr Thr Gly Gly Leu Pro Gly Leu Thr 595 600 605 Ala Leu Leu Leu Ile Thr Ser Gly Ser Thr Leu Gly Ser Gly Ile Pro 610 615 620 Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile 625 630 635 640 Ser Ser Leu Gly Pro Gly Ala Pro Ala Met Thr Thr Cys Gly Gly His 645 650 655 Ala Leu Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 660 665 670 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 675 680 685 Val Gly Leu Gly Gly Pro Gly Ala Gly Leu Val Ala Pro Gly Ala Ser 690 695 700 Val Leu Leu Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ser Thr Thr 705 710 715 720 Met Ala Thr Val Leu Gly Ala Pro Ala Gly Gly Leu Gly Thr Ile Gly 725 730 735 Ala Ile Ala Pro Thr Ala Ser Gly Thr His Thr Ala Gly Leu Pro Leu 740 745 750 Ala Leu Ala Ile Leu Thr Val Ala Leu Ser Ser Ser Thr Ala Thr Met 755 760 765 Gly Leu Ser Ser Leu Thr Ser Gly Ala Ser Ala Val Thr Thr Cys Ala 770 775 780 Ala Gly Ala Thr Ala Ala Thr Thr Gly Gly Gly Thr Thr Leu Thr Val 785 790 795 800 Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr 805 810 815 Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala 820 825 830 Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile 835 840 845 Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 850 855 860 Leu Val Ile Thr Leu Thr Cys Ala Ser Leu Ala Ser Ala Leu Leu His 865 870 875 880 Ser Ala Thr Met Ala Met Thr Pro Ala Ala Pro Gly Pro Thr Ala Leu 885 890 895 His Thr Gly Pro Thr Ala Pro Pro Ala Ala Pro Ala Ala Thr Ala Ser 900 905 910 Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met 915 920 925 Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro 930 935 940 Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala 945 950 955 960 Ser Ala Ala Ala Pro Ala Thr Leu Gly Gly Gly Ala Gly Leu Thr Ala 965 970 975 Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala 980 985 990 Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu Ala Pro 995 1000 1005 Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala 1010 1015 1020 Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His 1025 1030 1035 1040 Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala 1045 1050 1055 Ala Leu His Met Gly Ala Leu Pro Pro Ala 1060 1065 <210> 10 <211> 3217 <212> DNA <213> Artificial Sequence <400> 10 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc gcagatcgtg ctgacccaga gccccgccat catgagcgcc agccccggcg 120 agaaggtgac catcacctgc agcgccagca gcagcatcag ctacatgcac tggttccagc 180 agaagcccgg caccagcccc aagctgtgga tctacaccac cagcaacctg gccagcggcg 240 tgcccgccag attcagcggc agcggcagcg gcaccagcta cagcctgacc atcagcagaa 300 tggaggccga ggacgccgcc acctactact gccaccagag aagcacctac cccctgacct 360 tcggcagcgg caccaagctg gagctgaagg gagggggggg atccggggga ggaggctccg 420 gcggaggcgg aagccaggtg cagctgcagc agagcggcgc cgagctggcc aagcccggcg 480 ccagcgtgaa gatgagctgc aaggccagcg gctacacctt caccagctac agaatgcact 540 gggtgaagca gagacccggc cagggcctgg agtggatcgg ctacatcaac cccagcaccg 600 gctacaccga gtacaaccag aagttcaagg acaaggccac cctgaccgcc gacaagagca 660 gcagcaccgc ctacatgcag ctgagcagcc tgaccttcga ggacagcgcc gtgtactact 720 gcgccagagg cggcggcgtg ttcgactact ggggccaggg caccaccctg accgtgagca 780 gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc 840 tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac acgagggggc 900 tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc 960 tcctgtcact ggttatcacc ctttactgca ggagtaagag gagcaggctc ctgcacagtg 1020 actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac cagccctatg 1080 ccccaccacg cgacttcgca gcctatcgct ccaaacgggg cagaaagaaa ctcctgtata 1140 tattcaaaca accatttatg agaccagtac aaactactca agaggaagat ggctgtagct 1200 gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc agcaggagcg 1260 cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc aatctaggac 1320 gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag atggggggaa 1380 agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa gataagatgg 1440 cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag gggcacgatg 1500 gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt cacatgcagg 1560 ccctgccccc tcgcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg 1620 tggaggagaa ccctggacct atggccttac cagtgaccgc cttgctcctg ccgctggcct 1680 tgctgctcca cgccgccagg ccggacgtgc agatcaccca gagccccagc tacctggccg 1740 ccagccccgg cgagaccatc accatcaact gcagagccag caagagcatc agcaaggacc 1800 tggcctggta ccaggagaag cccggcaaga ccaacaagct gctgatctac agcggcagca 1860 ccctgcagag cggcatcccc agcagattca gcggcagcgg cagcggcacc gacttcaccc 1920 tgaccatcag cagcctggag cccgaggact tcgccatgta ctactgccag cagcacaaca 1980 agtaccccta caccttcggc ggcggcacca agctggagat caagggaggg gggggatccg 2040 ggggaggagg ctccggcgga ggcggaagcc aggtgcagct gcagcagccc ggcgccgagc 2100 tggtgagacc cggcgccagc gtgaagctga gctgcaaggc cagcggctac accttcacca 2160 gctactggat gaactgggtg aagcagagac ccgaccaggg cctggagtgg atcggcagaa 2220 tcgaccccta cgacagcgag acccactaca accagaagtt caaggacaag gccatcctga 2280 ccgtggacaa gagcagcagc accgcctaca tgcagctgag cagcctgacc agcgaggaca 2340 gcgccgtgta ctactgcgcc agaggcaact gggacgacta ctggggccag ggcaccaccc 2400 tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg 2460 cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc 2520 acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg gccgggactt 2580 gtggggtcct tctcctgtca ctggttatca ccctttactg caggagtaag aggagcaggc 2640 tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc cgcaagcatt 2700 accagcccta tgccccacca cgcgacttcg cagcctatcg ctccaaacgg ggcagaaaga 2760 aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 2820 atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt 2880 tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc tataacgagc 2940 tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg 3000 agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga 3060 aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca 3120 aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc 3180 ttcacatgca ggccctgccc cctcgctaag tttaaac 3217 <210> 11 <211> 985 <212> PRT <213> Artificial Sequence <400> 11 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Gly Ile Val Leu Thr Gly Ser Pro Ala Ile Met 20 25 30 Ser Ala Ser Pro Gly Gly Leu Val Thr Ile Thr Cys Ser Ala Ser Ser 35 40 45 Ser Ile Ser Thr Met His Thr Pro Gly Gly Leu Pro Gly Thr Ser Pro 50 55 60 Leu Leu Thr Ile Thr Thr Thr Ser Ala Leu Ala Ser Gly Val Pro Ala 65 70 75 80 Ala Pro Ser Gly Ser Gly Ser Gly Thr Ser Thr Ser Leu Thr Ile Ser 85 90 95 Ala Met Gly Ala Gly Ala Ala Ala Thr Thr Thr Cys His Gly Ala Ser 100 105 110 Thr Thr Pro Leu Thr Pro Gly Ser Gly Thr Leu Leu Gly Leu Leu Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val 130 135 140 Gly Leu Gly Gly Ser Gly Ala Gly Leu Ala Leu Pro Gly Ala Ser Val 145 150 155 160 Leu Met Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ser Thr Ala Met 165 170 175 His Thr Val Leu Gly Ala Pro Gly Gly Gly Leu Gly Thr Ile Gly Thr 180 185 190 Ile Ala Pro Ser Thr Gly Thr Thr Gly Thr Ala Gly Leu Pro Leu Ala 195 200 205 Leu Ala Thr Leu Thr Ala Ala Leu Ser Ser Ser Thr Ala Thr Met Gly 210 215 220 Leu Ser Ser Leu Thr Pro Gly Ala Ser Ala Val Thr Thr Cys Ala Ala 225 230 235 240 Gly Gly Gly Val Pro Ala Thr Thr Gly Gly Gly Thr Thr Leu Thr Val 245 250 255 Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr 260 265 270 Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala 275 280 285 Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile 290 295 300 Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 305 310 315 320 Leu Val Ile Thr Leu Thr Cys Leu Ala Gly Ala Leu Leu Leu Leu Thr 325 330 335 Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly Thr Thr Gly Gly Gly 340 345 350 Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly Gly Gly Gly Cys Gly 355 360 365 Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly 370 375 380 Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly 385 390 395 400 Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly 405 410 415 Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu 420 425 430 Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly 435 440 445 Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser 450 455 460 Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro 465 470 475 480 Pro Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu Leu Leu Gly Ala Gly 485 490 495 Ala Val Gly Gly Ala Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu 500 505 510 Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Ala Pro Ala Val Gly 515 520 525 Ile Thr Gly Ser Pro Ser Thr Leu Ala Ala Ser Pro Gly Gly Thr Ile 530 535 540 Thr Ile Ala Cys Ala Ala Ser Leu Ser Ile Ser Leu Ala Leu Ala Thr 545 550 555 560 Thr Gly Gly Leu Pro Gly Leu Thr Ala Leu Leu Leu Ile Thr Ser Gly 565 570 575 Ser Thr Leu Gly Ser Gly Ile Pro Ser Ala Pro Ser Gly Ser Gly Ser 580 585 590 Gly Thr Ala Pro Thr Leu Thr Ile Ser Ser Leu Gly Pro Gly Ala Pro 595 600 605 Ala Met Thr Thr Cys Gly Gly His Ala Leu Thr Pro Thr Thr Pro Gly 610 615 620 Gly Gly Thr Leu Leu Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly 625 630 635 640 Gly Ser Gly Gly Gly Gly Ser Gly Val Gly Leu Gly Gly Pro Gly Ala 645 650 655 Gly Leu Val Ala Pro Gly Ala Ser Val Leu Leu Ser Cys Leu Ala Ser 660 665 670 Gly Thr Thr Pro Thr Ser Thr Thr Met Ala Thr Val Leu Gly Ala Pro 675 680 685 Ala Gly Gly Leu Gly Thr Ile Gly Ala Ile Ala Pro Thr Ala Ser Gly 690 695 700 Thr His Thr Ala Gly Leu Pro Leu Ala Leu Ala Ile Leu Thr Val Ala 705 710 715 720 Leu Ser Ser Ser Thr Ala Thr Met Gly Leu Ser Ser Leu Thr Ser Gly 725 730 735 Ala Ser Ala Val Thr Thr Cys Ala Ala Gly Ala Thr Ala Ala Thr Thr 740 745 750 Gly Gly Gly Thr Thr Leu Thr Val Ser Ser Thr Thr Thr Pro Ala Pro 755 760 765 Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu 770 775 780 Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala 785 790 795 800 Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly 805 810 815 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Ala 820 825 830 Ser Leu Ala Ser Ala Leu Leu His Ser Ala Thr Met Ala Met Thr Pro 835 840 845 Ala Ala Pro Gly Pro Thr Ala Leu His Thr Gly Pro Thr Ala Pro Pro 850 855 860 Ala Ala Pro Ala Ala Thr Ala Ser Ala Val Leu Pro Ser Ala Ser Ala 865 870 875 880 Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu 885 890 895 Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly 900 905 910 Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly 915 920 925 Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr 930 935 940 Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala 945 950 955 960 Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala 965 970 975 Leu His Met Gly Ala Leu Pro Pro Ala 980 985 <210> 12 <211> 2974 <212> DNA <213> Artificial Sequence <400> 12 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc gcagatcgtg ctgacccaga gccccgccat catgagcgcc agccccggcg 120 agaaggtgac catcacctgc agcgccagca gcagcatcag ctacatgcac tggttccagc 180 agaagcccgg caccagcccc aagctgtgga tctacaccac cagcaacctg gccagcggcg 240 tgcccgccag attcagcggc agcggcagcg gcaccagcta cagcctgacc atcagcagaa 300 tggaggccga ggacgccgcc acctactact gccaccagag aagcacctac cccctgacct 360 tcggcagcgg caccaagctg gagctgaagg gagggggggg atccggggga ggaggctccg 420 gcggaggcgg aagccaggtg cagctgcagc agagcggcgc cgagctggcc aagcccggcg 480 ccagcgtgaa gatgagctgc aaggccagcg gctacacctt caccagctac agaatgcact 540 gggtgaagca gagacccggc cagggcctgg agtggatcgg ctacatcaac cccagcaccg 600 gctacaccga gtacaaccag aagttcaagg acaaggccac cctgaccgcc gacaagagca 660 gcagcaccgc ctacatgcag ctgagcagcc tgaccttcga ggacagcgcc gtgtactact 720 gcgccagagg cggcggcgtg ttcgactact ggggccaggg caccaccctg accgtgagca 780 gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc 840 tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac acgagggggc 900 tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc 960 tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc ctgtatatat 1020 tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc 1080 gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc aggagcgcag 1140 acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat ctaggacgaa 1200 gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg gggggaaagc 1260 cgcagagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa gataagatgg 1320 cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag gggcacgatg 1380 gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt cacatgcagg 1440 ccctgccccc tcgcggaagc ggagctacta acttcagcct gctgaagcag gctggagacg 1500 tggaggagaa ccctggacct atggccttac cagtgaccgc cttgctcctg ccgctggcct 1560 tgctgctcca cgccgccagg ccggacgtgc agatcaccca gagccccagc tacctggccg 1620 ccagccccgg cgagaccatc accatcaact gcagagccag caagagcatc agcaaggacc 1680 tggcctggta ccaggagaag cccggcaaga ccaacaagct gctgatctac agcggcagca 1740 ccctgcagag cggcatcccc agcagattca gcggcagcgg cagcggcacc gacttcaccc 1800 tgaccatcag cagcctggag cccgaggact tcgccatgta ctactgccag cagcacaaca 1860 agtaccccta caccttcggc ggcggcacca agctggagat caagggaggg gggggatccg 1920 ggggaggagg ctccggcgga ggcggaagcc aggtgcagct gcagcagccc ggcgccgagc 1980 tggtgagacc cggcgccagc gtgaagctga gctgcaaggc cagcggctac accttcacca 2040 gctactggat gaactgggtg aagcagagac ccgaccaggg cctggagtgg atcggcagaa 2100 tcgaccccta cgacagcgag acccactaca accagaagtt caaggacaag gccatcctga 2160 ccgtggacaa gagcagcagc accgcctaca tgcagctgag cagcctgacc agcgaggaca 2220 gcgccgtgta ctactgcgcc agaggcaact gggacgacta ctggggccag ggcaccaccc 2280 tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg 2340 tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg cccaccatcg 2340 cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc 2400 cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc 2400 acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg gccgggactt 2460 acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg gccgggactt 2460 gtggggtcct tctcctgtca ctggttatca ccctttactg caggagtaag aggagcaggc 2520 gtggggtcct tctcctgtca ctggttatca ccctttactg caggagtaag aggagcaggc 2520 tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc cgcaagcatt 2580 tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc cgcaagcatt 2580 accagcccta tgccccacca cgcgacttcg cagcctatcg ctccagagtg aagttcagca 2640 accagcccta tgccccacca cgcgacttcg cagcctatcg ctccagagtg aagttcagca 2640 ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 2700 ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 2700 taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 2760 taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 2760 ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 2820 ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 2820 ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg 2880 ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg 2880 ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc 2940 ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc 2940 acatgcaggc cctgccccct cgctaagttt aaac 2974 acatgcaggc cctgccccct cgctaagttt aaac 2974 <210> 13 <210> 13 <211> 546 <211> 546 <212> PRT <213> Artificial Sequence <400> 13 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ser Ala Ile Val Leu Thr Gly Ser Pro Ala Ser 20 25 30 Leu Ala Val Ser Leu Gly Gly Ala Ala Thr Ile Ser Cys Ala Ala Ser 35 40 45 Leu Ser Val Ser Thr Ser Gly Thr Ser Thr Leu His Thr Thr Gly Gly 50 55 60 Leu Pro Gly Gly Pro Pro Leu Leu Leu Ile Thr Leu Ala Ser Ala Leu 65 70 75 80 Gly Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala 85 90 95 Pro Thr Leu Ala Ile His Pro Val Gly Gly Gly Ala Ala Ala Thr Thr 100 105 110 Thr Cys Gly His Ser Ala Gly Leu Pro Pro Thr Pro Gly Ser Gly Thr 115 120 125 Leu Leu Gly Ile Leu Leu Ile Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly 165 170 175 Gly Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly Pro Ala Pro Ser Ala 180 185 190 Thr Thr Met Ser Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr 195 200 205 Ile Gly Gly Ile Ala Pro Thr Ser Ser Thr Ile Ala Pro Thr Pro Ser 210 215 220 Leu Leu Ala Leu Val Pro Ile Ser Ala Ala Ala Ala Leu Ala Thr Leu 225 230 235 240 Thr Leu Gly Met Ser Leu Val Ala Ser Gly Ala Thr Ala Leu Thr Thr 245 250 255 Cys Ala Ala Gly Ala Thr Thr Ala Thr Gly Ala Ala Met Ala Thr Thr 260 265 270 Gly Gly Gly Thr Ser Val Thr Val Ser Thr Thr Thr Pro Ala Pro Ala 275 280 285 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala 290 295 300 Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly 305 310 315 320 Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr 325 330 335 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Ala Ser 340 345 350 Leu Ala Ser Ala Leu Leu His Ser Ala Thr Met Ala Met Thr Pro Ala 355 360 365 Ala Pro Gly Pro Thr Ala Leu His Thr Gly Pro Thr Ala Pro Pro Ala 370 375 380 Ala Pro Ala Ala Thr Ala Ser Leu Ala Gly Ala Leu Leu Leu Leu Thr 385 390 395 400 Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly Thr Thr Gly Gly Gly 405 410 415 Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly Gly Gly Gly Cys Gly 420 425 430 Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly 435 440 445 Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly 450 455 460 Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly 465 470 475 480 Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu 485 490 495 Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly 500 505 510 Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser 515 520 525 Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro 530 535 540 Pro Ala 545 <210> 14 <211> 1657 <212> DNA <213> Artificial Sequence <400> 14 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc gagcgacatc gtgctgaccc agagccccgc cagcctggcc gtgagcctgg 120 gccagagagc caccatcagc tgcagagcca gcaagagcgt gagcaccagc ggctacagct 180 acctgcactg gtaccagcag aagcccggcc agccccccaa gctgctgatc tacctggcca 240 gcaacctgga gagcggcgtg cccgccagat tcagcggcag cggcagcggc accgacttca 300 ccctgaacat ccaccccgtg gaggaggagg acgccgccac ctactactgc cagcacagca 360 gagagctgcc cttcaccttc ggcagcggca ccaagctgga gatcaagaag atcagcggcg 420 gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cggcggcggc ggcagcggcg 480 gcggcggcag ccaggtgcag ctggtggaga gcggcggcgg cctggtgcag cccggcggca 540 gcctgaagct gagctgcgcc gccagcggct tcgacttcag cagatactgg atgagctggg 600 tgagacaggc ccccggcaag ggcctggagt ggatcggcga gatcaacccc accagcagca 660 ccatcaactt cacccccagc ctgaaggaca aggtgttcat cagcagagac aacgccaaga 720 acaccctgta cctgcagatg agcaaggtga gaagcgagga caccgccctg tactactgcg 780 ccagaggcaa ctactacaga tacggcgacg ccatggacta ctggggccag ggcaccagcg 840 tgaccgtgag caccacgacg ccagcgccgc gaccaccaac accggcgccc accatcgcgt 900 cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc gcagtgcaca 960 cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc gggacttgtg 1020 gggtccttct cctgtcactg gttatcaccc tttactgcag gagtaagagg agcaggctcc 1080 tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc aagcattacc 1140 agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc agaaagaaac 1200 tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg 1260 gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga gtgaagttca 1320 gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca 1380 atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga 1440 tggggggaaa gccgcagaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga 1500 aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca 1560 aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc 1620 ttcacatgca ggccctgccc cctcgctaag tttaaac 1657 <210> 15 <211> 490 <212> PRT <213> Artificial Sequence <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Ile Val Leu Thr Gly Ser Pro Ala Ser Leu 20 25 30 Ala Val Ser Leu Gly Gly Ala Ala Thr Ile Ser Cys Ala Ala Ser Leu 35 40 45 Ser Val Ser Thr Ser Gly Thr Ser Thr Leu His Thr Thr Gly Gly Leu 50 55 60 Pro Gly Gly Pro Pro Leu Leu Leu Ile Thr Leu Ala Ser Ala Leu Gly 65 70 75 80 Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro 85 90 95 Thr Leu Ala Ile His Pro Val Gly Gly Gly Ala Ala Ala Thr Thr Thr 100 105 110 Cys Gly His Ser Ala Gly Leu Pro Pro Thr Pro Gly Ser Gly Thr Leu 115 120 125 Leu Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly 145 150 155 160 Pro Gly Gly Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly Pro Ala Pro 165 170 175 Ser Ala Thr Thr Met Ser Thr Val Ala Gly Ala Pro Gly Leu Gly Leu 180 185 190 Gly Thr Ile Gly Gly Ile Ala Pro Thr Ser Ser Thr Ile Ala Pro Thr 195 200 205 Pro Ser Leu Leu Ala Leu Val Pro Ile Ser Ala Ala Ala Ala Leu Ala 210 215 220 Thr Leu Thr Leu Gly Met Ser Leu Val Ala Ser Gly Ala Thr Ala Leu 225 230 235 240 Thr Thr Cys Ala Ala Gly Ala Thr Thr Ala Thr Gly Ala Ala Met Ala 245 250 255 Thr Thr Gly Gly Gly Thr Ser Val Thr Val Ser Thr Thr Thr Pro Ala 260 265 270 Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser 275 280 285 Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys 325 330 335 Ala Ser Leu Ala Ser Ala Leu Leu His Ser Ala Thr Met Ala Met Thr 340 345 350 Pro Ala Ala Pro Gly Pro Thr Ala Leu His Thr Gly Pro Thr Ala Pro 355 360 365 Pro Ala Ala Pro Ala Ala Thr Ala Ser Ala Val Leu Pro Ser Ala Ser 370 375 380 Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly 385 390 395 400 Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala 405 410 415 Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro 420 425 430 Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala 435 440 445 Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His 450 455 460 Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala 465 470 475 480 Ala Leu His Met Gly Ala Leu Pro Pro Ala 485 490 <210> 16 <211> 1489 <212> DNA <213> Artificial Sequence <400> 16 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatcgtg ctgacccaga gccccgccag cctggccgtg agcctgggcc 120 agagggccac catcagctgc agggccagca agagcgtgag caccagcggc tacagctacc 180 tgcactggta ccagcagaag cccggccagc cccccaagct gctgatctac ctggccagca 240 acctggagag cggcgtgccc gccaggttca gcggcagcgg cagcggcacc gacttcaccc 300 tgaacatcca ccccgtggag gaggaggacg ccgccaccta ctactgccag cacagcaggg 360 agctgccctt caccttcggc agcggcacca agctggagat caagggaggg gggggatccg 420 ggggaggagg ctccggcgga ggcggaagcc aggtgcagct ggtggagagc ggcggcggcc 480 tggtgcagcc cggcggcagc ctgaagctga gctgcgccgc cagcggcttc gacttcagca 540 ggtactggat gagctgggtg aggcaggccc ccggcaaggg cctggagtgg atcggcgaga 600 tcaaccccac cagcagcacc atcaacttca cccccagcct gaaggacaag gtgttcatca 660 gcagggacaa cgccaagaac accctgtacc tgcagatgag caaggtgagg agcgaggaca 720 ccgccctgta ctactgcgcc aggggcaact actacaggta cggcgacgcc atggactact 780 ggggccaggg caccagcgtg accgtgagca ccacgacgcc agcgccgcga ccaccaacac 840 cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 900 cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc 960 ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcagga 1020 gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc 1080 ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca 1140 gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct 1200 ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc 1260 gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca 1320 atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg aaaggcgagc 1380 gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc accaaggaca 1440 cctacgacgc ccttcacatg caggccctgc cccctcgcta agtttaaac 1489 <210> 17 <211> 491 <212> PRT <213> Artificial Sequence <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Ile Val Leu Thr Gly Ser Pro Ala Ser Leu 20 25 30 Ala Val Ser Leu Gly Gly Ala Ala Thr Ile Ser Cys Ala Ala Ser Leu 35 40 45 Ser Val Ser Thr Ser Gly Thr Ser Thr Leu His Thr Thr Gly Gly Leu 50 55 60 Pro Gly Gly Pro Pro Leu Leu Leu Ile Thr Leu Ala Ser Ala Leu Gly 65 70 75 80 Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro 85 90 95 Thr Leu Ala Ile His Pro Val Gly Gly Gly Ala Ala Ala Thr Thr Thr 100 105 110 Cys Gly His Ser Ala Gly Leu Pro Pro Thr Pro Gly Ser Gly Thr Leu 115 120 125 Leu Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly 145 150 155 160 Pro Gly Gly Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly Pro Ala Pro 165 170 175 Ser Ala Thr Thr Met Ser Thr Val Ala Gly Ala Pro Gly Leu Gly Leu 180 185 190 Gly Thr Ile Gly Gly Ile Ala Pro Thr Ser Ser Thr Ile Ala Pro Thr 195 200 205 Pro Ser Leu Leu Ala Leu Val Pro Ile Ser Ala Ala Ala Ala Leu Ala 210 215 220 Thr Leu Thr Leu Gly Met Ser Leu Val Ala Ser Gly Ala Thr Ala Leu 225 230 235 240 Thr Thr Cys Ala Ala Gly Ala Thr Thr Ala Thr Gly Ala Ala Met Ala 245 250 255 Thr Thr Gly Gly Gly Thr Ser Val Thr Val Ser Thr Thr Thr Pro Ala 260 265 270 Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser 275 280 285 Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys 325 330 335 Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met 340 345 350 Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro 355 360 365 Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala 370 375 380 Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala 385 390 395 400 Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala 405 410 415 Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala 420 425 430 Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly 435 440 445 Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly 450 455 460 His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr 465 470 475 480 Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala 485 490 <210> 18 <211> 1492 <212> DNA <213> Artificial Sequence <400> 18 gcgatcgcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 60 ccgccaggcc ggacatcgtg ctgacccaga gccccgccag cctggccgtg agcctgggcc 120 agagggccac catcagctgc agggccagca agagcgtgag caccagcggc tacagctacc 180 tgcactggta ccagcagaag cccggccagc cccccaagct gctgatctac ctggccagca 240 acctggagag cggcgtgccc gccaggttca gcggcagcgg cagcggcacc gacttcaccc 300 tgaacatcca ccccgtggag gaggaggacg ccgccaccta ctactgccag cacagcaggg 360 agctgccctt caccttcggc agcggcacca agctggagat caagggaggg gggggatccg 420 ggggaggagg ctccggcgga ggcggaagcc aggtgcagct ggtggagagc ggcggcggcc 480 tggtgcagcc cggcggcagc ctgaagctga gctgcgccgc cagcggcttc gacttcagca 540 ggtactggat gagctgggtg aggcaggccc ccggcaaggg cctggagtgg atcggcgaga 600 tcaaccccac cagcagcacc atcaacttca cccccagcct gaaggacaag gtgttcatca 660 gcagggacaa cgccaagaac accctgtacc tgcagatgag caaggtgagg agcgaggaca 720 ccgccctgta ctactgcgcc aggggcaact actacaggta cggcgacgcc atggactact 780 ggggccaggg caccagcgtg accgtgagca ccacgacgcc agcgccgcga ccaccaacac 840 cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 900 cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac atctgggcgc 960 ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt tactgcaaac 1020 ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca gtacaaacta 1080 ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga ggatgtgaac 1140 tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc cagaaccagc 1200 tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac aagagacgtg 1260 gccgggaccc tgagatgggg ggaaagccgc agagaaggaa gaaccctcag gaaggcctgt 1320 acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg 1380 agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg 1440 acacctacga cgcccttcac atgcaggccc tgccccctcg ctaagtttaa ac 1492 <210> 19 <211> 997 <212> PRT <213> Artificial Sequence <400> 19 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Val Val Met Thr Gly Ser His Ala Pro Met 20 25 30 Ser Thr Ser Val Gly Ala Ala Val Ser Ile Thr Cys Ala Ala Ser Gly 35 40 45 Ala Val Ala Thr Ala Val Ser Thr Thr Gly Gly Leu Pro Gly Gly Ser 50 55 60 Pro Leu Leu Leu Ile Pro Ser Ala Ser Thr Ala Thr Thr Gly Val Pro 65 70 75 80 Ala Ala Pro Thr Gly Ser Gly Ser Gly Ala Ala Pro Thr Leu Thr Ile 85 90 95 Ser Ser Val Gly Ala Gly Ala Leu Ala Val Thr Thr Cys Gly Gly His 100 105 110 Thr Ser Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Leu 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Ile Gly Leu Val Gly Ser Gly Pro Ala Leu Leu Leu Pro Gly Gly Thr 145 150 155 160 Val Leu Leu Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ala Pro Gly 165 170 175 Met Ala Thr Val Leu Gly Ala Pro Gly Leu Gly Pro Leu Thr Met Ala 180 185 190 Thr Ile Ala Thr Thr Ala Thr Thr Gly Gly Ser Thr Pro Ala Ala Ala 195 200 205 Pro Leu Gly Ala Pro Ala Pro Ser Val Gly Thr Ser Ala Thr Thr Ala 210 215 220 Thr Leu Gly Ile Ala Ala Leu Leu Thr Gly Ala Thr Ala Thr Thr Pro 225 230 235 240 Cys Ala Ala Gly Gly Ile Thr Thr Gly Thr Ala Gly Gly Pro Ala Thr 245 250 255 Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala 260 265 270 Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser 275 280 285 Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys 325 330 335 Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met 340 345 350 Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro 355 360 365 Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala 370 375 380 Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala 385 390 395 400 Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala 405 410 415 Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala 420 425 430 Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly 435 440 445 Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly 450 455 460 His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr 465 470 475 480 Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr 485 490 495 Ala Pro Ser Leu Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly 500 505 510 Pro Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 515 520 525 His Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Ser Pro Ser Ser Leu 530 535 540 Ser Ala Ser Val Gly Ala Ala Val Thr Ile Thr Cys Leu Ala Ser Gly 545 550 555 560 Ala Val Gly Ile Ala Val Ala Thr Thr Gly Gly Leu Pro Gly Leu Val 565 570 575 Pro Leu Leu Leu Ile Thr Thr Ala Ser Thr Ala His Thr Gly Val Pro 580 585 590 Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile 595 600 605 Ser Ser Leu Gly Pro Gly Ala Val Ala Thr Thr Thr Cys Gly Gly Thr 610 615 620 Ser Ser Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Val Gly Ile Leu 625 630 635 640 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 645 650 655 Val Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser 660 665 670 Leu Ala Leu Ser Cys Ala Ala Ser Gly Pro Ala Pro Ser Ala Thr Thr 675 680 685 Met Ser Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Ile Gly 690 695 700 Gly Ile Ala Pro Ala Ser Ser Thr Ile Ala Thr Ala Pro Ser Leu Leu 705 710 715 720 Ala Leu Pro Ile Ile Ser Ala Ala Ala Ala Leu Ala Ser Leu Thr Leu 725 730 735 Gly Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys Ala 740 745 750 Ala Pro Ala Gly Ala Thr Thr Thr Pro Ala Val Thr Gly Gly Gly Thr 755 760 765 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr 770 775 780 Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala 785 790 795 800 Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro 805 810 815 Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val 820 825 830 Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Ala Ser Leu Ala Ser 835 840 845 Ala Leu Leu His Ser Ala Thr Met Ala Met Thr Pro Ala Ala Pro Gly 850 855 860 Pro Thr Ala Leu His Thr Gly Pro Thr Ala Pro Pro Ala Ala Pro Ala 865 870 875 880 Ala Thr Ala Ser Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala 885 890 895 Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala 900 905 910 Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly 915 920 925 Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr 930 935 940 Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly 945 950 955 960 Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly 965 970 975 Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly 980 985 990 Ala Leu Pro Pro Ala 995 <210> 20 <211> 2994 <212> DNA <213> Artificial Sequence <400> 20 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggatgtgg tgatgaccca gagccatcgc tttatgagca ccagcgtggg cgatcgcgtg 120 agcattacct gccgcgcgag ccaggatgtg aacaccgcgg tgagctggta tcagcagaaa 180 ccgggccaga gcccgaaact gctgattttt agcgcgagct atcgctatac cggcgtgccg 240 gatcgcttta ccggcagcgg cagcggcgcg gattttaccc tgaccattag cagcgtgcag 300 gcggaagatc tggcggtgta ttattgccag cagcattata gcaccccgtg gacctttggc 360 ggcggcacca aactggaaat taaaggaggg gggggatccg ggggaggagg ctccggcgga 420 ggcggaagcc agattcagct ggtgcagagc ggcccggatc tgaaaaaacc gggcgaaacc 480 gtgaaactga gctgcaaagc gagcggctat acctttacca actttggcat gaactgggtg 540 aaacaggcgc cgggcaaagg ctttaaatgg atggcgtgga ttaacaccac ccgctatacc 600 ggcgaaagct attttgcgga tgattttaaa ggccgctttg cgtttagcgt ggaaaccagc 660 gcgaccaccg cgtatctgca gattaacaac ctgaaaaccg aagataccgc gacctatttt 720 tgcgcgcgcg gcgaaattta ttatggctat gatggcggct ttgcgtattg gggccagggc 780 accctggtga ccgtgagcgc gaccacgacg ccagcgccgc gaccaccaac accggcgccc 840 accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900 gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960 gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020 aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080 gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140 gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140 aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200 aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200 gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260 gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260 cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 1320 cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 1320 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440 gacgcccttc acatgcaggc cctgccccct cgcggaagcg gagccaccaa cttcagcctg 1500 gacgcccttc acatgcaggc cctgccccct cgcggaagcg gagccaccaa cttcagcctg 1500 ctgaagcagg ccggcgacgt ggaggagaac cccggccccg ccttaccagt gaccgccttg 1560 ctgaagcagg ccggcgacgt ggaggagaac cccggccccg ccttaccagt gaccgccttg 1560 ctcctgccgc tggccttgct gctccacgcc gccaggccgg atattcagat gacccagagc 1620 ctcctgccgc tggccttgct gctccacgcc gccaggccgg atattcagat gacccagagc 1620 ccgagcagcc tgagcgcgag cgtgggcgat cgcgtgacca ttacctgcaa agcgagccag 1680 ccgagcagcc tgagcgcgag cgtgggcgat cgcgtgacca ttacctgcaa agcgagccag 1680 gatgtgggca ttgcggtggc gtggtatcag cagaaaccgg gcaaagtgcc gaaactgctg 1740 gatgtgggca ttgcggtggc gtggtatcag cagaaaccgg gcaaagtgcc gaaactgctg 1740 atttattggg cgagcacccg ccataccggc gtgccggatc gctttagcgg cagcggcagc 1800 atttattggg cgagcacccg ccataccggc gtgccggatc gctttagcgg cagcggcagc 1800 ggcaccgatt ttaccctgac cattagcagc ctgcagccgg aagatgtggc gacctattat 1860 tgccagcagt atagcagcta tccgtatacc tttggccagg gcaccaaagt ggaaattaaa 1920 ggaggggggg gatccggggg aggaggctcc ggcggaggcg gaagcgaagt gcagctggtg 1980 gaaagcggcg gcggcctggt gcagccgggc ggcagcctgc gcctgagctg cgcggcgagc 2040 ggctttgatt ttagccgcta ttggatgagc tgggtgcgcc aggcgccggg caaaggcctg 2100 gaatggattg gcgaaattaa cccggatagc agcaccatta actatgcgcc gagcctgaaa 2160 gataaattta ttattagccg cgataacgcg aaaaacagcc tgtatctgca gatgaacagc 2220 ctgcgcgcgg aagataccgc ggtgtattat tgcgcgcgcc cggatggcaa ctattggtat 2280 tttgatgtgt ggggccaggg caccctggtg accgtgagca gcaccacgac gccagcgccg 2340 cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 2400 tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 2460 tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 2520 ctttactgca ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 2580 cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 2640 gcctatcgct ccagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 2700 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 2760 aagagacgtg gccgggaccc tgagatgggg ggaaagccgc agagaaggaa gaaccctcag 2820 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 2880 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 2940 gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa 2994 <210> 21 <211> 997 <212> PRT <213> Artificial Sequence <400> 21 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Ala Pro Ala Val Val Met Thr Gly Ser His Ala Pro Met 20 25 30 Ser Thr Ser Val Gly Ala Ala Val Ser Ile Thr Cys Ala Ala Ser Gly 35 40 45 Ala Val Ala Thr Ala Val Ser Thr Thr Gly Gly Leu Pro Gly Gly Ser 50 55 60 Pro Leu Leu Leu Ile Pro Ser Ala Ser Thr Ala Thr Thr Gly Val Pro 65 70 75 80 Ala Ala Pro Thr Gly Ser Gly Ser Gly Ala Ala Pro Thr Leu Thr Ile 85 90 95 Ser Ser Val Gly Ala Gly Ala Leu Ala Val Thr Thr Cys Gly Gly His 100 105 110 Thr Ser Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Ala Ile Leu 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Ile Gly Leu Val Gly Ser Gly Pro Ala Leu Leu Leu Pro Gly Gly Thr 145 150 155 160 Val Leu Leu Ser Cys Leu Ala Ser Gly Thr Thr Pro Thr Ala Pro Gly 165 170 175 Met Ala Thr Val Leu Gly Ala Pro Gly Leu Gly Pro Leu Thr Met Ala 180 185 190 Thr Ile Ala Thr Thr Thr Gly Gly Ser Thr Pro Ala Ala Ala Pro Leu 195 200 205 Gly Ala Pro Ala Pro Ser Val Gly Thr Ser Ala Thr Thr Ala Thr Leu 210 215 220 Gly Ile Ala Ala Leu Leu Thr Gly Ala Thr Ala Thr Thr Pro Cys Ala 225 230 235 240 Ala Gly Gly Ile Thr Thr Gly Thr Ala Gly Gly Pro Ala Thr Thr Gly 245 250 255 Gly Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Ala 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala 275 280 285 Pro Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly 290 295 300 Leu Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu Ala 325 330 335 Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro 340 345 350 Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly 355 360 365 Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala 370 375 380 Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu 385 390 395 400 Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly 405 410 415 Ala Ala Pro Gly Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly 420 425 430 Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr 435 440 445 Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala 450 455 460 Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala 465 470 475 480 Leu His Met Gly Ala Leu Pro Pro Ala Gly Ser Gly Ala Thr Ala Pro 485 490 495 Ser Leu Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly Pro Met 500 505 510 Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His 515 520 525 Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Ser Pro Ser Ser Leu Ser 530 535 540 Ala Ser Val Gly Ala Ala Val Thr Ile Thr Cys Leu Ala Ser Gly Ala 545 550 555 560 Val Gly Ile Ala Val Ala Thr Thr Gly Gly Leu Pro Gly Leu Val Pro 565 570 575 Leu Leu Leu Ile Thr Thr Ala Ser Thr Ala His Thr Gly Val Pro Ala 580 585 590 Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile Ser 595 600 605 Ser Leu Gly Pro Gly Ala Val Ala Thr Thr Thr Cys Gly Gly Thr Ser 610 615 620 Ser Thr Pro Thr Thr Pro Gly Gly Gly Thr Leu Val Gly Ile Leu Gly 625 630 635 640 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val 645 650 655 Gly Leu Val Gly Ser Gly Gly Gly Leu Val Gly Pro Gly Gly Ser Leu 660 665 670 Ala Leu Ser Cys Ala Ala Ser Gly Pro Ala Pro Ser Ala Thr Thr Met 675 680 685 Ser Thr Val Ala Gly Ala Pro Gly Leu Gly Leu Gly Thr Ile Gly Gly 690 695 700 Ile Ala Pro Ala Ser Ser Thr Ile Ala Thr Ala Pro Ser Leu Leu Ala 705 710 715 720 Leu Pro Ile Ile Ser Ala Ala Ala Ala Leu Ala Ser Leu Thr Leu Gly 725 730 735 Met Ala Ser Leu Ala Ala Gly Ala Thr Ala Val Thr Thr Cys Ala Ala 740 745 750 Pro Ala Gly Ala Thr Thr Thr Pro Ala Val Thr Gly Gly Gly Thr Leu 755 760 765 Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro 770 775 780 Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys 785 790 795 800 Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala 805 810 815 Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 820 825 830 Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Leu Ala Gly Ala Leu Leu 835 840 845 Leu Leu Thr Ile Pro Leu Gly Pro Pro Met Ala Pro Val Gly Thr Thr 850 855 860 Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro Pro Gly Gly Gly Gly Gly 865 870 875 880 Gly Cys Gly Leu Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala 885 890 895 Thr Gly Gly Gly Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala 900 905 910 Ala Gly Gly Thr Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly 915 920 925 Met Gly Gly Leu Pro Gly Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr 930 935 940 Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly 945 950 955 960 Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly 965 970 975 Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly 980 985 990 Ala Leu Pro Pro Ala 995 <210> 22 <211> 3009 <212> DNA <213> Artificial Sequence <400> 22 cgatcgcatg gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc 60 cgccaggccg gatgtggtga tgacccagag ccatcgcttt atgagcacca gcgtgggaga 120 tcgagtgagc attacctgcc gcgcgagcca ggatgtgaac accgcggtga gctggtatca 180 gcagaaaccg ggccagagcc cgaaactgct gatttttagc gcgagctatc gctataccgg 240 cgtgccggat cgctttaccg gcagcggcag cggcgcggat tttaccctga ccattagcag 300 cgtgcaggcg gaagatctgg cggtgtatta ttgccagcag cattatagca ccccgtggac 360 ctttggcggc ggcaccaaac tggatattaa aggagggggg ggatccgggg gaggaggctc 420 cggcggaggc ggaagccaga ttcagctggt gcagagcggc ccggatctga aaaaaccggg 480 cgaaaccgtg aaactgagct gcaaagcgag cggctatacc tttaccaact ttggcatgaa 540 ctgggtgaaa caggcgccgg gcaaaggctt taaatggatg gcgtggatta acacctatac 600 cggcgaaagc tattttgcgg atgattttaa aggccgcttt gcgtttagcg tggaaaccag 660 cgcgaccacc gcgtatctgc agattaacaa cctgaaaacc gaagataccg cgacctattt 720 ttgcgcgcgc ggcgaaattt attatggcta tgatggcggc tttgcgtatt ggggccaggg 780 caccctggtg accgtgagcg cgaccacgac gccagcgccg cgaccaccaa caccggcgcc 840 caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg 900 cgcagtgcac acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc 960 cgggacttgt ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag 1020 aaagaaactc ctgtatatat tcaagcaacc atttatgaga ccagtacaaa ctactcaaga 1080 ggaagatggc tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt 1140 gaagttcagc aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa 1200 cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga 1260 ccctgagatg gggggaaagc cgcagagaag gaagaaccct caggaaggcc tgtacaatga 1320 actgcagaaa gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg 1380 gaggggcaag gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta 1440 cgacgccctt cacatgcagg ccctgccccc tcgcggaagc ggagccacca acttcagcct 1500 gctgaagcag gccggcgacg tggaggagaa ccccggcccc atggccttac cagtgaccgc 1560 cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccggatattc agatgaccca 1620 gagcccgagc agcctgagcg cgagcgtggg cgaccgcgtg accattacct gcaaagcgag 1680 ccaggatgtg ggcattgcgg tggcgtggta tcagcagaaa ccgggcaaag tgccgaaact 1740 gctgatttat tgggcgagca cccgccatac cggcgtgccg gatcgcttta gcggcagcgg 1800 cagcggcacc gattttaccc tgaccattag cagcctgcag ccggaagatg tggcgaccta 1860 ttattgccag cagtatagca gctatccgta tacctttggc cagggcacca aagtggaaat 1920 taaaggaggg gggggatccg ggggaggagg ctccggcgga ggcggaagcg aagtgcagct 1980 ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc ctgcgcctga gctgcgcggc 2040 gagcggcttt gattttagcc gctattggat gagctgggtg cgccaggcgc cgggcaaagg 2100 cctggaatgg attggcgaaa ttaacccgga tagcagcacc attaactatg cgccgagcct 2160 gaaagataaa tttattatta gccgcgataa cgcgaaaaac agcctgtatc tgcagatgaa 2220 cagcctgcgc gcggaagata ccgcggtgta ttattgcgcg cgcccggatg gcaactattg 2280 gtattttgat gtgtggggcc agggcaccct ggtgaccgtg agcagcacca cgacgccagc 2340 gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga 2400 ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga 2460 tatctacatc tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat 2520 caccctttac tgcaaacggg gcagaaagaa actcctgtat atattcaagc aaccatttat 2580 gagaccagta caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga 2640 agaaggagga tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca 2700 gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt 2760 tttggacaag agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa 2820 ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga 2880 gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct 2940 cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta 3000 agtttaaac 3009 <210> 23 <211> 423 <212> DNA <213> Artificial Sequence <400> 23 agctagctgc agtaacgcca ttttgcaagg catggaaaaa taccaaacca agaatagaga 60 agttcagatc aagggcgggt acatgaaaat agctaacgtt gggccaaaca ggatatctgc 120 ggtgagcagt ttcggccccg gcccggggcc aagaacagat ggtcaccgca gtttcggccc 180 cggcccgagg ccaagaacag atggtcccca gatatggccc aaccctcagc agtttcttaa 240 gacccatcag atgtttccag gctcccccaa ggacctgaaa tgaccctgcg ccttatttga 300 attaaccaat cagcctgctt ctcgcttctg ttcgcgcgct tctgcttccc gagctctata 360 aaagagctca caacccctca ctcggcgcgc cagtcctccg acagactgag tcgcccgggt 420 acc 423 <210> 24 <211> 165 <212> PRT <213> Artificial Sequence <400> 24 Met Ser Gly Leu Gly Ala Ser Ala Ala Gly Gly Ala Ser Ala Val Ala 1 5 10 15 Gly Gly Gly Ala Pro Pro Gly Gly Leu Thr Thr Gly Val Ala Met Ala 20 25 30 Ser Cys Pro Gly Gly Gly Thr Thr Ala Pro Leu Leu Gly Thr Cys Met 35 40 45 Ser Cys Leu Thr Ile Cys Ala His Gly Ser Gly Ala Thr Cys Ala Ala 50 55 60 Pro Cys Ala Ser Leu Ser Cys Ala Leu Gly Gly Gly Leu Pro Thr Ala 65 70 75 80 His Leu Leu Ala Ala Cys Ile Ser Cys Ala Ser Ile Cys Gly Gly His 85 90 95 Pro Leu Gly Cys Ala Thr Pro Cys Gly Ala Leu Leu Ala Ser Pro Val 100 105 110 Ala Leu Pro Pro Gly Leu Ala Ala Gly Ala Ser Gly Gly Val Gly Ala 115 120 125 Ala Ser Ala Ala Ser Gly Ala Thr Gly Gly Leu Gly His Ala Gly Ser 130 135 140 Gly Ala Ser Pro Ala Leu Pro Gly Leu Leu Leu Ser Ala Ala Gly Val 145 150 155 160 Ala Leu Val Thr Ser 165 <210> 25 <211> 54 <212> PRT <213> Artificial Sequence <400> 25 Met Leu Gly Met Ala Gly Gly Cys Ser Gly Ala Gly Thr Pro Ala Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gly Leu Ala Cys Ser Ser Ala Thr 20 25 30 Pro Pro Leu Thr Cys Gly Ala Thr Cys Ala Ala Ser Val Thr Ala Ser 35 40 45 Val Leu Gly Thr Ala Ala 50 <210> 26 <211> 204 <212> PRT <213> Artificial Sequence <400> 26 Ser Gly Pro Val Leu Gly Leu Val Gly Ser Val Gly Gly Ala Val Thr 1 5 10 15 Pro Pro Leu Leu Ser Leu Val Leu Gly Val Ala Ser Ile Val Thr Thr 20 25 30 Pro Ala Thr Thr Pro Leu Val Thr Ile Gly Pro Gly Gly Gly Thr Ile 35 40 45 Ile Val Thr Gly Ala Ala Ala Ala Gly Ala Val Ala Pro Pro Ala Gly 50 55 60 Gly Thr Ser Leu Leu Leu Ser Leu Leu Leu Leu Ala Ala Ser Gly Ile 65 70 75 80 Thr Thr Val Gly Ile Thr Ser Ser Ser Leu Gly Gly Pro Ser Thr Gly 85 90 95 Gly Thr Val Leu His Val Thr Gly His Leu Ser Leu Pro Leu Val Thr 100 105 110 Met Gly Leu Gly Ser Ala Leu Ala Gly Thr Cys Val Thr Ala Leu Thr 115 120 125 Cys Cys Met Gly His Gly Gly Gly Ala Val Ile Thr Thr Thr Leu Ala 130 135 140 Leu Gly Gly Ala Ala Ala Gly Ser His Ala Gly Ser Ile Leu Pro Ile 145 150 155 160 Ser Thr Ala Thr Gly Gly Ser Ala Met Thr Pro Ile Cys Val Ala Ala 165 170 175 Ala Pro Val Ser Ala Ala Pro Ser Ser Pro Ile Leu Ala Ala Leu Leu 180 185 190 Cys Gly Gly Ala Ala Ala Ala Pro Ala Ser Ser Met 195 200 <210> 27 <211> 46 <212> PRT <213> Artificial Sequence <400> 27 Met Ala Ala Ser Thr Gly Ala Gly Gly Ser Ala Leu Thr Ser Cys Leu 1 5 10 15 Leu Leu Ala Gly Gly Met Leu Leu Leu Gly Cys Val Ser Ile Leu Pro 20 25 30 Ala Leu Gly Ser Pro Ser Val Ala Ser Ser Leu Ala Gly Leu 35 40 45 <210> 28 <211> 242 <212> PRT <213> Artificial Sequence <400> 28 Ala Pro Ala Pro Thr Leu Gly Val Gly Gly Ser Val Thr Val Gly Gly 1 5 10 15 Gly Leu Cys Val Leu Val Pro Cys Thr Pro Pro His Pro Ile Pro Thr 20 25 30 Thr Ala Leu Ala Ser Pro Val His Gly Thr Thr Pro Ala Gly Gly Ala 35 40 45 Ile Ile Ser Ala Ala Ser Pro Val Ala Thr Ala Leu Leu Ala Gly Gly 50 55 60 Val Gly Gly Gly Thr Gly Gly Ala Pro Ala Leu Leu Gly Ala Pro Ser 65 70 75 80 Ala Ala Ala Cys Ser Leu Ser Ile Val Ala Ala Ala Ala Ala Ala Ala 85 90 95 Gly Ser Thr Pro Pro Ala Met Gly Ala Gly Ser Thr Leu Thr Ser Thr 100 105 110 Leu Ser Pro Gly Leu Ser Val His Val Thr Ala Leu Thr His Ala Pro 115 120 125 Leu Ile Leu Ile Pro Gly Thr Leu Gly Pro Gly His Ser Leu Ala Leu 130 135 140 Thr Cys Ser Val Ser Thr Ala Cys Gly Gly Gly Thr Pro Pro Ile Pro 145 150 155 160 Ser Thr Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Ala Thr Thr His 165 170 175 Ser Ser Val Leu Ile Ile Thr Pro Ala Pro Gly Ala His Gly Thr Ala 180 185 190 Leu Thr Cys Gly Val Leu Pro Ala Gly Ala Gly Val Thr Thr Gly Ala 195 200 205 Thr Ile Gly Leu Ala Val Thr Thr Val Pro Gly Ala Pro Thr Thr Gly 210 215 220 Ile Pro Pro Gly Ala Gly Ser Gly Leu Gly Gly Thr Ala Ala Gly Val 225 230 235 240 Val His <210> 29 <211> 287 <212> PRT <213> Artificial Sequence <400> 29 Thr Leu Gly Ala Pro Ala Pro Pro Ile Thr Ala Leu Ala Met Leu Ala 1 5 10 15 Leu Ala Gly Gly Leu Thr Thr Ala Leu Ala Ala Ala Val Thr Ala Ile 20 25 30 Gly Cys Val Leu Ala Ala Ala Thr Ser Met Pro Ala Val Ala Ala Ser 35 40 45 Thr Cys Gly Pro Gly Ala Ile Ser Leu Cys Gly Val Thr Ala Thr Thr 50 55 60 Val Ala Val Ala Ala Pro Pro Pro Ser Thr Thr Ile Leu Pro Pro Gly 65 70 75 80 Ala Ser Gly Leu Pro Thr Ala Gly Ala Gly Ala Leu Thr Cys Thr Ile 85 90 95 His Ala Val Ala Pro Leu Ser Cys Ser Thr Ala Val Gly Pro Gly Ala 100 105 110 Pro Ala Ala Val Gly Thr Ala Leu Thr Leu Ala Val Ala Ala Ala Ala 115 120 125 Gly Gly Thr Gly Cys Leu His Thr Leu Thr Ala Ala Gly Gly Thr Ala 130 135 140 Ile Gly Cys Ala Pro Ala Ala Ile Ser Ala Leu Ser Ser Gly Ser Gly 145 150 155 160 Ser Ser His Ile Leu Val Ala Gly Ala Ser Ala Ala Pro Gly Ile Pro 165 170 175 Cys Thr Ala Leu Pro Val Val Pro Ser Gly Ile Gly Ile Leu Thr Pro 180 185 190 Pro Ala Met Thr Ala Leu Cys Ala Leu Thr His Ser Pro Met His Thr 195 200 205 Leu Met Ala Ser His Pro Ala Ala Leu Pro Ala Thr Gly Leu Gly Ile 210 215 220 Gly Leu Ala Met Gly Pro Val Ile Thr Gly Gly Val Ala Ala Ala Thr 225 230 235 240 Ser Pro Gly Leu Leu Ala Pro Gly Thr Thr Thr Val Gly Ile Ala Ala 245 250 255 Ala Gly Ala Val Thr Gly Pro Leu Ser Ala Thr Ser Thr Pro Gly Ala 260 265 270 Pro Gly Cys Ala Gly Gly Gly Gly Ala Ala Thr Ala Ala Thr Ala 275 280 285 <210> 30 <211> 272 <212> PRT <213> Artificial Sequence <400> 30 Pro Gly Gly Pro Leu Val Val Leu Val Gly Gly Gly Ala Ala Ala Val 1 5 10 15 Leu Gly Cys Leu Leu Gly Thr Ser Ala Gly Pro Thr Gly Gly Leu Thr 20 25 30 Thr Ser Ala Gly Ser Pro Leu Leu Pro Pro Leu Leu Leu Ser Leu Gly 35 40 45 Leu Pro Gly Leu Gly Ile His Met Ala Pro Leu Ala Ile Thr Leu Pro 50 55 60 Ile Pro Ala Val Ser Gly Gly Met Gly Gly Pro Thr Leu Cys Gly Pro 65 70 75 80 Gly Pro Pro Ser Gly Leu Ala Thr Gly Pro Gly Thr Thr Val Ala Val 85 90 95 Gly Gly Ser Gly Gly Leu Pro Ala Thr Ala Val Ser Ala Leu Gly Gly 100 105 110 Leu Gly Cys Gly Leu Leu Ala Ala Ser Ser Gly Gly Pro Ser Ser Pro 115 120 125 Ser Gly Leu Leu Met Ser Pro Leu Leu Thr Val Thr Ala Leu Ala Ala 130 135 140 Pro Gly Ile Thr Gly Gly Gly Pro Pro Cys Leu Pro Pro Ala Ala Ser 145 150 155 160 Leu Ala Gly Ser Leu Ser Gly Ala Leu Thr Met Ala Pro Gly Ser Thr 165 170 175 Leu Thr Leu Ser Cys Gly Val Pro Pro Ala Ser Val Ser Ala Gly Pro 180 185 190 Leu Ser Thr Thr His Val His Pro Leu Gly Pro Leu Ser Leu Leu Ser 195 200 205 Leu Gly Leu Leu Ala Ala Ala Pro Ala Ala Ala Met Thr Val Met Gly 210 215 220 Thr Gly Leu Leu Leu Pro Ala Ala Thr Ala Gly Ala Ala Gly Leu Thr 225 230 235 240 Thr Cys His Ala Gly Ala Leu Thr Met Ser Pro His Leu Gly Ile Thr 245 250 255 Ala Ala Pro Val Leu Thr His Thr Leu Leu Ala Thr Gly Gly Thr Leu 260 265 270 <210> 31 <211> 6 <212> PRT <213> Artificial Sequence <400> 31 Pro Ile Cys Val Thr Val 1 5 <210> 32 <211> 47 <212> PRT <213> Artificial Sequence <400> 32 Leu Ile Ser His Pro Leu Leu Met Gly Ser Leu Ala Pro Ile Ala Ala 1 5 10 15 His Thr Pro Thr Ile Ala Ile Thr Ala Cys Gly Pro Ala Ala Pro Ser 20 25 30 Gly Leu Ala Ser Pro Ser Thr Gly Thr Cys Thr Ser Ile Gly Ser 35 40 45 <210> 33 <211> 480 <212> PRT <213> Artificial Sequence <400> 33 Ala Ser Ser Leu Thr Val Pro Gly His Pro Gly Thr Leu Thr Ala Thr 1 5 10 15 Gly Gly Ala Cys Val Thr Ile Pro Cys Thr Thr Ala Ala Leu Ala Gly 20 25 30 Ala Leu Gly Ser Pro Ile Leu Pro His Ala Pro Gly Thr Ala Leu Ala 35 40 45 Thr Ser Leu Pro Ala Gly Thr Ala Leu Thr Gly Ser Thr Leu Ala Gly 50 55 60 Leu Val Pro Ser Gly Gly Leu Ala Val Gly Pro Leu Gly Ala Leu Ala 65 70 75 80 Leu Ala Cys Thr Leu Ser Ile His Pro Val His Leu Ala Ala Ser Gly 85 90 95 Gly Leu Gly Leu Ala Met Gly Ser Leu Thr Gly Leu Thr Met Gly Ala 100 105 110 Ile His Leu Ala Val Ser Gly Ala Pro Pro Pro Pro His Ile Gly Leu 115 120 125 Pro Pro Gly Ile Gly Gly Ser Gly Gly Val Thr Leu Thr Cys Leu Leu 130 135 140 Ala Pro Ser Cys Thr Gly Thr Pro Ile Gly Leu Gly Thr Leu Leu Gly 145 150 155 160 Gly Val Pro Met Ala Gly Ala Ala Val Thr Ser Thr Ser Leu Thr Ile 165 170 175 Leu Ser Val Pro Thr Ala Ser Gly Leu Leu Pro Ser Pro Gly Thr Ser 180 185 190 His His Gly Leu Ile Val Thr Cys Gly Leu Gly Ala Ala Ala Gly Leu 195 200 205 Pro Leu Ser Ala Ala Thr Val Gly Leu Ala Val Leu His Thr Pro Leu 210 215 220 Leu Gly Ile Leu Val Thr Pro Ser Ala Ala Ile Val Ala Gly Gly Ala 225 230 235 240 Ser Val Thr Met Thr Cys Gly Val Ser Ser Ser Ala Pro Gly Thr Thr 245 250 255 Thr Val Ser Thr Leu Leu Ala Gly Thr Ser Leu Leu Leu Gly Ala Thr 260 265 270 Pro Thr Leu Ala Leu Ala Gly Val Thr Leu Ala Gly Ser Gly Leu Thr 275 280 285 Cys Cys Gly Val Ser Ala Ala Val Gly Pro Gly Ala Ser Gly Gly Val 290 295 300 Pro Leu Gly Val Gly Thr Ala Pro Gly Pro Ser Thr Val Gly Ile Leu 305 310 315 320 His Ser Pro Ala Val Gly Gly Ser Gly Val Gly Pro Leu Cys Met Ser 325 330 335 Leu Ala Ala Pro Leu Pro Thr Ala Thr Thr Thr Thr His Ala Gly Leu 340 345 350 Gly Met Gly Gly Ala Thr Gly Gly Leu Val His Ile Pro Leu Ile Leu 355 360 365 Pro Thr His Ala Gly Thr Thr Ser Cys Val Ala Gly Ala Ile Leu Gly 370 375 380 Thr Gly Gly Ala Gly Pro Gly Ala Gly Leu Ala Val Gly Thr Pro Pro 385 390 395 400 Leu Leu Val Thr Thr Val Ile Gly Ala Pro Met Pro Ile Ala Gly Gly 405 410 415 Ala Thr Val Thr Leu Ser Cys Ala Thr Ala Ser Ser Ala Pro Ser Val 420 425 430 Thr Ala Thr Gly Thr Leu Pro His Gly Ala Thr Gly Gly Pro Ser Leu 435 440 445 Gly Val Leu Leu Ile Gly Ala Val Gly Thr Ala Ala Thr Thr Ile Ala 450 455 460 Cys Ala Ala Cys Ala Ser Thr Cys Ser Thr Ala Ser Pro Val Ala Leu 465 470 475 480 <210> 34 <211> 480 <212> PRT <213> Artificial Sequence <400> 34 Gly Ser Pro Thr Pro Ser Pro Thr Gly Leu Thr Thr Ala Leu Met Pro 1 5 10 15 Ser Val Pro Leu Ser Ser Ala Pro Leu Pro Thr His Thr Thr Ala Pro 20 25 30 Ser Pro Ala Ser Thr Pro Gly Ala Gly Ala Ala Pro Ser Gly Thr Thr 35 40 45 Thr Ser Leu Ser Pro Ala Ala Thr Ser Thr Gly Val Ser Pro Ala Ser 50 55 60 Leu Ala Ala Ala Ser Ala Pro Ala Thr Thr Gly Val Ser Ser Val Gly 65 70 75 80 Thr Pro His Leu Pro Thr His Ala Ala Ser Gly Thr Pro Ser Ala Gly 85 90 95 Thr Ala Thr Gly Thr Pro Ser Gly Ser Ala Ala Ala Ala Leu Leu Ala 100 105 110 Pro Thr Pro Gly Ser Ala Ala Ile Ser Ala Val Pro Gly Gly Ala Ser 115 120 125 Thr Ala Ser Thr Pro Pro Thr Ala Pro Val Ser Pro Leu Thr Thr Thr 130 135 140 Leu Ser Leu Ala His His Ser Ser Ala Ala Leu Pro Ala Ala Thr Ser 145 150 155 160 Ala Thr Thr Ile Thr Ala Ala Thr Ser Ala Ala Thr Leu Ala Ala Ser 165 170 175 Gly Thr Thr Thr Leu Ser Pro Ser Gly Ser Ala Val Ile Ser Thr Thr 180 185 190 Thr Ile Ala Thr Thr Pro Ser Leu Pro Thr Cys Ala Gly Leu Thr Ala 195 200 205 Ala Ile Thr Val Ala Thr Leu Thr Ala Leu Gly Thr Leu Leu Pro Thr 210 215 220 Ala Leu Leu Ala Val Ala Gly Ala Val Gly Cys Gly Ala Ala Thr Cys 225 230 235 240 Thr Ala Ala Gly Val His Ala Leu Thr Gly Cys Leu Ala Ala Ser Val 245 250 255 Ser Ile Ser His Ala Ser Cys Thr Ala Pro Ala Leu Thr Leu Ile Leu 260 265 270 Ala Val Pro Pro Gly Val Gly Leu Pro Gly Leu His Ala Cys Thr Gly 275 280 285 Val Gly Leu Ala Ala Thr Thr Ile Cys Leu Leu Thr Leu Ala Ile Gly 290 295 300 Thr Pro Thr Cys Ala Thr Gly Ala Ile Thr Thr Ala Pro Gly Cys Gly 305 310 315 320 Ala Met Ile Pro Ala Ala Leu Gly Ile Leu Leu Gly Ala Leu Gly Pro 325 330 335 Gly His Gly Thr Leu Cys Ala Ser Gly Ile Leu Thr Ala Ala His Leu 340 345 350 Pro Thr Ala Ala Ser Leu Ile Ile Leu Thr Ala Pro Gly Ser Pro Gly 355 360 365 Gly Pro Gly Ile Ile Pro Cys Ala Ser Gly Ala Ala His Gly Gly Val 370 375 380 Ile Thr Thr Ala Pro Pro Gly Ala Ser Pro His Ala Pro Thr Leu Cys 385 390 395 400 Thr Ile Leu Gly Thr Gly Leu Ala Cys Leu Ala Leu Ala Leu Ala Leu 405 410 415 Ile Leu Thr Ala Leu Gly Ala Leu Leu Pro Thr Thr Leu Thr Val Leu 420 425 430 Ser Leu His Ala Thr Ile Ile Ala Leu Val Gly Ala Ala Gly Ser Ala 435 440 445 Ala Met Cys His Pro Thr Thr Leu Ser Ala Pro Pro Ser Gly Val Thr 450 455 460 Ala Met Thr Val Ser Met Thr Ser Ala Ala Ser Met His Val Leu Cys 465 470 475 480 <210> 35 <211> 65 <212> PRT <213> Artificial Sequence <400> 35 Ile Thr Cys Pro Pro Pro Met Ser Val Gly His Ala Ala Ile Thr Val 1 5 10 15 Leu Ser Thr Ser Leu Thr Ser Ala Gly Ala Thr Ile Cys Ala Ser Gly 20 25 30 Pro Leu Ala Leu Ala Gly Thr Ser Ser Leu Thr Gly Cys Val Leu Ala 35 40 45 Leu Ala Thr Ala Val Ala His Thr Thr Thr Pro Ser Leu Leu Cys Ile 50 55 60 Ala 65 <210> 36 <211> 153 <212> PRT <213> Artificial Sequence <400> 36 Met Thr Ala Met Gly Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Ala Ser Gly Ile His Val Pro Ile Leu Gly Cys Pro Ser Ala 20 25 30 Gly Leu Pro Leu Thr Gly Ala Ala Thr Val Ala Val Ile Ser Ala Leu 35 40 45 Leu Leu Ile Gly Ala Leu Ile Gly Ser Met His Ile Ala Ala Thr Leu 50 55 60 Thr Thr Gly Ser Ala Val His Pro Ser Cys Leu Val Thr Ala Met Leu 65 70 75 80 Cys Pro Leu Leu Gly Leu Gly Val Ile Ser Leu Gly Ser Gly Ala Ala 85 90 95 Ser Ile His Ala Thr Val Gly Ala Leu Ile Ile Leu Ala Ala Ala Ser 100 105 110 Leu Ser Ser Ala Gly Ala Val Thr Gly Ser Gly Cys Leu Gly Cys Gly 115 120 125 Gly Leu Gly Gly Leu Ala Ile Leu Gly Pro Leu Gly Ser Pro Val His 130 135 140 Ile Val Gly Met Pro Ile Ala Thr Ser 145 150

Claims

1. An engineered cell comprising an engineered chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide, said chimeric antigen receptor polypeptide comprising: A signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signal transduction domain; and wherein the antigen recognition domain is a CD45 antigen recognition domain, and the cell has the gene encoding the cell surface CD45 antigen knocked out by genetic engineering so that the surface antigen CD45 is absent.

2. The engineered cell according to claim 1, wherein the CD45 antigen recognition domain comprises a binding portion or variable region of a monoclonal antibody selective for CD45.

3. The engineered cell according to claim 1, wherein the CD45 antigen recognition domain comprises the CD45 antibody binding domain.

4. The engineered cell according to claim 1, wherein the CD45 antigen recognition domain comprises a polypeptide selective for SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and the corresponding polynucleotide sequences SEQ ID NO 14, SEQ ID NO. 16, SEQ ID NO.

18.

5. Use of a medicament for treating CD45-related cell proliferative diseases, using engineered cells comprising a polynucleotide encoding a chimeric antigen receptor polypeptide, said polynucleotide comprising: A signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signal transduction domain, administering the medicament to a patient in need thereof; and selectively detecting CD45-positive cells associated with a cell proliferative disease; wherein the engineered cell is a T cell or NK cell comprising a CD45 antigen recognition domain, and when the engineered cell comprises an endogenous cell surface antigen targeted as the antigen recognition domain, the cell has the gene encoding the cell surface antigen knocked out by genetic engineering so that the surface antigen CD45 is absent, wherein the related cell proliferative diseases are acute leukemia, chronic leukemia, B and T cell lymphoma, myeloid leukemia, acute lymphoblastic lymphoma or leukemia, primary effusion lymphoma, reticulohistiocytoma, transient myeloproliferative disorder of Down's syndrome, lymphocyte-predominant Hodgkin's lymphoma, myeloid leukemia or sarcoma, dendritic cell tumor, histiocytic sarcoma, giant cell tumor of the tendon sheath, interdigitating dendritic cell sarcoma, post-transplant lymphoproliferative disorder.

6. A method for reducing the number of CD45-positive cell leukemia cells in vitro, the method comprising the steps of: i. contacting CD45-positive cell leukemia cells or positive cell lymphoma cells with an effective amount of an engineered cell, the engineered cell comprising a polynucleotide encoding a chimeric antigen receptor polypeptide, the polynucleotide comprising: a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signal transduction domain; and ii. Analyze and detect CD45-positive T leukemia cells or CD45-positive lymphoma cells as needed; wherein the engineered cells are T cells or NK cells comprising a CD45 antigen recognition domain, and when the engineered T cells comprise an endogenous cell surface antigen targeted as the antigen recognition domain, the cells are genetically engineered to knockout the gene encoding the cell surface antigen so that the surface antigen CD45 is absent.

7. The use according to claim 5, wherein the engineered cells can be used for pretreatment before a patient receives bone marrow and stem cell transplantation.

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