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

Abstract

The present invention provides chimeric antigen receptor polypeptides having antigen recognition domains against CD2, CD3, CD4, CD5, CD7, CD8, and CD52 antigens, and their encoding. The present invention also provides cells engineered to express polynucleotides or polypeptides. In some embodiments, the present invention provides methods for treating diseases associated with CD2, CD3, CD4, CD5, CD7, CD8, and CD52 antigens.

Description

[0001] This application is a divisional application of a patent for invention with an application date of February 26, 2016, an application number of 201680011435.3, and an invention title of "Chimeric Antigen Receptors (CARs) Targeting Hematological Malignancies, Compositions Thereof, and Methods of Use".

[0002] Cross - reference to related applications

[0003] This application is an international PCT application claiming priority to U.S. Provisional Application No. 62 / 121,842, which was filed on February 27, 2015, and is incorporated herein by reference in its entirety. Background of the Invention

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

[0005] When genetically engineered, T cells can produce a special receptor (called a chimeric antigen receptor (CAR)) on their surfaces. A CAR is a protein that allows T cells to recognize a specific protein (antigen) in the cytoplasm of a tumor cell. These CAR T cells are 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 shown that chimeric antigen receptor (CAR) T cells have great promise in hematologic malignancies resistant to standard chemotherapy. Most notably, certain CD19 CAR T cell therapies have had remarkable effects, 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] Although CAR therapy has been successful in B-cell leukemia and lymphoma, the application of CAR therapy for T-cell malignancies has not been established. Given that therapies for T-cell malignancies have significantly worse outcomes than those for B-cell malignancies (Abramson, Feldman et al., 2014), CAR therapy has greater potential to further address clinical needs in this regard.

[0008] More than 80% of T-cell acute lymphoblastic leukemia expresses CD5. Since T-cell leukemia or T-cell lymphoma cells express the CD5 surface molecule, one treatment option is to treat patients with anti-CD5 antibodies. However, these attempts have only had limited success.

[0009] Therefore, there is still a need for improved chimeric antigen receptor therapies that are more effective, safer, and more efficient in targeting T-cell-related malignancies. SUMMARY OF THE INVENTION

[0010] The present invention provides chimeric antigen receptors (CARs) that target malignancies, compositions thereof, and methods of use.

[0011] In one embodiment, the present invention provides a modified chimeric antigen receptor polypeptide that comprises: a signal peptide, a CD2 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0012] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide that comprises: a signal peptide, a CD3 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0013] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide, which polypeptide comprises: a signal peptide, a CD4 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0014] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide, which polypeptide comprises: a signal peptide, a CD5 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0015] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide, which polypeptide comprises: a signal peptide, a CD7 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0016] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide, which polypeptide comprises: a signal peptide, a CD8 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0017] In another embodiment, the present invention provides a modified chimeric antigen receptor polypeptide, which polypeptide comprises: a signal peptide, a CD52 antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0018] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD2.

[0019] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD3.

[0020] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD4.

[0021] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD5.

[0022] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD7.

[0023] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD8.

[0024] In one embodiment, the present invention provides a modified chimeric antigen receptor polynucleotide encoding a chimeric antigen receptor polypeptide having an antigen recognition domain selective for CD52.

[0025] In one embodiment, the present invention provides a modified cell expressing any one of the above chimeric antigen receptor polypeptides.

[0026] In another embodiment, the present invention provides a modified cell expressing any one of the above chimeric antigen receptor polypeptides.

[0027] In another embodiment, the present invention provides a method for producing a modified cell expressing a chimeric antigen receptor polypeptide or polynucleotide having an antigen recognition domain selective for CD2, CD3, CD4, CD5, CD7, CD8 or CD52. The method comprises (i) providing peripheral blood cells or cord blood cells; (ii) introducing the above polynucleotide into the above cells; (iii) amplifying the cells of step (ii); and isolating the cells of step (iii) to provide the modified cell.

[0028] In another embodiment, the present invention provides a method for producing a modified cell expressing a chimeric antigen receptor polypeptide or polynucleotide having an antigen recognition domain selective for CD2, CD3, CD4, CD5, CD7, CD8 or CD52. The method comprises (i) providing peripheral blood cells or cord blood cells; (ii) introducing the above polynucleotide into the above cells; (iii) amplifying the cells of step (ii); and isolating the cells of step (iii) to provide the modified cell.

[0029] In another embodiment, the present invention provides a method for producing a modified cell expressing a chimeric antigen polypeptide or polynucleotide having an antigen recognition domain selective for CD2, CD3, CD4, CD5, CD7, CD8 or CD52. The method comprises (i) providing placental cells, embryonic stem cells, induced pluripotent stem cells or hematopoietic stem cells; (ii) introducing the above polynucleotide into the cells of step (i); (iii) amplifying the cells of step (ii); and (iv) isolating the cells of step (iii) to provide the modified cell.

[0030] In one embodiment, the present invention provides a method for conferring anti-leukemia or anti-lymphoma immunity against CD4-positive T cell leukemia or CD4-positive T cell lymphoma to a patient in need thereof. The method comprises (i) administering to the patient in need thereof an effective therapeutic amount of a modified cell expressing a CAR polypeptide having a CD4 antigen recognition domain; and (ii) optionally, analyzing the immunity of the patient against T cell leukemia or T cell lymphoma.

[0031] In another embodiment, the present invention provides a method for reducing the number of CD4-positive T-cell leukemia cells or CD4-positive T-cell lymphoma cells. The method comprises (i) contacting the CD4-positive T-cell leukemia cells or CD4-positive T-cell lymphoma cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD4 antigen recognition domain; and (ii) optionally, analyzing the CD4-positive T-cell leukemia cells or CD4-positive T-cell lymphoma cells.

[0032] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having a CD2 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD2 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0033] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having a CD3 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD3 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0034] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having a CD4 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD4 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0035] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having a CD5 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD5 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0036] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having a CD7 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD7 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0037] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having the CD8 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD8 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0038] In another embodiment, the present invention provides a method for reducing the number of immunomodulatory cells having the CD52 antigen. The method comprises (i) contacting the immunomodulatory cells with an effective amount of engineered cells expressing a CAR polypeptide having a CD52 antigen recognition domain; and (ii) optionally, analyzing the reduction in the number of immunomodulatory cells.

[0039] In one embodiment, the present invention provides a method for treating a cell proliferative disease. The method comprises (i) administering to a patient in need thereof a therapeutically effective amount of engineered cells expressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8 or CD52 antigen recognition domain.

[0040] In one embodiment, the present invention provides a method for treating an autoimmune disease. The method comprises (i) administering to a patient in need thereof an effective therapeutic amount of engineered cells expressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8 or CD52 antigen recognition domain.

[0041] In one embodiment, the present invention provides engineered cells expressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8 or CD52 antigen recognition domain for treating a cell proliferative disease. Its use includes administering the engineered cells to a patient in need thereof.

[0042] In some embodiments, the CAR generally comprises at least one of an intracellular signaling domain, a hinge domain and / or a transmembrane domain. The first generation CAR comprises the intracellular signaling domain CD3z, while the second generation CAR comprises a single co-stimulatory domain derived from (e.g., but not limited to) CD28 or 4-1BB. The third generation CAR includes two co-stimulatory domains, such as (but not limited to) CD28, 4-1BB (also known as CD137) and OX-40 and any other co-stimulatory molecules.

[0043] In some embodiments, a CAR having a CD2, CD3, CD4, CD5, CD7, CD8 or CD52 antigen recognition domain is part of an expression construct. In some preferred embodiments, the expression gene or expression construct may comprise an auxiliary gene or a tag or a part thereof. The auxiliary gene may be an inducible suicide gene or a part thereof, including (but not limited to) the caspase 9 gene. The elimination mechanism of the "suicide gene" improves the safety of the gene therapy and kills cells only when activated by a specific compound or molecule. In some embodiments, the epitope tag is a c-myc tag, a streptavidin-binding peptide (SBP), a truncated EGFR gene (EGFRt) or a part or combination thereof.

[0044]

Brief Description of the Drawings

[0045] Figure 1: CD4 CAR expression. (A), An exemplary representative diagram of a recombinant lentiviral vector encoding CD4 CAR. CD4 CAR expression is initiated by the SFFV (spleen focus-forming virus) promoter. The third-generation CD4 CAR contains a leader sequence, an anti-CD4 scFv, a hinge domain (H), a transmembrane domain (TM) and the following intracellular signaling domains: CD28, 4-1BB (both co-stimulatory molecules) and CD3z. (B): 293FT cells were transfected with a lentiviral plasmid against GFP (lane 1) and CD4 CAR (lane 2) for Western blot analysis 48 h after transfection and probed with a mouse anti-human CD3z antibody. (C): Display of the components of a third-generation chimeric antigen receptor T cell targeting CD4 expression in the cytoplasm.

[0046] Figure 2: Generation of CD4 CAR T cells. (A), Experimental design. (B), Buffy coat cells were activated with anti-CD3 antibody and IL-2. Cells were transduced with GFP (middle) or CD4 CAR (right) lentiviral supernatants. After 7 days of culture, cells were analyzed by flow cytometry using biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody and streptavidin-PE. Untransduced labeled CB cells are shown on the left. (C): CD4 CAR T cells depleted the CD4+ population during T cell expansion. Buffy coat cells were activated with anti-CD3 antibody and IL-2 for two days. Buffy coat cells contain two distinct populations of T cells, CD8+ cytotoxic T cells and CD4+ helper T cells (left). Cells were transduced with GFP (middle) or CD4 CAR (right) lentiviral supernatants. After 3 days of culture, cells were analyzed by flow cytometry using mouse-anti-human CD4 (FITC) and CD8 (APC) antibodies. Untransduced PBMCs were also labeled (left). (D): Most CD4 CAR T cells had a central memory phenotype. Buffy coat cells were activated with anti-CD3 antibody for 2 days. Cells were transduced with CD4 CAR lentiviral supernatant. After 6 days of expansion, the CD62L, CD45RO, and CD45RA phenotypes of CD8+ cells were analyzed by flow cytometry (N = 3).

[0047] Figure 3: CD4 CAR T cells eliminated T cell leukemia cells in co-culture assays. (A), CD4 CAR T cells eliminated KARPAS 299 T cell leukemia cells in co-culture. Activated human buffy coat cells transduced with GFP (middle) or CD4 CAR (right) lentiviral supernatants were cultured with KARPAS 299 cells at a ratio of 2:1. After 24 hours of co-culture, cells were stained with mouse anti-human CD4 (APC) and CD8 (PerCp) antibodies and analyzed for T cell subsets by flow cytometry (N = 3). (B) and (C): CD4 CAR T cells eliminated primary T cell leukemia cells in co-culture. Activated human buffy coat cells transduced with GFP (middle) or CD4 CAR (right) lentiviral supernatants were cultured with SP53 mantle cell lymphoma cells pre-stained with the cell membrane dye CMTMR at a ratio of 2:1. After 24 hours of co-culture, cells were stained with mouse anti-human CD3 (PerC™) and then analyzed by flow cytometry (N = 2). SP53 cells pre-stained with CMTMR were also labeled alone (left).

[0048] Figure 4: CD4 CAR T cells derived from PBMCs potently enhance CD8+ T cells and specifically kill leukemia cells expressing the CD4 antigen. (A) CD4 CAR T cells derived from PBMCs potently enhance CD8+ T cells. PBMC buffy coat cells composed of CD4+ and CD8+ T cells were activated with anti-CD3 antibody and IL-2 for 2 days and then transduced with GFP (middle) or CD4 CAR (right) lentiviral supernatants. After 3 days of culture, the labeled cells were analyzed for T cell subsets by flow cytometry. Un-transduced PBMCs were also labeled (left). (B): CD4 CAR T cells specifically target and kill KARPAS 299 cells. PBMC T cells transduced with GFP control or CD4 CAR lentiviral supernatants were cultured with CFSE-stained KARPAS299 at ratios of 2:1, 5:1, and 10:1, respectively. After overnight culture at 37 °C, the dye 7AAD was added, and the cells were analyzed by flow cytometry. The killing rate of target cells was measured by comparing the survival of target cells relative to that of negative control cells (SP53 cells, a B cell lymphoma cell line stained with CMTMR).

[0049] Figure 5: CD4 CAR T cells efficiently mitigate the anti-leukemia effect in vivo using different regimens. NSG mice received 2.5 Gy sublethal irradiation. Twenty-four hours after irradiation, 1x10 6 (in A) or 0.5x10 6 KARPAS 299 cells (in B and C) were injected subcutaneously into the mice. The injected mice were treated with different regimens of CD4 CAR T cells or control T cells. Each group had N = 5 injected mice. (A): A low dose (2x10 6 ) of CD4 CAR T cells was injected on day 3. After accelerated tumor growth was detected on day 22, a high dose (8x10 6 ) of CD4 CAR T cells was injected. (B): Two high doses of CD4 CAR T cells (8x10 6 and 5.5x10 6 ) were injected on days 3 and 10, respectively. (C): The same low dose (2.5x10 6 ) of CD4 CAR T cells was injected every 5 days for a total of four times. (D): Surviving mice treated with the indicated numbers of CD4 CAR T cells or control GFP T cells. N = 10.

[0050] Figure 6: CD4CAR is expressed on the surface of HEK-293 cells. HEK-293 cells were transduced with CD4CAR or GFP control virus supernatant for 6 hours. After culturing for 3 days, the cells were analyzed by flow cytometry.

[0051] Figure 7 : Compare the cell growth of activated PMBC leukocyte layer cells transduced with lenti-GFP and CD4CAR viruses. Activated PMBC leukocyte layer cells were transduced with GFP control or CD4-CAR lentivirus supernatant on day 0. The cells were washed on day 1 and medium was added on days 3 and 5.

[0052] Figure 8: CD4CAR construct. (A) Schematic representative diagram of a lentiviral vector encoding a third-generation CD4CAR driven by the spleen focus-forming virus (SFFV) promoter. The construct contains a leader sequence, anti-CD4 scFv, hinge domain (H), transmembrane domain (TM), and signaling domains CD28, 4-1BB, and CD3ζ. (B): HEK293FT cells were transfected with GFP vector control (lane 1) and CD4CAR (lane 2) lentiviral plasmids. Forty-eight hours after transfection, the cells were removed and used for Western blot analysis with a mouse anti-human CD3z antibody. (C) Illustration of third-generation CAR NK cells targeting cells expressing CD4.

[0053] Figure 9 : Generation of CD4CAR NK cells. (A, upper panel) Expression level of CD4CAR on NK cells before sorting by FACS (N = 3); (A, lower panel) CD4CAR expression on NK cells before co-culture experiments (N = 3) after sorting and expansion.

[0054] Figure 10: CD4 CAR NK cells eliminate CD4+ leukemia and lymphoma cells in co-culture assays. Co-culture experiments were performed at a 2:1 effector cell to target cell ratio for 24 hours and analyzed directly by flow cytometry for CD56 and CD4 (panels A and B). Each analysis consisted of target cells alone control (left), target cells co-cultured with vector control (center), or NK cells transduced with CD4CAR (right) lentivirus supernatant. Top row, panel A: Karpas 299 (N = 3). Middle row, panel A: HL-60T cells (N = 2). Bottom row, panel A: CCRF CEM cells (N = 2). CD4CAR NK cells eliminate primary T cell leukemia cells from patients with CD4+ T cell lymphoma / Sézary syndrome (N = 2) and pediatric T cell ALL expressing CD4 (N = 2). (C): Bar graph summarizing the results of co-culture assays at 2:1 and 5:1 E:T ratios.

[0055] Figure 11 : Co-culture specificity and dose-response killing curve table. CD4CAR NK cells lysed leukemia cell lines expressing CD4 in a dose-dependent and specific manner. CD4CAR NK and vector control cells were cultured with equal ratios of CFSE-stained "target" (Karpas 299 or CCRF-CEM) cells and CMTMR-stained "off-target" MOLT4 cells at effector-to-target cell ratios of 1:4, 1:2, and 1:1. After 24 hours, 7-AAD dye was added and the remaining live cells were analyzed by flow cytometry. The killing rate of target cells was measured by comparing the cell viability of CD4+ Karpas 299 or CCRF-CEM in CD4CAR NK cell co-cultures with that of CD4+ Karpas 299 or CCRF-CEM in vector control NK cell co-cultures.

[0056] Figure 12: CD4CAR NK cells eliminated CD4+ T cells isolated from human umbilical cord blood at an effector-to-target cell ratio of 2:1, but did not affect the output of the hematopoietic stem cell / progenitor compartment. (A) Co-culture analysis was performed at an effector-to-target cell ratio of 2:1 for 24 hours, after which the cells were stained with mouse anti-human CD56 and CD4 antibodies. Target cells were cultured alone as a control (left). NK cells were transduced with vector control (center) or CD4CAR (right) lentiviral supernatants and co-cultured with CD4+ T cells isolated from human umbilical cord blood. (N = 2) (B) CD4CAR NK cells were co-cultured with 500 CD34+ cord blood cells in NK cell medium supplemented with IL-2 at effector:target cell ratios of 2:1 and 5:1 for 24 hours, respectively. The experimental controls used were CD34+ cells alone, and untransduced NK cells co-cultured with CD34+ CB cells at their respective 2:1 and 5:1 effector:target cell ratios. Hematopoietic compartment output was assessed on day 16 by the formation of the number of burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte / monocyte (CFU-GM). CFU statistical analysis was performed by two-way ANOVA with α set at 0.05.

[0057] Figure 13: CD4CAR NK cells demonstrated an anti-leukemia effect in vivo. NSG mice were sub-lethally irradiated and injected intradermally with luciferase-expressing Karpas 299 cells (day 0) to induce the formation of measurable tumors. On day 1 and every 5 days for a total of 6 courses, the mice were injected intravenously with 5x10 6CD4 CAR NK cells or vector control group NK control cells. (A) On days 7, 14, and 21, mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging. (B): The average light intensity of mice injected with CD4 CAR NK was compared with the average light intensity of mice injected with vector control group NK. (C) On day 1 and every other day thereafter, tumor size area was measured and the average tumor size between the two groups was compared. (D) The survival percentage of mice was measured and compared between the two groups.

[0058] Figure 14 : CD4 CAR NK cells eliminated CD4-positive leukemia and lymphoma cells in co-culture assays. All assays shown were performed with a 5:1 effector cell to target cell ratio for 24-hour co-culture, after which cells were stained with mouse anti-human CD56 and CD4 antibodies. Each assay consisted of NK cells transduced with vector control group (central) or CD4CAR (right) lentiviral supernatants and co-cultured with target cells, and target cells cultured alone as a control (left). CD4CAR NK cells eliminated Karpas 299 leukemia T cells (A), HL-60 T cells (B), and CCRF-CEM cells (C). CD4CAR NK cells eliminated primary T cell leukemia cells from patients with T cell leukemia / Sézary syndrome (E) expressing CD4 and pediatric T cell ALL (F) expressing CD4.

[0059] Figure 15 : NK cells were transduced with vector control group or CD4CAR lentiviral supernatants or cultured for an untransduced control group. After 7 days of culture, cells were harvested and analyzed by flow cytometry using biotinylated goat anti-mouse F(Ab’)2 and streptavidin-PE in sequence. After sorting, >85% of the NK cells were CD4CAR+.

[0060] Figure 16: CD4CAR NK cells did not lyse CD4-, CD5+ MOLT4 negative control. (A) The immunophenotype of MOLT4 cells was confirmed to be almost entirely CD4- and CD5+. (B): By comparison with vector control group NK cell tumor lysis (upper grid) assessment, CD4CAR NK cells did not lyse MOLT4 cells at 0 h, 4 h, 8 h, and 24 h (lower grid) at a 5:1 effector cell to target cell ratio. (C) Anti-CD4 CDCAR NK anti-tumor activity was confirmed at 4 h with a CD4+ Karpas 299 positive control at a 5:1 E:T ratio.

[0061] Figure 17: Generation of CD5 CAR. A: DNA gene construct and translated protein construct of CD5 CAR, and sketches of the generation and function of the anchored CD5 scFv antibody and the displayed CD5 CAR. The DNA construct of the third-generation CD5 CAR construct reads from 5' to 3': leader sequence, anti-CD5 extracellular single-chain antibody (anti-CD5 ScFv), hinge region, transmembrane region, and three intracellular signaling domains that define this construct as a third-generation CAR; CD28, 4-1BB, and CD3 ζ. The DNA construct of the anchored CD5 scFv antibody is the same as the CD5 CAR construct without intracellular signaling domains, as is the translated protein product used to anchor the CD5 scFv antibody. These translated protein constructs contain an anti-CD5 ScFv that will bind to the CD5 target cell, a hinge region that allows the anti-CD5 ScFv to be properly positioned to allow optimal binding to the CD5 target cell, and a transmembrane region. The complete CD5 CAR protein also contains two co-stimulatory domains and the intracellular domain of the CD3ζ chain. This construct is considered a third-generation CAR: CD28, 4-1BB, and CD3ζ. B: Western blot analysis confirmed CD5 CAR expression in HEK293 cells. HEK293 cells that had been transduced with GFP (as a negative control group) or CD5 CAR lentivirus for 48 h were used for Western blot analysis using a CD3ζ antibody to determine CD5 CAR expression. Left lane, GFP control group HEK293 cells, with no band as expected. The right lane shows a band of approximately 50 kDa, the expected molecular weight, based on the molecular weight of the CD5 CAR construct. C: Flow cytometry analysis of CD5 CAR expression on the surface of lentivirus-transduced CD5 CAR T cells. This analysis was performed on doubly transduced CD5 CAR T cells on the 8th day after the second lentivirus transduction. Left: Isotype control group T cell population (negative control); Right: Using goat anti-mouse F(AB')2-PE, 20.53% CD5 CAR expression was measured on the transduced T cells by flow cytometry.

[0062] Figure 18: Schematic diagram of the study on the transduction of CD5 CAR T cells. A: Steps to generate CD5 CAR T cells by single transduction. B: Steps to generate CD5 CAR T cells by double transduction. C: Comparison of the downregulation of CD5 expression on T cells by single transduction and double transduction using CD5 CAR lentivirus. Analysis of the downregulation of extracellular CD5 protein and GFP T cell control group within 8 days after lentiviral transduction. The singly transduced CD5 CAR T cells did not show complete downregulation of CD5 on the cell surface at day 8, and the CD5 protein expression decreased maximally at day 6. In the doubly transduced population, the absolute number of CD5+, CD3+ double-positive CD5 CAR T cells decreased over time, decreasing from 24.44% at day 0 to near complete absence of CD5 expression at day 4. In contrast, the GFP T cell control group maintained a CD5+, CD3+ double-positive population greater than 95% from day 2 to day 8.

[0063] Figure 19: Downregulation of CD5 expression on T cells 7 days after lentiviral transduction with an anchored CD5 scFv antibody. A: Schematic diagram of the study on transduction (single transduction) with an anchored CD5 scFv lentivirus. B: The anchored CD5 scFv downregulates or reduces the amount of surface CD5 expression on T cells. Flow cytometry analysis confirmed a significant decrease in CD5 protein expression (∼32%) after single transduction of CD5 scFv and 7-day culture. Elimination of CD5 expression was observed, but it was not complete after 7 days, and subsequent studies are currently being completed on the doubly transduced anchored CD5 scFv antibody.

[0064] Figure 20: CD5 CAR cells effectively lyse T-ALL cell lines expressing CD5 and do not lyse T leukemia cell lines not expressing CD5. A: Flow cytometry analysis of T-ALL cell lines alone (left column), co-cultured with T cells transduced with a GFP vector (middle column), and co-cultured with T cells transduced with CD5 CAR (right column). Each cell line is shown in each row, and CD5+ T-ALL cell lines (CCRF-CEM and Molt-4) are in the top and middle rows, and CD5-negative cell lines are in the bottom row (KARPAS 299). KAEPAS299 is a CD5-negative T cell lymphoma. All co-culture times were 24 hours, and the effector cell:target cell ratio was 5:1. Compared with the GFP control group, the cell lysis of the two CD5 T-ALL leukemia cell lines exceeded 78% compared with the GFP control group. B: This bar graph indicates that CD5 CAR T cells achieved such T cell lysis when co-cultured with GFP T cells described in Figure 20A No lysis was observed in the CD5 CAR T cell co-cultures when co-cultured with CD5-negative KARPAS 299 (n = 3 independent experiments were performed in duplicate).

[0065] Figure 21: CD5CAR cells effectively lyse T-cell acute lymphoblastic leukemia cells from patient samples expressing CD5. A: Flow cytometry analysis of T-ALL cells alone (left column), co-cultured with GFP T cells (middle column), and co-cultured with CD5CAR T cells (right column). Each patient's cells are given a row and numbered to maintain patient confidentiality. All co-culture times are 24 hours, and the effector cell:target cell ratio is 5:1. Compared to the control group, the cell lysis of TALL-1 was more than 71.3% higher than that of the GFP control group. The remaining cell lines also confirmed positive cell lysis, but to a lesser extent, between 33% and 47%. This can be related to each leukemia sample expressing CD5 and is discussed in the following text. B: This bar graph indicates that CD5CAR T cells achieve T-cell lysis when co-cultured compared to the GFP T cells described in Figure 21A . All experiments were done in duplicate. C: Flow cytometry analysis data confirmed the expression levels of CD3 and CD5 of the patient T-cell ALL samples analyzed in Figure 21A . T-ALL 1 and T-ALL 3 had different CD5 positivity. D. Flow cytometry analysis of the CD5 expression levels of four patient sample T-ALL cell populations on the same template. The difference in mean fluorescence intensity (MFI) was measured by flow cytometry analysis ( Figure 21C ).

[0066] Figure 22 : Detailed analysis of the killing of CD5CAR T cells against patient T-ALL cells (T-ALL-8). Flow cytometry analysis confirmed the ability of CD5CAR T cells to kill patient TALL cells. Co-culture of the control group GFP-T cells and T-ALL-8 cells is shown on the left, and co-culture of CD5CAR and T-ALL 8 is shown on the right. All CD5-positive cells (CD34-positive (red circle) and CD34-negative (green circle, T cells)) strongly lysed the target cells, and CD5-negative cells did not lyse the target cells. When compared to the GFP control group, CD5CAR T cells lysed at least 93.1% of CD5-positive TALL-8 cells. The experiment was done in duplicate. In addition, CD5CAR T cells basically eliminated the T-cell population (CD5+CD34-, green circle).

[0067] Figure 23: CD5 CAR T cells effectively eliminate normal GFP-labeled T cells. A: CD5 CAR T cells kill normal T cells in a dose-dependent manner. CD5 CAR T cells or CD123 CAR T cells (control group) were co-cultured with GFP-labeled T cells at effector cell to target cell ratios of 0.25:1, 0.5:1, and 1:1. After 24 hours, the remaining live GFP T cells were analyzed by flow cytometry. The killing rate of target cells was measured by comparing the survival of GFP T cells in the CD5 co-cultures relative to the survival of GFP T cells in the control group CD123 CAR T cells (since T cells do not express CD123). B: Co-culture killing curve graph based on the data in A.

[0068] Figure 24: T cells continuously express CD5 when co-cultured with CD5 CAR or anchored CD5 scFv T cells. A: Steps for generating CD5 CAR T cells or anchored CD5 scFv T cells and CD123 CAR T cells (control group). B: Degree of CD5 expression on T cells transduced with different CARs (3 days after the second transduction). Activated T cells were transduced with lentiviruses expressing CD5 CAR or anchored CD5 scFv and CD123 CAR. Three days after transduction, CD5 expression was analyzed by flow cytometry.

[0069] Figure 25: Co-culture analysis was performed to determine whether normal T cells continuously express CD5 when co-cultured at a 1:1 ratio with CD5 CAR or anchored CD5 scFv T cells or CD123 CAR (control group) for 2 days ( Figure 25A ) or 4 days ( Figure 25B ). GFP-labeled T cells were co-cultured with CD5 CAR T cells or anchored CD5 scFv T cells or CD123 CAR T (control group) cells and the CD5 expression and live cells of the GFP-labeled T cells after co-culture were analyzed by flow cytometry. C( Figure 25C ), CCRF-CEM or Molt-4 T-ALL cells transduced with CD5 CAR- or anchored CD5 scFv showed downregulation of CD5 expression. CCRF-CEM or Molt-4 T-ALL cells were transduced with lentiviruses expressing CD5 CAR or anchored CD5 scFv. After the second transduction, the CD5 expression of the transduced leukemia cells was analyzed by flow cytometry.

[0070] Figure 26: CD5 CAR T cells demonstrate a profound anti-leukemia effect in vivo. NSG mice were irradiated sub-lethally 24 hours before intravenous injection of 1x10 6CCRF-CEM cells expressing luciferase (day 0) were used to initiate measurable tumor formation. On days 3 and 4, 5x10 6 CD5 CAR T cells or vector control T cells were injected intravenously into the mice. These injections were repeated on days 6 and 7, for a total of 2.0x10 7 cells injected per mouse. A: On days 5, 8, 10, and 13, the mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging. B: The average light intensity of the mice injected with CD5 CAR T cells was compared to the average light intensity of the mice injected with vector control T cells. C: The killing rate of tumor cells in the mice treated with CD5 CAR T cells compared to the control group of mice. D: On day 15, peripheral blood was drawn from the mice and the percentage of leukemia cells was measured and compared to the percentage of leukemia cells in the vector control or normally injected mice.

[0071] Figure 27: CD5 CAR NK cells (NK-92) effectively eliminate the CCRF CEM T-ALL cell line in vitro. A and B: The T-lymphoblastic cell line CCRF CEM expressing CD5 was co-cultured with CD5 CAR NK cells at the indicated E:T (effector cell:target cell) cell ratios for 24 hours. NK-CAR and target cell populations were separated using CD56 and CD5 respectively and the target population was quantified by flow cytometry. Cell survival expression of the transduced vector control NK cells and each bar represents the mean statistical data of N = 2 replicate samples. C: CD5 CAR NK cells eliminate CCRF-CEM cells in a dose-dependent manner. The T-lymphoblastic cell line CCRF CEM expressing CD5 was co-cultured with CD5 CAR NK cells at the indicated E:T (effector cell:target cell) cell ratios and the lower bound of the E:T ratio was decreased. Saturation was achieved at an E:T ratio of 2:1 and co-culture at decreasing ratios showed a dose-dependent manner of CD5 elimination. Complete elimination of CCRF-CEM was achieved at 5:1.

[0072] Figure 28: CD5 CAR NK cells effectively lyse two CD5+ T-ALL cell lines, MOLT-4 and Jurkat. A: CD5 CAR NK cells were co-cultured with MOLT-4 cells at the indicated E:T (effector cell:target cell) cell ratios for 24 hours. Cell survival relative to the expression of the transduced vector control NK cells and each bar represents the mean statistical data of N = 2 experimental replicate samples. B: CD5 CAR NK cells were co-cultured with Jurkat cells at the indicated E:T (effector cell:target cell) cell ratios for 24 hours. Cell survival relative to the expression of the transduced vector control NK cells and each bar represents the mean statistical data of N = 2 experimental replicate samples.

[0073] Figure 29: CD5CAR NK cells effectively eliminate invasive CD5+ T-ALL cells in human samples. A: T-ALL cells from patient T-ALL#1 were co-cultured with CD5CAR NK cells for 24 hours at the indicated E:T (effector cell: target cell) ratios. B: T-ALL cells from patient T-ALL#2 were co-cultured with CD5CAR NK cells for 24 hours at the indicated E:T (effector cell: target cell) ratios. The gated target population was screened by flow cytometry using the cell cytotracker dye (CMTMR) to quantify T-ALL patient samples. Data show mean statistics of replicate samples. The target CD5+CD34+ cell population was gated using an isotype control. Cell viability is shown in the bar graph, relative to control NK cells transduced with the vector. From left to right, the bar graph shows data for CD34+CD5+ (left) and CD5+cd34- (right) at each ratio. CD5CAR NK shows nearly complete lysis of the highly expressed CD5+ target population with activity against the low CD5+CD34+ potential tumor stem cell population. Saturation was achieved at 2:1, indicating the need to dilute the E:T ratio. Figure 29C and 29D : Leukemia cells from patient #3 (PTCL) and patient #4 (Sezary syndrome) were co-cultured with CD5CAR NK cells for 24 hours at the indicated E:T (effector cell: target cell) ratios, respectively.

[0074] Figure 30 : CD5NK-CAR specifically eliminates cord blood T cells. T cells were isolated from cord blood (UCB) T cells and co-cultured with CD5CAR NK cells for 24 hours at the indicated E:T (effector cell: target cell) ratios. NK-CAR and T cell populations were isolated using CD56 and CD5 respectively and quantified by flow cytometry. Cell viability is expressed relative to vector-transduced control NK cells and each bar graph represents mean statistics of replicate samples.

[0075] Figure 31: CD5CAR NK cells effectively eliminate CD5+ mantle cell lymphoma and chronic lymphocytic leukemia. CD5CAR NK cells were co-cultured with Jeko cells ( Figure 31A ) and cells from patients with mantle cell lymphoma ( Figure 31B ) and chronic lymphocytic leukemia ( Figure 31CCo-culture was performed with leukemia cells from patients

[0076] Figure 32 : Bar graph summarizing CD5CAR NK cell co-culture studies.

[0077] Figure 33: CD5CAR NK cells demonstrate potent anti-leukemia effects in vivo. NSG mice were irradiated sub-lethally 24 hours prior to intravenous injection of 1 x 10 6 luciferase-expressing CCRF-CEM cells (day 0) to induce measurable tumor formation. On days 3 and 4, mice were injected intravenously with 5 x 10 6 CD5CAR NK cells or vector control NK cells. These injections were repeated on days 6 and 7, for a total of 2.0 x 10 7 cells per mouse. A: On day 5, mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging. B: Tumor cell killing rate in mice treated with CD5CAR NK cells relative to the control group.

[0078] Figure 34: Structure and expression of CD3CAR. A: Schematic representation of the CD3CAR structure in a lentiviral vector. CAR expression is driven by the SFFV (spleen focus-forming virus) promoter and is a third-generation construct that contains a leader sequence, anti-CD3 scFv, hinge domain (H), transmembrane domain (TM), two co-stimulatory domains CD28 and 4-1BB, and the intracellular signaling domain CD3ζ. B: HEK-293FT cells were transduced with lentiviral plasmids encoding GFP (lane 1) and CD3CAR (lane 2) for western blot analysis 48 hours post-transduction and probed with a mouse anti-human CD3ζ antibody.

[0079] Figure 35: CD3CAR NK cells eliminate T-ALL cell lines expressing CD3 in vitro. A: Approximately 80% of the T-lymphocytic cell line Jurkat expressing CD3 was co-cultured with CD3CAR NK cells at the indicated E:T (effector cell: target cell) ratios for 6 hours. B: Sorted (CCRF-CD3) or unsorted CCRF-CEM (CCRFCEM) cells were co-cultured with CD3CAR NK cells for 24 hours. NKCAR and target cell populations were separated using CD56 and CD3 respectively and the target population was quantified by flow cytometry. Cell viability was expressed relative to the transduced vector control NK cells and each bar represents the mean statistics of replicate samples from N = 2 experiments.

[0080] Figure 36: These CD3CAR NK cells show strong killing against primary CD3+ leukemia cells from patient samples. A: SPT-1 (Sezary syndrome) patient cells were CD3 positive and co-cultured with CD3CAR NK cells at the indicated E:T (effector cell: target cell) ratios for 24 hours. NK-CAR and target cell populations were separated using CD56 and CD3 respectively and the target population was quantified by flow cytometry. Although SPT-1 is a heterogeneous cell population, the broad population expressing CD3+ was still eliminated by CD3NK-CAR. B: PT4 (unclassified PTCL) patient cells were CD3+CD7- and co-cultured with CD3CAR NK cells at the indicated E:T (effector cell: target cell) ratios for 24 hours. The gated target population was quantified as shown in the schema. PT4 leukemia cells were of the CD3+CD7- type and were effectively eliminated by CD3CAR NK cells. The broad CD3+ population was also affected by CD3CAR NK cells.

[0081] Figure 37 : CD3CAR NK cells can lyse normal T cells as expected. Normal T cells were isolated from cord blood and transduced with lentivirus expressing GFP. These transduced GFP T cells were co-cultured with CD3CAR NK cells. The co-culture was performed in NK cell medium with 2.5% serum. The co-culture was incubated for 24 hours and labeled for flow cytometry analysis. The ability of CD3CAR NK cells to lyse target T cells was evaluated by comparing the number of remaining CD3+GFP T cells after co-culture. Importantly, over time, the target CD3+GFP T cells were shown to be lysed with an efficiency of more than 80% at an effector cell to target cell ratio of 5:1.

[0082] Figure 38: CD3CAR NK cells demonstrate a profound anti-leukemia effect in vivo. A: NSG mice were irradiated sub-lethally 24 hours prior and then injected intravenously with 1x10 6Jurkat cells expressing luciferase (day 0) were used to induce measurable tumor formation. On days 3 and 4, mice were injected intravenously with 5x10 6 CD3CAR NK cells or vector control NK cells per day. These injections were repeated on days 6 and 7, and again on day 10, for a total of 2.5x10 7 cells per mouse. (A) On days 4, 7, 9, and 13, mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging. B: Comparison of the average light intensity of mice injected with CD3CAR NK with that of mice injected with vector control NK cells. C: Tumor cell killing rate of mice treated with CD3CAR NK cells relative to the control group.

[0083] Figure 39 : Steps for generating CAR T or NK cells targeting T cell lymphoma or T cell leukemia.

[0084] Figure 40 : Three pairs of sgRNAs designed with CHOPCHOP to target CD2, CD3, CD5, and CD7. Three pairs of sgRNAs were designed with CHOPCHOP to target the designated genes. The specific gene sgRNAs were cloned into a lentiviral vector (Lenti U6-sgRNASFFV-Cas9-puro-wpre) expressing human Cas9 and puromycin resistance gene linked by an E2A self-cleaving linker. The U6-sgRNA gene cassette is located in front of the Cas9 element. The expression of sgRNA and Cas9puro is driven by the U6 promoter and the SFFV promoter, respectively.

[0085] Figure 41: Generation of stable CD5-deficient CCRF-CEM and MOLT-4 T cells using the CRISPR / Cas9 lentiviral system. A: Flow cytometry analysis confirmed that CCRF-CEM T cells with CRISPR / Cas9 KD after using two different sgRNAs (Lenti-U6-sgCD5a-SFFV-Cas9puro (sgCD5A) and Lenti-U6-sgCD5b-SFFV-Cas9puro (sgCD5B)) lost CD5 expression after puromycin selection. Wild-type controls are seen in the majority of the scatter plots on the left. This population (indicated by the blue circle and arrow) was selected for Figure 41BSorting, purification, and analysis in the middle. B: Flow cytometry analysis data indicate the percentage of pure sorted and stable CD5-negative CCRF-CEM cells transduced with the scCD5A CRISPR / Cas9 technology. We noted >99% purity of CD45-positive, CD5-negative CCRF sgCD5A T cells. C: Flow cytometry analysis confirmed that MOLT-4 T cells with CRISPR / Cas9 KD using two different sgRNA sequences (sequences CD5A and CD5B, middle and right columns) lost CD5 expression after puromycin treatment. Wild-type controls are seen in the majority of the left scatter plots. Since the CRISPR / Cas9 KD technology with primer CD5A was more successful in downregulating CD5 protein, this population (indicated by the blue circle and arrow) was selected for Figure 41D Sorting, purification, and analysis in the middle. D: Flow cytometry analysis data indicate the percentage of pure sorted and stable CD5-negative MOLT-4 cells transduced with the scCD5A CRISPR / Cas9 technology. We noted >99% purity of CD45-positive, CD5-negative MOLT-4 sgCD5A T cells.

[0086] Figure 42: Stable CD7 loss generated using the CRISPR / Cas9 lentiviral system and cell sorted in CCRF CEM cells or NK-92 cells. CD7 loss percentage in CCRF-CEM ( Figure 42A and B) or NK-92 ( Figure 42C and D) using sgCD7A (Lenti-U6-sgCD7a-SFFV-Cas9-puro) and sgCD7B (Lenti-U6-sgCD7b-SFFV-Cas9-puro) was determined by flow cytometry analysis using CD45 and CD7 antibodies after puromycin treatment. The values embedded in the schema show the percentage of positive and negative expression of CD45 or CD7. The right square indicates the percentage purity of sorted stable CD7-negative cells in CCRF-CEM (B) or NK-92 cells (D) prepared from CD7-negative cells transduced with sgCD7A or sgCD7D CRISPR lentivirus.

[0087] Figure 43: CD7CAR NK7--92 cells effectively lysed T cell ALL cell lines expressing CD7. To avoid suicide, CD7-deficient NK-92 (NK 7--92) cells were generated and transduced with CD7CAR. Their killing ability was tested using two transduced CD7CAR NK7--92 cells #A and #B. A: Flow cytometry analysis of CCRF-CEM cells alone (left column), co-cultured with GFP NK-92 cells (middle column), and co-cultured with CD7CAR-NK-92-cells #A and B# (right column). B: Bar graph of data obtained from A.

[0088] Figure 44 : CD3 multi-subunit protein complex. CD3 contains a protein complex and is composed of four different chains described in the figure above. The complex contains the CD3δ chain, CD3γ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) composed of αβ chains. Detailed description

[0089] The present invention provides chimeric antigen receptor (CAR) compositions, methods for their manufacture, and methods of using such CAR compositions.

[0090] Composition

[0091] Chimeric antigen receptor polypeptide

[0092] In one embodiment, the present invention provides a chimeric antigen receptor (CAR) polypeptide having a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0093] 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 includes the sequence of a protein or the sequence of a peptide, with no maximum amino acid number limit. A polypeptide includes any peptide or protein having two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, which are also commonly referred to in the art as (for example) peptides, oligopeptides, and oligomers; and long chains, which are also commonly referred to in the art as proteins (which have many types). "Polypeptide" includes (for example) bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. These polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0094] A "signal peptide" includes a peptide that directs intracellular transport and localization of any associated polypeptide to, for example, a cellular organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0095] The signal peptide is a peptide of any secretory or transmembrane protein that directs the polypeptide of the present invention 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, wherein 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.

[0096] 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, type II, type III or type IV human protein. In one embodiment, the signal peptide includes an immunoglobulin heavy chain signal peptide.

[0097] "Antigen recognition domain" includes a polypeptide that is selective for a target antigen, target receptor, target peptide ligand or target protein ligand, or target polypeptide.

[0098] The target-specific antigen recognition domain preferably includes an antigen-binding domain derived from an antibody against the target antigen; or a peptide that binds the target antigen; or a peptide or protein that binds an antibody that binds the target antigen; or a peptide or protein ligand (including but not limited to growth factors, interleukins or hormones) that binds a receptor on the target; or a domain derived from a receptor (including but not limited to a growth factor receptor, an interleukin receptor or a hormone receptor) that binds a peptide or protein ligand on the target. The targets include CD2, CD3, CD4, CD5, CD7, CD8 and CD52. In another embodiment, the target includes any portion of CD2, CD3, CD4, CD5, CD7, CD8 and CD52. In one embodiment, the target includes the surface-exposed portion of the CD2, CD3, CD4, CD5, CD7, CD8 and CD52 polypeptides.

[0099] In another embodiment, the target is the extracellular domain of CD2 (SEQ ID NO.19). In another embodiment, the target is the extracellular domain of the CD3ε chain (SEQ ID NO.20). In another embodiment, the target is the extracellular domain of CD4 (SEQ ID NO.21). In another embodiment, the target is the extracellular domain of CD5 (SEQ ID NO.22). In another embodiment, the target is the extracellular domain of CD7 (SEQ ID NO.23). In another embodiment, the target is the extracellular domain of the CD8α chain (SEQ ID NO.24). In another embodiment, the target is the extracellular domain of the CD8β chain (SEQ ID NO.25). In another embodiment, the target is the CD52 CAMPATH-1 antigen (SEQ ID NO.26).

[0100] In one embodiment, the antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody specific for the target (selective for the target).

[0101] In one embodiment, the antigen recognition domain comprises a fragment antigen-binding fragment (Fab). In another embodiment, the antigen recognition domain comprises a single-chain variable fragment (scFv). An scFv is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are joined by a short linker peptide.

[0102] 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 or VHH antibody found in camelids. The VHH antibody of camelids (e.g., camels, dromedaries, llamas, and alpacas) refers to the variable fragment of a camelid single-chain antibody (see Nguyen et al., 2001; Muyldermans, 2001), and also includes isolated VHH antibodies of camelids, recombinant VHH antibodies of camelids, or synthetic VHH antibodies of camelids.

[0103] In another embodiment, the antigen recognition domain comprises a ligand that binds to its cognate receptor. In another embodiment, the antigen recognition domain is humanized.

[0104] The antigen recognition domain may include some variability in 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 are within the scope of the present invention.

[0105] In another embodiment, the antigen recognition domain is selective for SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, or SEQ ID NO.25, or SEQ ID NO.26.

[0106] The hinge region is located in a sequence, for example (including but not limited to), between the chimeric antigen receptor and at least one co-stimulatory domain and signaling domain. The hinge sequence can be obtained (including, for example) from any combined sequence or a portion thereof from any genus (including humans). Such hinge regions are known in the art. In one embodiment, the hinge region comprises the 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, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0107] In one embodiment, the hinge region comprises a CD8 hinge region.

[0108] In some embodiments, the hinge region comprises a hinge region selected from (but not limited to) immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0109] 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 cytoplasm).

[0110] The transmembrane domain can be in the form of an alpha helix or a beta barrel or a combination thereof. The transmembrane domain can comprise a polytopic protein that has multiple transmembrane segments, each an alpha helix, beta sheet, or a combination thereof.

[0111] In one embodiment, a transmembrane domain that binds to one of the domains in the native CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins such that interaction with other members of the receptor complex is minimized.

[0112] For example, the transmembrane domain comprises the transmembrane domain of the T cell receptor alpha or beta chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0113] Artificially designed transmembrane domains mainly comprise polypeptides of hydrophobic residues (such as leucine and valine). In one embodiment, a phenylalanine, tryptophan, and valine triplet is found at each end of the synthetic transmembrane domain.

[0114] In one embodiment, the transmembrane domain is a CD8 transmembrane domain. In another embodiment, the transmembrane domain is a CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0115] The signaling domain and co-stimulatory domain comprise polypeptides that provide activation of immune cells to stimulate or activate at least some aspects of the immune cell signaling pathway.

[0116] In one embodiment, the signaling domain includes a polypeptide of a functional signaling domain of CD3ζ, common FcRγ (FCER1G), FcγRlla, FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, and a combination thereof. Such signaling domains are known in the art.

[0117] In one embodiment, the CAR polypeptide further comprises one or more co-stimulatory domains. In one embodiment, the co-stimulatory domain is from a functional signaling domain of a protein including 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 binding to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), an active fragment thereof, a functional derivative thereof, and a combination thereof.

[0118] In one embodiment, the CAR polypeptide is CD2CAR and includes SEQ ID NO.10 or SEQ ID NO.11. In one embodiment, the CAR polypeptide is CD3CAR and includes SEQ ID NO.12. In one embodiment, the CAR polypeptide is CD4CAR and includes SEQ ID NO.13 or SEQ ID NO.14. In one embodiment, the CAR polypeptide is CD5CAR and includes SEQ ID NO.15. In one embodiment, the CAR polypeptide is CD7CAR and includes SEQ ID NO.17. In one embodiment, the CAR polypeptide is CD52CAR and includes SEQ ID NO.18.

[0119] A polynucleotide encoding a chimeric antigen receptor

[0120] The present invention further provides a polynucleotide encoding the above chimeric antigen receptor polypeptide. The polynucleotide encoding the CAR can be readily prepared from the specified amino acid sequence of the CAR by any conventional method. The underlying sequence encoding the amino acid sequence can be obtained from the above NCBI RefSeq ID or GenBank accession number of the amino acid sequence of each domain, and the nucleic acids of the present invention can be prepared using standard molecular biology and / or chemical procedures. For example, based on the underlying sequence, polynucleotides can be synthesized, and the polynucleotides of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0121] In one embodiment, the polynucleotides disclosed herein are part of a gene, or an expression or cloning cassette.

[0122] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Polynucleotides include DNA and RNA. In addition, nucleic acid is a polymer of nucleotides. Thus, the terms nucleic acid and polynucleotide are used interchangeably herein. Those skilled in the art have the common knowledge that nucleic acids are polynucleotides which can be hydrolyzed into monomeric "nucleotides". These monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include (but are not limited to) all nucleic acid sequences obtained by any method available in the art, including (but not limited to) recombinant methods (i.e., selecting nucleic acid sequences from recombinant libraries or cellular genomes using common cloning techniques and polymerase chain reaction (PCR), etc.) and by synthetic methods.

[0123] In one embodiment, the polynucleotide comprises the CD2CAR polynucleotide of SEQ ID NO.1 or SEQ ID NO.2. In one embodiment, the polynucleotide comprises the CD3CAR polynucleotide of SEQ ID NO.3. In one embodiment, the polynucleotide comprises the CD4CAR polynucleotide of SEQ ID NO.4 or SEQ ID NO.5. In one embodiment, the polynucleotide comprises the CD5CAR polynucleotide of SEQ ID NO.6. In one embodiment, the polynucleotide comprises the CD7CAR polynucleotide of SEQ ID NO.8. In one embodiment, the polynucleotide comprises the CD52CAR polynucleotide of SEQ ID NO.9.

[0124] Polynucleotide vector

[0125] The above polynucleotides can be cloned into a vector. A "vector" comprises an isolated polynucleotide and a composition of matter that can be used to deliver the isolated polynucleotide into the interior of a cell. Many vectors are known in the art, including (but not limited to) linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, phagemids, cosmids, and viruses. Viruses include bacteriophages, bacteriophage derivatives. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This 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.

[0126] In one embodiment, the vector includes a cloning vector, an expression vector, a replication vector, a probe-forming vector, an integration vector, and a sequencing vector.

[0127] 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 modified cell is transduced with the virus to express the polynucleotide sequence.

[0128] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or in vitro to the cells of an individual. 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.

[0129] Viral vector technology is well known and described in the art (e.g., 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 at least one origin of replication function in an organism, a promoter sequence, conventional restriction endonuclease sites, and one or more selectable markers (e.g., WO01 / 96584; WO 01 / 29058 and U.S. Patent No. 6,326,193).

[0130] Expression of the chimeric antigen receptor polynucleotide can be achieved using, for example, expression vectors that include, but are not limited to, at least one of the SFFV or human elongation factor 11α (EF) promoter, CAG (chicken β-actin promoter with CMV enhancer) promoter, human elongation factor 1α (EF) promoter. Examples of promoters with less strength / weaker expression that can be utilized 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 a portion thereof. Inducible expression of the chimeric antigen receptor can be achieved using, for example, a tetracycline-responsive promoter that includes, but is not limited to, TRE3GV (Tet-responsive element, including all generations and preferably the third generation), an inducible promoter (Clontech Laboratories, Mountain View, CA) or a portion or combination thereof.

[0131] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can be operably linked to any polynucleotide sequence thereof to achieve a high level of expression. Another example of a suitable promoter is elongation factor-1α (EF-1α). 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) the 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 inducible promoters should also be considered a part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is needed or turn off the expression when expression is not needed. Examples of inducible promoters include (but not limited to) the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0132] An "expression vector" refers to a vector containing a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0133] Additional promoter elements (e.g., enhancers) regulate the frequency of transcriptional initiation. Generally, these promoter elements are located in the 30-100 bp region upstream of the start site, however many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, such that promoter function is retained when the elements in the thymidine kinase (tk) promoter are inverted or moved relative to one another, and the spacing between promoter elements can be increased to a 50 bp separation after activity begins to decline. Depending on the promoter, individual elements may appear to act cooperatively or independently to activate transcription.

[0134] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector to be introduced into the cells may also contain a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells transfected or infected by the viral vector. In other embodiments, such selectable markers may be carried on separate DNA fragments and used in co-transfection procedures. The selectable marker and the reporter gene may both be flanked by appropriate regulatory sequences to effect expression in the host cell. Suitable selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0135] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or not expressed by the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some easily detectable property (e.g., enzyme activity). The expression of the reporter gene is analyzed at a combined time after the DNA has been introduced into the recipient cells. Exemplary reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared or obtained commercially using known techniques. Generally, a construct having the minimal 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to evaluate the ability of an agent to modulate transcription driven by the promoter.

[0136] Methods for introducing genes into cells and expressing those genes in the cells are known in the art. In the context of an expression vector, the vector can be readily introduced into host cells such as mammalian, bacterial, yeast, or insect cells by any method in the art. For example, the expression vector can be transferred into the host cells by physical, chemical, or biological methods.

[0137] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing the vector and / or exogenous nucleic acid are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0138] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors and, in particular, retroviral vectors have become the most widely used method for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0139] Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). In cases where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids (in vitro, ex vivo, or in vivo) into host cells. In another aspect, the nucleic acid can be bound to a lipid. The nucleic acid bound to a lipid can be encapsulated within the aqueous interior of a liposome; dispersed within the lipid bilayer of a liposome; bound to a liposome via a linking molecule that binds to both the liposome and the oligonucleotide; entrapped within a liposome; complexed with a liposome; dispersed in a solution containing a lipid; mixed with a lipid; combined with a lipid; contained within a lipid as a suspension; contained within a micelle or complexed with a micelle; or in other forms of binding to a lipid. Lipid, lipid / DNA, or lipid / expression vector binding compositions are not limited to any particular structure in solution. For example, they can exist as micelles in a bilayer structure or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that are naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally occurring in the cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0140] The applicable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). The stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. Chloroform is only used as a solvent because it evaporates more easily than methanol.

[0141] "Liposome" is a general term that encompasses various single and multi-layer lipid carriers formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when the phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 191 Glycobiology 5; 505-10). However, also encompassed are compositions having a structure different from the normal vesicle structure in solution. For example, the lipids can assume a micellar structure or exist only as an inhomogeneous aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0142] Regardless of the method used to introduce exogenous polynucleotides into host cells or otherwise expose the cells to the polynucleotides of the present invention, various analyses can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such analyses include, for example, "molecular biology" analyses well known to those skilled in the art, such as blotting hybridization and Western blotting, RT-PCR and PCR; "biochemical" analyses, such as, for example, detecting the presence or absence of a specific peptide by immunological methods (ELISA and Western blotting) or by identifying agents falling within the scope of the present invention by the analyses described herein.

[0143] Engineered cells

[0144] In another embodiment, the present invention provides engineered cells that express the above-described chimeric antigen receptor polypeptides or express polynucleotides encoding such chimeric antigen receptor polypeptides, as described above.

[0145] "Modified cell" means any cell of any organism that has been modified, transformed or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells and genetically modified 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 chimeric antigen receptor complex and express the chimeric receptor on the cell surface. The isolated host cells and modified cells can be used, for example, to enhance NK cell activity or T lymphocyte activity; treat cancer; and treat infectious diseases.

[0146] Any cell that can express the chimeric antigen receptor polypeptide as disclosed herein and / or can integrate the chimeric antigen receptor polypeptide as disclosed herein into the cell membrane can be used.

[0147] In one embodiment, the modified cell includes 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).

[0148] In one embodiment, the modified cell includes natural killer cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK-92 cells. Other examples of NK cell lines include NKG, YT, NKYS, HANK-1, YTS cells and NKL cells.

[0149] NK cells mediate anti-tumor effects without the risk of GvHD and have a shorter survival period compared to T cells. Therefore, NK cells will be immediately depleted after destroying cancer cells, thus reducing the need for an inducible suicide gene for the CAR construct of the modified cells.

[0150] In one embodiment, the modified cell can include more than one type of chimeric antigen receptor polypeptide described herein. Embodiments in which the modified cell includes at least two of CD2CAR, CD3CAR, CD4CAR, CD5CAR, CD7CAR, CD8CAR and CD52CAR have been envisioned. For example, the modified cell can include a CD4 chimeric antigen receptor polypeptide (CD4CAR) and a CD5 chimeric antigen receptor polypeptide (CD5CAR).

[0151] As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain. CDX as used herein can be any one of CD2, CD3, CD4, CD5, CD7, CD8 and CD52.

[0152] TCR-deficient T cells carrying CAR

[0153] In one embodiment, the engineered cells (specifically, allogeneic T cells obtained from a donor) can be modified to inactivate the components of the TCR (T cell receptor) involved in MHC recognition. Thus, TCR-deficient T cells will not cause graft-versus-host disease (GVHD).

[0154] T-antigen-deficient T and NK cells

[0155] T cell lymphoma or T cell leukemia expresses specific antigens, which can be useful targets for such diseases. For example, T cell lymphoma or leukemia expresses CD7, CD2, CD3, and CD5. However, CD7, CD2, CD3, and CD5 are also expressed in CAR T or NK cells (except for CD3 and CD5), which offsets their ability to target these antigens. Suicide can occur in T cells or NK cells with CARs targeting any of these antigens. This makes it difficult to generate CARs targeting these antigens. Therefore, when T or NK cells are used as targets equipped with CARs, it may be necessary to inactivate the endogenous antigens in the T or NK cells.

[0156] In another embodiment, the engineered cells are further modified to inactivate cell surface polypeptides to prevent the engineered cells from acting on other engineered cells. For example, one or more of the endogenous CD2, CD3, CD4, CD5, and CD7 genes in the engineered cells can be knocked out or inactivated. In a preferred embodiment, the engineered cell line has natural killer cells with at least one of the endogenous CD2 and CD7 genes inhibited or inactivated.

[0157] In another preferred embodiment, the engineered cell line has T cells with at least one of the endogenous CD2, CD3, CD4, CD5, CD7, and CD8 genes inhibited or inactivated. In another preferred embodiment, the engineered cells have NK cells with at least one of the endogenous CD2 and CD7 genes knocked out or inactivated.

[0158] In one embodiment, the gene expressing the antigen in the engineered cells expressing a CAR with a specific antigen recognition domain has been inactivated or knocked out. For example, T cells with a CD2 CAR will have the CD2 antigen gene inactivated or knocked out. In another embodiment, engineered cells (e.g., NK cells or T cells) with a CAR containing a CD4 antigen recognition domain will be modified such that the CD4 antigen is not expressed on their cell surface. In another embodiment, in engineered cells (e.g., NK cells or T cells) having one CAR containing a CD2 antigen recognition domain and another CAR containing a CD7 antigen recognition domain, both the CD2 antigen gene and the CD7 antigen gene may have been knocked out or inactivated.

[0159] Natural killer cell T cell CD2 + + CD4 - + CD3 - + CD5 - + CD7 + + CD8 - +

[0160] Methods for knocking out genes or inactivating genes are well known in the art. For example, the CRISPR / Cas9 system, zinc finger nucleases (ZFNs), TALE nucleases (TALENs), and meganucleases can be used to knock out or inactivate the CD2, CD3, CD4, CD5, CD7, CD8, and CD52 genes in engineered cells.

[0161] Cell source

[0162] The engineered cells can be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymus tissue. The host cells can include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. The cells can be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. The cells can be obtained from established cell lines.

[0163] The above cells can be obtained by any conventional method. The cells can be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the engineered cells.

[0164] The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0165] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some aspects, allogeneic materials from individuals of the same species can be antigenically different enough to interact.

[0166] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0167] The term "syngeneic" refers to genetic similarity or identity that is extremely close, particularly with respect to antigens or immune responses. Syngeneic systems include, for example, models in which organs and cells (e.g., cancer cells and their non-cancer counterparts) are from the same individual and / or models in which organs and cells are from different individual animals having the same inbred strain.

[0168] Suicide system

[0169] The modified cells of the present invention may also include a suicide system. The suicide system provides a mechanism by which the above-mentioned modified cells can be deactivated or destroyed. This feature allows for precise therapeutic control of any treatment in which such modified cells are used. As used herein, the suicide system provides a mechanism by which cells having the suicide system can be deactivated or destroyed. Suicide systems are well known in the art.

[0170] In one embodiment, the suicide system includes a gene that can be pharmacologically activated, if desired, to eliminate the cells it contains. In a particular aspect, the suicide gene is not immunogenic to the host having the polynucleotide or cell. In one example, the suicide system includes a gene that causes CD20 to be expressed on the cell surface of the modified cells. Accordingly, administration of rituximab can be used to destroy the modified cells containing the gene.

[0171] In some embodiments, the suicide system includes an epitope tag. Examples of epitope tags include the c-myc tag, streptavidin-binding peptide (SBP), and truncated EGFR gene (EGFRt). In this embodiment, the epitope tag is expressed in the modified cells. Accordingly, administration of an antibody against the epitope tag can be used to destroy the modified cells containing the gene.

[0172] In another embodiment, the suicide system includes a gene that causes truncated epidermal growth factor receptor to be expressed on the surface of the modified cells. Accordingly, administration of cetuximab can be used to destroy the modified cells containing the gene.

[0173] In another embodiment, the suicide gene may include the caspase 8 gene, caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450.

[0174] Examples of other suicide systems include those described by Jones et al. (Jones BS, Lamb LS, Goldman F, and Di Stasi A (2014) Improving the safety of cell therapy products by suicide gene transfer. Front. Pharmacol. 5:254. doi:10.3389 / fphar.2014.00254), which is incorporated herein by reference in its entirety.)

[0175] CD2CAR is a cell surface antigen that expresses CD2 adhesion molecule on all peripheral blood T cells and natural killer cells but not on B lymphocytes. The extracellular domain of CD2 contains an immunoglobulin-like domain that can mediate homodimerization.

[0176] Binding of CD58 (LFA-3) or CD48 to CD2 helps T cells adhere to antigen-presenting cells and triggers a signal transduction pathway that enhances signaling through the T cell receptor against antigens. CD2 knockout mice show normal immune function, and the function of CD2 is believed to be similar to that of other T cell co-stimulatory receptors such as CD28.

[0177] CD2 is expressed on cells of T-ALL, T cell lymphoma / leukemia, acute promyelocytic leukemia (microparticle variant), systemic mastocytosis, mast cell disease, thymoma, and acute myeloid lymphoma (M0) and NK cell leukemia.

[0178] In one embodiment, the present invention provides a chimeric antigen receptor polypeptide having an antigen recognition domain specific for the CD2 antigen, and a modified cell expressing the chimeric antigen receptor polypeptide.

[0179] In another embodiment, the present invention provides a chimeric antigen receptor polypeptide having a variant of the sequence of an antigen recognition domain specific for the CD2 antigen, and a modified cell expressing the chimeric antigen receptor polypeptide.

[0180] In one embodiment, the CD2 CAR comprises at least one co-stimulatory domain. In another embodiment, the CD2 CAR comprises at least two co-stimulatory domains.

[0181] In one embodiment, the CD2CAR comprises SEQ ID NO.10 and SEQ ID NO.11.

[0182] The CD3CAR CD3 consists of a protein complex and is composed of four different chains as described in the figure above. This complex contains the CD3δ chain, the CD3γ chain, and two CD3ε chains. These chains bind to the T cell receptor (TCR) composed of αβ chains.

[0183] The TCR / CD3 complex is a unique marker of T lineage cells. Various monoclonal antibodies against this complex have been developed. One such monoclonal antibody is the murine monoclonal antibody OKT3 against surface CD3. CD3 is a common marker for T cells and T cell malignancies. OKT3 against CD3ε is a common antibody used to identify T cells. Anti-CD3 monoclonal antibodies for therapeutic use include: (1) acute renal, cardiac, or hepatic allograft rejection; (2) depletion of T cells from donor bone marrow before transplantation; (3) new-onset type I diabetes. CD3 against the CD3ε chain is the most specific T cell antibody for identifying T cells in both benign and malignant diseases. CD3 is found in 86% of peripheral T cell lymphomas.

[0184] In some embodiments, the present invention includes a method for generating CD3CAR. In other embodiments, the CD3CAR includes an scFv antibody that specifically binds to a surface protein of CD3.

[0185] In some embodiments, the CD3CAR includes an scFv molecule that specifically binds to the TCR / CD3 complex.

[0186] In some embodiments, the scFv in the CAR can be a molecule that specifically binds to the extracellular domain of the αβ TCR that binds to CD3.

[0187] CD4CAR

[0188] In one embodiment, the chimeric antigen receptor of the present invention includes a CD4 antigen recognition domain (CD4CAR).

[0189] In one embodiment, the CD4 CAR contains at least one co-stimulatory domain. In another embodiment, the CD4CAR contains at least two co-stimulatory domains.

[0190] In one embodiment, the CD4CAR contains SEQ ID NO.13 and SEQ ID NO.14.

[0191] CD5CAR

[0192] In another embodiment, the present invention provides a chimeric antigen receptor polypeptide specifically targeting the CD5 antigen recognition domain, and a modified cell expressing the chimeric antigen receptor polypeptide.

[0193] In one embodiment, the CD5 CAR comprises at least one co-stimulatory domain. In another embodiment, the CD5 CAR comprises at least two co-stimulatory domains.

[0194] CD7 CAR CD7 is a transmembrane protein and is a member of the immunoglobulin superfamily. This protein is expressed on the surface of mature T cells. It is the earliest surface antigen expressed on cells of the T cell lineage. CD7 is an excellent marker for T-ALL and over 90% of T-ALL expresses CD7.

[0195] CD7 is also expressed in NK lymphoma, T cell lymphoma / leukemia, chronic myeloid leukemia, acute myeloid leukemia, and lymphocyte-rich thymoma.

[0196] In one embodiment, the present invention provides a chimeric antigen receptor polypeptide having an antigen recognition domain specifically targeting CD7, and a modified cell expressing the chimeric antigen receptor polypeptide.

[0197] In one embodiment, the CD7 CAR comprises at least one co-stimulatory domain. In another embodiment, the CD7 CAR comprises at least two co-stimulatory domains.

[0198] Method

[0199] Method for manufacturing a modified cell

[0200] In one embodiment, the present invention also provides a method for manufacturing the above-mentioned modified cell.

[0201] In this embodiment, the above-mentioned cells are obtained or isolated. These cells can be isolated by any known method. These cells include peripheral blood cells or umbilical cord blood cells. In another embodiment, these cells are placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0202] The polynucleotide encoding the above-mentioned chimeric antigen receptor polypeptide is introduced into peripheral blood cells or umbilical cord blood cells by any known method. In one example, the polynucleotide encoding the above-mentioned chimeric antigen receptor polypeptide is introduced into the cells by means of a viral vector.

[0203] The polynucleotide encoding the above-mentioned chimeric antigen receptor polypeptide is introduced into placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells by any known method. In one example, the polynucleotide encoding the above-mentioned chimeric antigen receptor polypeptide is introduced into the cells by means of a viral vector.

[0204] In other embodiments, the chimeric antigen receptor polynucleotide can be constructed into a "biodegradable derivative" modified by transient RNA. The RNA-modified derivative can be introduced into T cells or NK cells by electroporation.

[0205] In another embodiment, the chimeric antigen receptors described herein can be constructed in a transposon system (also known as the "Sleeping Beauty") that integrates the chimeric antigen receptor polynucleotide into the host genome without a viral vector.

[0206] Once the above polynucleotides are introduced into cells to provide modified cells, the modified cells are expanded. The modified cells containing the above polynucleotides are expanded by any known method.

[0207] The expanded cells are isolated by any known method to provide the isolated modified cells of the present invention.

[0208] Methods of Use

[0209] The present invention provides methods for killing immunomodulatory cells, reducing the number of immunomodulatory cells, or depleting immunomodulatory cells. In another embodiment, the present invention provides methods for killing cells having at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52, reducing the number of cells having at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52, or depleting cells having at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0210] As used herein, "reducing the number" includes reducing by at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 99%, or 100%.

[0211] As used herein, "depleting" includes reducing by at least 75%, at least 80%, at least 90%, at least 99%, or 100%.

[0212] In one embodiment, the present invention includes a method for reducing the number of immunomodulatory cells having CD2 by contacting the immunomodulatory cells having CD2 with an effective amount of the above modified cells expressing a chimeric antigen receptor peptide having a CD2 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD2 can be determined by any cell death assay known in the art.

[0213] As used herein, the immunomodulatory cells can be present in a patient; can be present in a cell culture or be isolated.

[0214] As used herein, "patient" includes mammals. The mammals referred to herein can be any mammals. 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). Such mammals can be from the order Carnivora, including the Felidae (cats) and Canidae (dogs). Such mammals can be from the order Artiodactyla (including the Bovidae (cows) and Suidae (pigs)) or Perissodactyla (including the Equidae (horses)). Such mammals can be of the order Primates, Ceboid or Simoid (monkeys) or can be of the order Anthropoidea (humans and apes). Preferably, the mammal is a human.

[0215] In certain embodiments, the patient is a human of 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.

[0216] As used herein, the terms "effective amount" and "therapeutically effective amount" of the modified cells mean an amount of the modified cells sufficient to provide the desired therapeutic or physiological effect or result. Such effect or result includes a reduction or improvement in the symptoms of the cell disease. Undesired effects (e.g., side effects) sometimes accompany the desired therapeutic effect; thus, the practitioner should balance the potential benefits and potential risks in determining the "effective amount" appropriate. The precise amount required will vary from individual to individual, depending on the species, age and general condition of the individual, mode of administration, etc. Thus, it may not be possible to specify an exact "effective amount". However, the "effective amount" appropriate in any particular case can be determined by one of ordinary skill in the art using only routine experimentation. Generally, the (such) modified cells are given in an amount and under conditions sufficient to reduce the proliferation of the target cells.

[0217] In one embodiment, the invention includes a method of reducing the number of such immunomodulatory cells by contacting immunomodulatory cells having CD2 with an effective amount of the above-described modified cells expressing a chimeric antigen receptor peptide having a CD2 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD2 can be determined by any cell death assay known in the art.

[0218] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD3 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD3 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD3 can be determined by any cell death assay known in the art.

[0219] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD4 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD4 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD4 can be determined by any cell death assay known in the art.

[0220] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD5 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD5 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD5 can be determined by any cell death assay known in the art.

[0221] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD7 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD7 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD7 can be determined by any cell death assay known in the art.

[0222] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD8 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD8 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD8 can be determined by any cell death assay known in the art.

[0223] In one embodiment, the present invention includes a method of reducing the number of immunomodulatory cells by contacting immunomodulatory cells having CD52 with an effective amount of the above-modified cells expressing a chimeric antigen receptor peptide having a CD52 antigen recognition domain. Optionally, the reduction in the number of immunomodulatory cells having CD52 can be determined by any cell death assay known in the art.

[0224] Treatment methods

[0225] In another embodiment, the present invention provides a method for treating a cell proliferative disease. The method comprises administering to a patient in need thereof a therapeutically effective amount of the engineered cells.

[0226] A cell proliferative disease is any one of cancer, a neoplastic disease, or any disease involving uncontrolled cell proliferation (e.g., formation of a cell mass) where the cells do not differentiate into specific different cells.

[0227] Cell proliferative diseases also include malignant or pre-cancerous conditions (such as myelodysplastic syndromes or pre-leukemia or pre-lymphoma).

[0228] According to the method disclosed herein, the cancer can be any cancer, including any one of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma, malignant mesothelioma, liposarcoma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia and Burkitt's lymphoma, extranodal NK / T-cell lymphoma, NK-cell leukemia / lymphoma, post-transplant lymphoproliferative disorder, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer. Preferably, the cancer is a hematological malignancy (e.g., leukemia or lymphoma, including (but not limited to) Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphocytic cancer, acute myeloid leukemia, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt's lymphoma), thymic cancer, diffuse large cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL), T-cell lymphoma and peripheral T-cell lymphoma.

[0229] The present invention provides a method for treating acute organ rejection by depleting T and NK cells associated with CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0230] In one embodiment, the present invention includes a method for treating acute or chronic graft-versus-host disease (GVHD) by depleting T cells and NK cells associated with at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0231] In one embodiment, the present invention provides a method for preventing organ rejection by administering an effective amount of engineered cells having CD3CAR to a patient who has undergone or will undergo an organ transplant.

[0232] In another embodiment, the present invention provides a method for preventing or treating GVHD by administering an effective amount of engineered cells having CD3CAR to a patient in need thereof.

[0233] In one embodiment, the present invention includes a method for using CAR T or NK cells in vivo for stem cell transplantation to deplete or reduce donor and host T or NK cells. This can be accomplished by administering CAR T or NK cells to the patient immediately prior to infusion of the bone marrow stem cell graft.

[0234] The present invention provides immunotherapy as a regulatory or pre-transplant bridging strategy or for independently treating proliferative diseases associated with at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0235] The present invention provides a method for treating proliferative diseases associated with at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0236] In another embodiment, the present invention provides a method for treating non-cancer-related diseases associated with the expression of at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

[0237] In some embodiments, CARs having antigen recognition domains for CD2, CD3, CD4, CD5, CD7, CD8, or CD52 for treating proliferative diseases are combined with checkpoint blockade (such as CTLA-4 and PD1 / PD-L1). This can result in enhanced tumor eradication.

[0238] The presence of an immunosuppressive microenvironment can limit the full function of CAR T / NK cells. In some embodiments, the combination of CD4CAR with checkpoint blockade (such as CTLA-4 and PD1 / PD-L1) can result in enhanced tumor eradication.

[0239] Currently, checkpoint blockade is being tested in combination with CAR T cells in clinical trials.

[0240] In some embodiments, CARs having antigen recognition domains of CD2, CD3, CD4, CD5, CD7, CD8, or CD52 are used as a strategy to deepen, remove, reduce, resist, and / or prolong the response to initial chemotherapy, or when used in combination with other adjuvant therapies. All adjuvant therapies available for treating or preventing disease conditions are considered part of and within the scope of the present invention.

[0241] In some embodiments, NK cell CARs having antigen recognition domains of CD2, CD3, CD4, CD5, CD7, CD8, or CD52 are "off-the-shelf" administered to any mammal suffering from cancer and / or an autoimmune disease.

[0242] CD3CAR

[0243] In some embodiments, NK cells carrying CD3 CAR exhibit anti-tumor immunity and exert efficacy in killing CD3-expressing leukemia / lymphoma.

[0244] The present invention provides methods for using CD3CAR NK cells to delete or reduce abnormal or malignant T cells in bone marrow, blood, and organs. In some embodiments, CD3-positive malignancies can include (but are not limited to) precursor T lymphoblastic leukemia / lymphoma, mature T cell lymphoma / leukemia, EBV-positive T cell lymphoproliferative disease, adult T cell leukemia / lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disease, peripheral T cell lymphoma (not otherwise specified), angioimmunoblastic T cell lymphoma, and anaplastic large cell lymphoma.

[0245] In some embodiments, CD3CAR NK cells can be used to treat T-leukemia / lymphoma patients who are ineligible for stem cell therapy or have undergone numerous intensive chemotherapy regimens but have never achieved remission. In other embodiments, CD3CAR NK cells can be used as a component of a conditioning regimen for bone marrow transplantation or as a bridging therapy before bone marrow transplantation.

[0246] CD4CAR

[0247] In one embodiment, the engineered cell with CD4 CAR exhibits anti-tumor immunity when the antigen recognition domain of the CAR binds to its corresponding antigen. In a preferred embodiment, the CD8 T cells comprising the CAR are effective in killing CD4-expressing leukemia / lymphoma cells.

[0248] The present invention includes methods for deleting, reducing, treating, preventing or eliminating abnormal or malignant T cells found in, including but not limited to, bone marrow, blood and / or organs. In some embodiments, malignant CD4-expressing cells are present in patients with: precursor T lymphoblastic leukemia / lymphoma, mature T cell lymphoma / leukemia cells, such as (for example) T cell prolymphocytic leukemia, EBV-positive T cell lymphoproliferative disease, adult T cell leukemia / lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disease, peripheral T cell lymphoma (not otherwise specified), angioimmunoblastic T cell lymphoma, and anaplastic large cell lymphoma.

[0249] In some embodiments, the CD4CAR cells are used to treat T-leukemia / lymphoma cells in patients who do not qualify for stem cell therapy or have undergone numerous intensive chemotherapy regimens but have never achieved remission.

[0250] In some embodiments, the CD4CAR cells are used to treat acute myelomonocytic leukemia, acute monoblastic leukemia, monocytic leukemia, and chronic myelomonocytic leukemia expressing CD4.

[0251] In some embodiments, the CD4CAR T cells can be expanded in a T cell culture medium and subpopulations, such as central memory T cells or naive T cells, can be isolated and used to improve engraftment. These cells can retain and support memory T cell functions, which will make them ideal candidates for the long-term control of cancer.

[0252] The presence of an immunosuppressive microenvironment can limit the full function of CAR T / NK cells. In some embodiments, the combination of CD4CAR with checkpoint blockade, such as CTLA-4 and PD1 / PD-L1, can lead to enhanced tumor eradication.

[0253] In some embodiments, the CD4CAR cells are used as a strategy to deepen, remove, reduce, resist, and / or prolong the response to initial chemotherapy, or are used when combined with other adjuvant therapies. All adjuvant therapies available for treating or preventing disease conditions are considered part of and within the scope of the present invention. Chemotherapy includes (but is not limited to) CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or any other multi-drug regimen. In a preferred embodiment, the CD4CAR cells are used to treat or prevent residual disease after stem cell transplantation and / or chemotherapy.

[0254] In one embodiment, cells comprising CD4CAR exhibit depletion of immunomodulatory cells when the antigen recognition domain of the CAR binds to its corresponding antigen. For example, cells comprising CD4CAR include (but are not limited to) at least one of CD8 T cells, NK cells, or NK-92 cells. Any other combined cells having CD4CAR that exhibit and / or exert efficient deletion of such CD4 helper cells when encountering CD4 helper cells, thereby preventing organ transplant rejection or controlling or alleviating autoimmune diseases, are considered part of and within the scope of the present invention.

[0255] There is no concern about persistent CAR-related side effects observed in CAR T cells. In some embodiments, CD4CAR NK cells can be administered to patients with autoimmune diseases in an acute or definitive clinical setting to rapidly deplete immunomodulatory cells (such as CD4 helper T cells), and thereby enable or allow the regeneration of naive or non-memory CD4 helper T cells.

[0256] The present invention includes a method for generating CD4CAR. In some embodiments, CD4CAR is generated using T cells. In other embodiments, CD4CAR is generated using NK cells or NK-92 cells such that they are "off-the-shelf" for administration to any mammal with cancer and / or autoimmune diseases. In some embodiments, CD4CAR NK-92 or NK cells can kill cells, reduce, deplete, and / or prevent specific CD4+ T cells or CD4-expressing cancer cells.

[0257] In some embodiments, CD4CAR NK-92 cells with a high expression level of CD4CAR can be generated by flow cytometry using goat-anti-mouse Fab antibody or a portion thereof. Any other type of antibody generated using any other genus is considered part of and within the scope of the present invention.

[0258] In some embodiments, CD4CAR NK-92 cells can be used in a therapy when there is minimal residual disease after stem cell transplantation or chemotherapy.

[0259] In some embodiments, the CD4CAR is part of an expression gene or an expression cassette. In a preferred embodiment, the expression gene or the expression cassette may also contain a helper gene or an epitope tag or a part thereof in addition to the CD4CAR. The helper gene can be an inducible suicide gene or a part thereof, including (but not limited to) caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450 29. The "suicide gene" elimination method improves the safety of gene therapy and kills cells only when activated by a specific compound or molecule. In some embodiments, the suicide gene is inducible and is activated using a specific chemical inducer of dimerization (CID).

[0260] In some embodiments, the helper tag is a c-myc tag, a truncated EGFR gene (EGFRt), or a part thereof or a combination thereof. The helper tag can be used as a nonimmunogenic selection tool or as a tracking marker.

[0261] In some embodiments, the host cells expressing CD4CAR can be administered to a mammal (e.g., a human) together with one or more additional therapeutic agents. In this regard, a composition containing the host cells or vectors containing CD4CAR can be administered first, and one or more additional therapeutic agents can be administered second, or vice versa.

[0262] The present invention encompasses within its scope the administration to a mammal of a typical amount of host cells expressing CD4CAR, which typical amount can be, for example, in the range of 500,000 to 1 billion cells. All subranges and ranges outside the ranges indicated above are considered part of the present invention and are within the scope of the present invention.

[0263] In a preferred embodiment, the SFFV promoter is used to redirect CD8 T cells to CD4-expressing target cells and drive CD4CAR expression. In some embodiments, the CAR includes functional characteristics such as extracellular expression of scFv and exerting a strong immune response when encountering CD4-expressing cells.

[0264] In one embodiment, the cells containing CD4CAR are selected from the group including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. In a preferred embodiment, the cells having CAR include (but not limited to) CD8 T cells, NK cells, and NK-92 cells.

[0265] In some embodiments, the CD4CAR can be used in conjunction with a drug conjugate (including a DNA / nucleic acid conjugate, a peptide, a chemical entity, and / or a small molecule) to provide enhanced efficacy and safety.

[0266] HIV-1 infection control in HIV patients can be achieved using a combination of antiretroviral therapies. However, the viral load increases after discontinuation. The source or reservoir of the re-emerging HIV-1 is memory CD4 T cells. In one embodiment, the CD4CAR of the present invention is used to deplete memory CD4 T cells, thereby achieving a sterilizing cure for HIV infection. In another embodiment, the CD4CAR helps to block the entry of the HIV virus, and the CD4CAR binds to the CD4 protein (a protein necessary for HIV entry).

[0267] Accordingly, the present invention provides a method for preventing organ transplant rejection by depleting CD4 T cells. The method includes administering to a patient in need thereof a therapeutically effective amount of a modified cell having a chimeric antigen receptor polypeptide containing a CD4 antigen recognition domain.

[0268] CD5CAR

[0269] In another embodiment, a CAR polypeptide having a CD5 antigen recognition domain (CD5CAR) is used for the treatment of rheumatoid arthritis. In another embodiment, CD5CAR can be used to prevent graft-versus-host disease after bone marrow transplantation therapy (BMT). In another embodiment, CD5CAR can be used to modify CD5 expression in the treatment of autoimmune diseases and malignancies.

[0270] In some embodiments, the modified cells of the present invention having a chimeric antigen receptor selective for CD5 can serve as a bridging therapy for bone marrow transplantation for patients who no longer respond to chemotherapy or have minimal residual disease and are not eligible for bone marrow transplantation. In other embodiments, CD5CAR can eliminate CD5-positive leukemia cells, followed by bone marrow stem support to support lymphopenia.

[0271] In certain embodiments, CD5CAR T or NK cells target cells expressing CD5. The target cells can be (but are not limited to) cancer cells, such as T cell lymphoma or T cell leukemia, precursor acute T cell lymphoblastic leukemia / lymphoma, B cell chronic lymphocytic leukemia / small lymphocytic lymphoma, mantle cell lymphoma, CD5-positive diffuse large B cell lymphoma, and thymic carcinoma.

[0272] In one embodiment, CD5CAR can be used to treat non-blood diseases, including (but not limited to) rheumatoid arthritis, graft-versus-host disease, and autoimmune diseases.

[0273] Such engineered or modified T cells can be expanded in the presence of IL-2 or / and both IL-7 and IL-15 or using other molecules.

[0274] The introduction of the CAR can be accomplished before or after inactivation of CD5 by expanding the engineered T cells in vitro before administration to the patient.

[0275] In certain embodiments, inactivation of CD5 can be achieved by any of the following methods:

[0276] (1) Expressing an anti-CD5 scFv on the surface of the T cell linked via a hinge region to the transmembrane domain. This can result in the conversion of CD5-positive T cells into CD5-negative T cells.

[0277] (2) Expressing an anti-CD5 scFv that specifically binds to the CD5 protein or its negative regulator of CD5 or a fragment or domain thereof.

[0278] In some embodiments, the anti-CD5 scFv (single-chain antibody) is derived from a monoclonal or polyclonal antibody that binds to intracellular CD5 and blocks the transport of the CD5 protein to the cell surface. In a preferred embodiment, the anti-CD5 scFv includes the ER (endoplasmic reticulum) retention sequence KDEL. When the anti-CD5 scFv is expressed intracellularly and retained in the ER or Golgi, it traps CD5 in the secretory pathway, which results in preventing the cell surface localization of CD5 itself in T cells.

[0279] In some embodiments, the CD5 CAR T cells are co-administered with immunomodulatory drugs (such as, but not limited to, CTLA-4 and PD-1 / PD-L1 blockade) or cytokines (such as IL-2 and IL12) or inhibitors of colony-stimulating factor-1 receptor (CSF1R) (such as FPA008), which results in better therapeutic outcomes.

[0280] In another embodiment, the present invention provides a method for conferring, assisting, increasing, or promoting anti-leukemia or anti-lymphoma immunity.

[0281] A therapeutic agent comprising an engineered cell expressing a CAR as an active ingredient can be administered intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or into the afferent lymphatics, by parenteral administration (e.g., by injection or infusion), although the route of administration is not limited.

[0282] Any method of the present invention can further comprise the step of administering to the individual an additional cancer therapy (such as surgery, radiotherapy, hormone therapy, chemotherapy, immunotherapy, or a combination thereof).

[0283] Chemotherapy includes, but is not limited to, CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or any other multi-drug regimen.

[0284] In a preferred embodiment, the CD54CAR cells are used to treat or prevent residual disease after stem cell transplantation and / or chemotherapy.

[0285] In another embodiment, any method of the invention may further include antiviral therapy: cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients.

[0286] In other aspects, the T cells of the invention can be combined with chemotherapy, radiotherapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immune depletion agents such as CAMPATH, anti-CD3 antibody or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation in a treatment regimen. Drugs that inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (which is important for signaling induced by growth factors (rapamycin)) can also be used (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immunol. 5:763-773, 1993).

[0287] In another aspect, the cell compositions of the invention are administered to a patient in combination with (e.g., before, simultaneously with, or after administration) bone marrow transplantation, T cell depletion therapy using chemotherapeutic agents (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide) or antibodies (such as OKT3 or CAMPATH). In one aspect, the cell compositions of the invention are administered after B-cell depletion therapy (such as an agent reactive with CD20, e.g., Rituxan). For example, in one embodiment, an individual may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the individual receives an infusion of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0288] As used herein, the term "autoimmune disease" is defined as a disease caused by an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include (but are not limited to) Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0289] The invention may be better understood with reference to the examples set forth below. The following examples are presented to provide 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 limiting of the invention.

[0290] After a delivery system for treating, inhibiting, or preventing cancer, the efficacy of the therapeutically modified cells can be evaluated by a variety of methods well known to those of ordinary skill in the art. For example, one of ordinary skill will understand that therapeutically modified cells delivered in combination with a chemical adjuvant are effective in treating or inhibiting cancer in an individual by observing that the therapeutically modified cells reduce the cancer cell load or prevent further increase in the cancer cell load. The cancer cell load 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 identifying certain cancer cell markers in the blood, using, for example, antibody assays to detect the presence of the markers in a sample (e.g., but not limited to, blood) from the individual or patient, or by measuring the level of circulating cancer cell antibodies in the patient.

[0291] Throughout this specification, ranges are defined by, and are inclusive of, 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.

[0292] As used herein, the terms "one embodiment," "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," "an example," or "an example" throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and / or sub-combination in one or more embodiments or examples. Additionally, it should be understood that the drawings provided herein are for illustrative purposes to those of ordinary skill in the art and are not necessarily drawn to scale.

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

[0294] In addition, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or 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 schematics given herein should not be construed as in any way limiting or representing the definition of any term using them. On the contrary, such examples or schematics should be regarded as being described for a specific embodiment and being merely illustrative. One of ordinary skill in the art will understand that any term using such examples or schematics will encompass other embodiments that may or may not be given subsequently or elsewhere in this 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 schematics includes (but is not limited to): "for example", "for instance", "e.g.", and "in one embodiment". In this specification, various parameter groups containing multiple members are described. In a group of parameters, each member can be combined with any one or more of the other members to create additional subgroups. For example, if the members in a group are a, b, c, d, and e, then additional subgroups that are explicitly contemplated include any one, two, three, or four of those members, e.g., a and c; a, d, and e; b, c, d, and e, etc.

[0296] Example

[0297] Targeting human T-cell malignancies using T cells engineered with a CD4-specific chimeric antigen receptor (CAR)

[0298] Materials and methods

[0299] Blood donors, primary tumor cells, and cell lines Human lymphoma cells and peripheral blood mononuclear cells were obtained from residual samples. Umbilical cord blood cells were obtained from donors at Stony Brook University Hospital. The SP53 and KARPAS 299 lymphoma cell lines were obtained from ATCC (Manassas, VA).

[0300] Lentivirus production and transduction of T cells To produce virus supernatant, 293FT cells were co-transfected with pMD2G and pSPAX virus packaging plasmids and the pRSC.CD4.3G or GFP lentiviral vector using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the experimental procedures provided by the manufacturer. Before lentiviral transduction, umbilical cord blood or peripheral blood mononuclear cell buffy coat cells were activated for two days in the presence of 300 IU / mL IL-2 and 1 μg / mL anti-human CD3 (Miltenyi Biotec, Germany).

[0301] T cell expansion

[0302] Expand CAR-transduced T cells in T cell medium supplemented with IL-2 (50% AIMV, 40% RPMI 1640, 10% FBS, and 1x penicillin / streptomycin; all from Gibco) for 7 days. Count cells daily and add medium every 2 to 3 days to keep the T cell count below 2 x 106 cells / mL.

[0303] CAR immunophenotype

[0304] To analyze the CAR cell immunophenotype, after 7 days of expansion, stain CD4 CAR T cells and GFP control cells with CD45RO, CD45RA, CD62L, and CD8 (all from BD Biosciences) for flow cytometry analysis.

[0305] Co-culture target cell elimination assay

[0306] In 1 mL of T cell medium without IL-2, co-culture CD4 CAR T cells or GFP T cells (control group) with target cells at ratios of 2:1, 5:1, and 10:1 (200,000, 500,000, or 1 million effector cells to 100,000 target cells, respectively) for 24 h. Target cells are KARPAS 299 cells (anaplastic large T cell lymphoma expressing CD4), leukemic cells from patients with CD4+ T cell leukemia (Sezary syndrome), and leukemic cells from patients with CD4+ PTCL lymphoma. As a negative control group, in a separate 1 mL reaction, also co-culture CD4 CAR T cells and GFP T cells with SP53 (mantle cell lymphoma) cells that do not express CD4 at the same ratios. After 24 h of co-culture, stain the cells with mouse anti-human CD8 and CD4 antibodies.

[0307] In experiments with SP53 cells, the SP53 cell line is labeled with CMTMR (Life Technologies) before co-culture with T cells, and T cells are labeled with mouse anti-human CD3 (PerCp) after co-culture.

[0308] In vivo murine xenograft model

[0309] NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) from the Jackson Laboratory were used according to an agreement approved by the Stony Brook University IACUC. All mice were male and between 8 and 12 weeks of age. Three in vivo experiments were conducted without blinding. For each group, 10 mice were irradiated with a sublethal (2.5 Gy) dose of γ-rays and randomly assigned to the treatment or control group. Twenty-four hours later, the mice were injected intradermally with 0.5x10 6 or 1.0x10 6 KARPAS 299 cells to form measurable subcutaneous tumors within 7 days. Tumor size area was measured every other day. In the first group, three days after injection of 1 million KARPAS 299 cells, 2 million CD4CAR T (5 mice) or 2 million GFP T control group cells (5 mice) were injected intravenously (by tail vein injection) into the mice. A second dose of 8 million cells was injected intravenously on day 22. In the second group, 10 NSG mice were irradiated and injected with 0.5x10 6 KARPAS 299 cells. On day 2, a course of 8 million CD4CAR T cells (5 mice) and 8 million GFP T control group cells (5 mice) were injected intravenously into the mice. A second dose of 5.5 million cells was injected intravenously on day 10. In the third group, 10 NSG mice were irradiated and injected with 0.5x10 6 KARPAS 299 cells. On day 1, 2.5x10 6 CD4CAR T cells or GFP T control group cells (5 mice per group) were injected intravenously into the mice. Intravenous injection was repeated every 5 days for a total of four courses.

[0310] Results

[0311] Generation of the third-generation CD4CAR

[0312] The nucleotide sequence of the scFv (single-chain variable fragment) against the CD4 molecule was derived from the humanized monoclonal ibalizumab (also known as Hu5A8 or TNX-355). This monoclonal antibody has been used in various phase I or II clinical trials. To improve signal transduction through CD4CAR, the intracellular domains of the CD28 and 4-1BB co-stimulatory factors were fused to the CD3ζ signaling domain. In addition, the CD8 leader sequence was introduced to efficiently express the CD4CAR molecule on the cell surface. In fact, the anti-CD4 scFv was linked by a CD8-derived hinge (H) and transmembrane (TM) region ( Figure 1A) is connected to the intracellular signaling domain. The CD4CAR DNA molecule is subcloned into a lentiviral plasmid. Since there are two co-stimulatory domains (CD28 and 4-1BB), the CD4CAR is regarded as a third-generation CAR. CD4CAR expression is controlled by a strong SFFV (spleen focus-forming virus) promoter and is suitable for blood applications.

[0313] Characterization of CD4CAR

[0314] To confirm the CD4CAR construct, transfected 293-FT cells were analyzed by Western blotting. Immunoblotting with an anti-CD3ζ monoclonal antibody showed a band of the expected size corresponding to the CD4CAR CD3ζ fusion protein ( Figure 1B ). As expected, no CD3ζ expression was observed for the GFP control vector ( Figure 1B ). The transduction efficiency of the generated CD4CAR lentivirus in HEK293 cells was also tested by flow cytometry against the scFv ( Figure 6 ). Thus, we confirmed that our generated third-generation CD4CAR contains the CD3ζ intracellular domain at the intracellular end and the scFv at the extracellular end, meaning that all other elements are present: the CD8 hinge domain and transmembrane domain and the CD28 and 4-1BB co-stimulatory domains ( Figure 1C ). For preclinical characterization of CD4CAR expression and function in T cells, human T cells were activated with anti-CD3 antibody and IL-2 and then transduced with CD4CAR and GFP control lentiviral supernatants respectively. The T cells were expanded for 7 days after transduction.

[0315] CD4CAR T cells derived from umbilical cord blood are highly enriched for CD8+ T cells and most of them have a central memory T cell immunophenotype.

[0316] Human umbilical cord blood (CB) is an alternative source for allogeneic T cell therapy. Human CB buffy coat layer cells were activated and transduced with CD4CAR or control (GFP) lentivirus. After transduction, CD4CAR T cells and GFP T cells were expanded for 7 days, and a 20-fold increase in cell count was observed for both CD4CAR and GFP T cells (Figure 7). At day 7, cells were analyzed by flow cytometry for T cell subsets ( Figure 2A ). Flow cytometry analysis showed that ~54% of the T cells expressed CD4CAR ( Figure 2B ). In addition, we analyzed the CD4 and CD8 subsets during the process of T expansion after CD4CAR transduction. Consistent with previous findings, a small subset of CD8 cells was induced to express CD4 during T cell activation with anti-CD3 and co-stimulatory molecules ( Figure 2C)。As expected, compared to the GFP control group (where ~33% of the cells retained CD4+( Figure 2C ))), this CD4+ T subset was almost completely depleted within 3 or 4 days after CD4CAR transduction. These data indicate that CD4CAR T cells display potent in vitro anti-CD4 activity during T cell expansion.

[0317] We also evaluated the immune phenotype of CD4CAR T cells at the end of each culture. After stimulation, naïve T cells lose CD45RA and acquire CD45RO to become central memory T cells. Flow cytometry analysis from 3 representative experiments showed that 96% of the expanded T cells were CD45RO+, ~83% were CD62L+ and ~80% were CD8+CD45RO+CD62L+, whereas less than 4% were CD45RA+( Figure 2D ). This CD8+CD45RO+CD62L+ immune phenotype is consistent with the acquisition of a central memory-like phenotype, and the low CD45RA+ expression confirms the loss of the naïve T cell state.

[0318] CD4CAR T cells derived from cord blood specifically kill CD4-expressing leukemia / lymphomas, including anaplastic large cell lymphoma, Sézary syndrome, and unclassified PTCL lymphoma.

[0319] Generate CD4CAR T cells highly enriched for CD8+ T cells( Figure 2C ). Then, the anti-leukemia function of these cells was tested in vitro using the KARPAS 299 cell line. The KARPAS 299 cell line was originally established from the peripheral blood of a patient with anaplastic large T cell lymphoma expressing CD4. Cytogenetic analysis had previously shown that KARPAS 299 cells have numerous cytogenetic abnormalities. During the co-culture experiment, CD4CAR cells showed profound leukemia cell killing( Figure 3A ). First, the ability of CB-derived CD4CAR T cells to eliminate KARPAS 299 cells was tested. Indeed, after 24 h of culture and at a low E:T (effector cell: target cell) of 2:1, CD4CAR cells successfully eliminated KARPAS 299 cells. As a control, the ability of these CD4CAR T cells to eliminate CD4-negative lymphoma cells was also tested. The SP53 mantle cell lymphoma cell line is a human B-cell lymphoma cell line that does not express CD4. Flow cytometry analysis showed that CD4CAR T cells were unable to lyse or eliminate the SP53 mantle cell lymphoma( Figure 3D ).

[0320] Studies were also conducted using patient samples. Patient 1 had an aggressive form of CD4+ T cell leukemia (Sezary syndrome) that did not respond to standard chemotherapy. Patient 2 had an unspecified CD4+ PTCL lymphoma. Flow cytometry analysis of both patient samples showed strong and uniform CD4 expression, and almost all leukemia cells expressed CD4( Figure 3B and C). As visualized by flow cytometry analysis, co-culture of the patient samples with CD4CAR for 24 hours led to the rapid and complete elimination of CD4+ malignancies, and once again, approximately 98% elimination was observed for both co-cultures of Sezary syndrome and PTCL, which was consistent with the elimination of KARPAS shown previously( Figure 3B and 3C ). Thus, we showed that in co-culture assays, CD4CAR T cells efficiently eliminated two different types of aggressive CD4+ lymphoma / leukemia cells directly from patient samples even at a low E:T ratio of 2:1( Figure 3B and 3C ). These data support that CD4 is a promising therapeutic target for CD4-positive T cell leukemia and lymphoma, similar to the role of CD19 in targeting B-cell malignancies via anti-CD19 CAR. Thus, our patient sample and CD4CAR co-culture analysis extends the concept of using CAR to target CD4-positive malignancies.

[0321] CD4CAR T cells derived from PBMCs clearly killed tumor cell lines expressing CD4.

[0322] Since autologous CAR T therapy is commonly used in the clinic, we tested CD4CAR T cells derived from PBMCs (peripheral blood mononuclear cells). PBMCs were activated and transduced with CD4CAR lentivirus. These CD4 and CD8 populations were monitored by flow cytometry during cell expansion and compared with the monitoring results of cells transduced with the control GFP. Similar to what was observed for CD4CAR T cells derived from CB, PBMC-derived CD4CAR T cells were also highly enriched for CD8+ T cells( Figure 4A), this indicates the role of CD4CAR in the depletion of CD4+. The ability of PBMC-derived CD4CAR cells to eliminate CD4-positive leukemia / lymphoma cells was subsequently tested using the KARPAS 299 cell line. This elimination assay involved co-culturing CD4CAR T cells or GFP T cells with KARPAS 299 cells and with a negative control of the SP53-coated cell lymphoma cell line. The reaction was stopped after 24 hours: dead cells were stained with 7-AAD (7-aminoactinomycin D) and live cells were analyzed by flow cytometry. KARPAS 299 cells co-cultured overnight with CD4CAR T cells were eliminated at rates of 38%, 62%, and 85% at E:T ratios of 2:1, 5:1, and 10:1, respectively ( Figure 4B ). These combined data confirm a strong dose-response relationship. When the target cells were co-cultured with GFP control T cells, no killing of KARPAS 299 cells was observed. These results confirm that CD4CAR T cells are specific for eliminating CD4+ targets.

[0323] CD4CAR T cells show significant in vivo anti-tumor activity.

[0324] To evaluate in vivo anti-tumor activity, we developed a xenogeneic mouse model using the KARPAS 299 cell line. Multiple different settings were used to test the efficacy of CD4CAR T cells in vivo. We first tested the ability of CD4CAR T cells to delay the appearance of leukemia in NSG mice with a single low dose. Before injection, as confirmed by flow cytometry analysis, the modified T cells showed that ~40 to 50% of the cells expressed CD4CAR. The mice received an intradermal injection of KARPAS 299 cells and then a single systemic injection (intravenous administration) of a low dose (2 million) of CD4CAR T cells. Single low-dose systemic administration of CD4CAR T cells to mice with leukemia caused only transient shrinkage of the leukemia mass or delayed the appearance of the leukemia mass ( Figure 5A ). When leukemia growth began to accelerate, additional courses of administration of 8x10 6 CD4CAR T cells significantly inhibited leukemia growth ( Figure 5A ).

[0325] To further test the efficacy of CD4CAR anti-leukemia activity, we administered two courses of relatively large doses of CD4CAR T cells. Similarly, two injections of a total of 13.5x10 6 CD4CAR T cells caused deeper inhibition of leukemia growth compared to lower CD4CAR doses, but ultimately the leukemia cell population recovered ( Figure 5B ). Finally, we studied low-dose CD4CAR T cells (2.5x10 each)6 The efficacy of multiple-course injections of (cells). After a total of 4 injections every 4 or 5 days, we treated mice with subcutaneous leukemia by repeated intravenous injection of CD4CAR T cells. After four courses of CD4CAR T cell administration, one of the four treated mice had no tumors and showed no toxic appearance. Compared with a single dose, mice treated with multiple doses of CD4CAR T cells showed a more significant anti-leukemia effect ( Figure 5C and 5A). In addition, compared with treatment with GFP-transduced control T cells, treatment with CD4CAR T cells significantly prolonged the survival of mice with KARPAS 299 lymphoma ( Figure 5D ).

[0326] Methods for highly efficient targeting of T cell malignancies by anti-CD4 chimeric antigen receptor (CD4CAR) NK cells in preclinical models Materials

[0327] Primary tumor cells and cell lines

[0328] Human leukemia cells were obtained from residual samples according to an agreement approved by the Institutional Review Board of Stony Brook University. Umbilical cord blood cells were also obtained from donors at Stony Brook University Hospital according to an agreement. Written informed consent was obtained from all donors. The Karpas 299, HL-60, CCRF-CEM, MOLT4, and NK-92 cell lines were obtained from ATCC (Manassas, VA). NK-92 cells were cultured in filtered NK cell medium, which, unless otherwise stated, was defined as α-MEM supplemented with IL-2 (300 IU / mL) without ribonucleosides and deoxyribonucleosides but containing the following: 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 12.5% heat-inactivated horse serum, 12.5% heat-inactivated FBS, 1X Pen / Strep, 0.2% inositol, 0.02% folic acid, and 50 μM β-mercaptoethanol. Karpas 299, CCRF-CEM, and MOLT4 cells were cultured in RPMI, 10% FBS, 1x Pen / Strep (Gibco, Waltham, MA, USA). HL-60 cells were cultured in IMDM, 10% FBS, 1x Pen / Strep (Gibco, Waltham, MA, USA).

[0329] CAR construct generation

[0330] The CD4-targeted CAR (pRSC.SFFV.CD4.3G) is designed to contain an intracellular CD28 domain upstream of the 4-1BB and CD3ζ domains, making this construct a third-generation CAR.

[0331] Lentivirus production and transduction

[0332] To generate virus supernatant, Lipofectamine 2000 (Life Technologies, Carlsbad, CA) was used according to the experimental procedures provided by the manufacturer to co-transfect 293FT cells with the pMD2G and pSPAX virus packaging plasmids and the pRSC.CD4.3G or GFP lentiviral vector. Before transduction with the virus supernatant, NK cells were cultured in the presence of 300 IU / mL IL-2 for at least 2 days. The transfection and transduction procedures are further described in the Supplementary Materials.

[0333] Detection of CAR on transduced NK cells

[0334] To determine CAR expression, 3 days after transduction, NK cells were washed and resuspended in FAC buffer (0.2% BSA in DPBS). Normal goat IgG (Jackson Immunoresearch, West Grove, PA) was used to block non-specific binding. Each NK cell sample was probed with biotinylated polyclonal goat anti-mouse F(Ab’)2 (1:250, Jackson Immunoresearch, West Grove, PA) for 30 minutes at 4°C. The cells were washed once and resuspended in FAC buffer. Then the cells were stained with PE-conjugated streptavidin (1:250, Jackson Immuno Research, West Grove, PA) for 30 minutes at 4°C. The cells were washed with FAC buffer and resuspended in 2% formalin. Flow cytometry was performed using a FACCalibur instrument (Becton Dickinson, Franklin Lakes, NJ) and the results were analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0335] Co-culture analysis

[0336] CD4CAR or vector control group NK cells were cultured with CD4-expressing Karpas 299 cells (anaplastic large T-cell lymphoma), HL-60 cells (acute promyelocytic leukemia), CCRF-CEM cells (T-cell acute lymphoblastic leukemia: T-ALL), CD4+ T cells isolated from human umbilical cord blood, or primary human leukemia cells expressing CD4 (adult Sézary syndrome and pediatric T-ALL) at ratios of 2:1 and 5:1 (200,000 and 500,000 effector cells to 100,000 target cells respectively) in 1 mL of NK-cell medium without IL-2. After 24 hours of co-culture, the remaining live cells were harvested and stained with mouse anti-human CD56 and CD4 antibodies and incubated at 4°C for 30 minutes. CD56+ single positivity indicates NK cells and CD4+ single positivity indicates target cells. All cells were washed with FAC buffer, suspended in 2% formalin and analyzed by flow cytometry.

[0337] Cytotoxicity assay

[0338] CD4CAR or vector control group NK cells were cultured with a 50:50 mixture of target cells (CFSE-stained Karpas 299 cells and CMTMR-stained CCRF-CEM cells) and off-target MOLT4 cells labeled with CMTR at effector cell:target cell ratios of 1:1, 1:2, and 1:4 in 1 mL of NK-cell medium without IL-2. After 24 hours, the cells were stained with 7-AAD (BioLegend, San Diego, CA), washed with FAC buffer and analyzed by flow cytometry for live 7-AAD negative cells.

[0339] Colony-forming unit (CFU) assay

[0340] CD4CAR NK cells and 500 CD34+ CB cells were co-cultured at co-culture effector cell:target cell ratios of 2:1 and 5:1 in NK cell medium supplemented with IL-2 for 24 hours. The controls used were CD34+ cells alone and untransduced NK cells co-cultured with CD34+ CB cells at effector cell:target cell ratios of 2:1 and 5:1. Hematopoietic compartment output was assessed by the number of erythroid burst-forming units (BFU-E) and granulocyte / monocyte colony-forming units (CFU-GM) formed on day 16. CFU statistical analysis was performed by two-way ANOVA with α set at 0.05.

[0341] Xenogeneic mouse model

[0342] Male 12-week-old NSG mice (NOD.Cg-Prkdcsid Il2rgtm1Wjl / SzJ) were purchased from Jackson Laboratory (Bar Harbor, ME) and used under an agreement approved by the Stony Brook University IACUC. The NSG mice were irradiated with a sublethal (2.5 Gy) dose of γ-rays. Twenty-four hours later, the mice were injected intradermally with 0.5 x 10 6 Karpas 299 cells that had been stably transduced to express luciferase to induce the formation of measurable subcutaneous tumors. On day 1, 24 hours after injection of Karpas 299 cells, 5 x 10 6 CD4CAR NK cells or vector control NK cells (N = 4 per group) were injected intravenously into the mice via the tail vein. The intravenous injection was repeated every 5 days for a total of 6 cycles. The tumor size area was measured every other day. On days 7, 14, and 21 after injection of Karpas 299 cells, the mice were injected subcutaneously with 100 μL of RediJect D-luciferin (Perkin Elmer, Waltham, MA) and subjected to IVIS imaging (Perkin Elmer, Waltham, MA). The imaging was analyzed using Caliper Life Sciences software (Perkin Elmer, Waltham, MA).

[0343] Statistics

[0344] The xenogeneic model sample sizes were evaluated using a two-sample two-sided equal-probability analysis (90% probability (power) and < 5% significance). An unpaired Student's t-test was used to determine the significance of the tumor size area and light intensity. Survival curves were constructed using the Kaplan-Meier method and statistical analysis of survival was performed using the log-rank (Mantel-Cox) test with P < 0.05 considered significant. Statistical analysis was performed using GraphPad Prism 6 software. It was determined that the variance between the treatment group and the control group was similar before the unpaired Student's test.

[0345] Results

[0346] Generation of the third-generation CD4CAR

[0347] The nucleotide sequence of the single-chain variable fragment (scFv) against CD4 molecule is derived from the humanized monoclonal antibody ibalizumab (Hu5A8 or TNX-355), the safety and efficacy of which have been well studied in HIV clinical trials. To improve signal transduction, the CD4CAR is designed to have CD28 and 4-1BB domains fused to the CD3ζ signaling domain, making it a third-generation CAR. Third-generation CAR T cells targeting CD19 have been previously used in clinical trials and have shown excellent efficacy. For efficient expression of the CD4CAR molecule on the surface of NK cells, the strong spleen focus-forming virus promoter (SFFV) is used and the CD8 leader sequence is incorporated into the construct. The anti-CD4 scFv is separated from the intracellular signaling domain by a CD8-derived hinge (H) and transmembrane (TM) region ( Figure 8A and 8C ). The CD4CAR DNA molecule is then subcloned into a lentiviral plasmid.

[0348] Characterization of CD4CAR

[0349] To verify the CD4CAR construct, HEK293-FT cells were transfected with the CD4CAR lentiviral plasmid or a vector control plasmid, and the cells were harvested 48 hours later for Western blot analysis. Immunoblotting with an anti-CD3ζ monoclonal antibody showed a band of the expected size corresponding to the CD4CAR-CD3ζ fusion protein ( Figure 8B ). As expected, no CD3ζ expression was observed for the GFP vector control protein ( Figure 8B ).

[0350] Generation of CD4CAR NK cells

[0351] The CD4CAR NK transduction efficiency was determined to be 15.9%, as measured by flow cytometry ( Figure 9 upper panel). Subsequently, fluorescence-activated cell sorting (FACS) was used to further enrich for CD4CAR+ NK cells. After sorting, the collected CD4CAR high NK cells were confirmed to be more than 85% CD4CAR positive ( Figure 15 ). After FACS collection of the CD4CAR high cells, the CD4CAR expression level remained consistently stable at 75 - 90% on NK cells during up to 10 passages of expansion and after cryopreservation. Indeed, at the start of the co-culture experiment, the expanded CD4CAR high NK cells expressed the CAR at 85% ( Figure 9The CD4CAR NK cells lysed CD4+ blood cancer cells, including anaplastic large T cell lymphoma (Karpas 299), acute myeloid leukemia (HL-60), and T cell acute lymphoblastic leukemia (CCRF-CEM).

[0352] The following CD4+ cells were used to test the in vitro anti-lymphoma activity of CD4CAR NK cells: Karpas 299, HL-60, and CCRF-CEM. The Karpas 299 cell line was established from the peripheral blood of a 25-year-old patient with anaplastic large T cell lymphoma. The HL-60 cell line was established from the peripheral blood of a 36-year-old patient with acute promyelocytic leukemia. The CCRF-CEM cell line was established from the peripheral blood of a 4-year-old patient with T cell acute lymphoblastic leukemia (TALL).

[0353] During the 24-hour co-culture experiment, CD4CAR NK cells showed profound killing of CD4-positive leukemia / lymphoma cells at low effector-to-target cell ratios (E:T) of 2:1 ( Figure 10A ) and at a standard 5:1 ratio ( Figure 10C ) of effector-to-target cell ratio (E:T). In the co-culture cytotoxicity assay, target tumor cells were identified by the CD4+, CD56- immunophenotype (marked in blue on the flow cytometry plot). As expected, the vector control NK cells showed some NK cell-intrinsic non-specific tumor cell killing, but as expected, the vector control NK cells were far less effective against CD4+ tumor cells compared to CD4CAR NK cells. Analysis of Karpas 299 cells alone confirmed 99.1% CD4+ expression ( Figure 10A upper panel), significantly, at an E:T ratio of 2:1, CD4CAR NK cells completely eliminated 100% of Karpas 299 cells compared to the vector control (N = 2) ( Figure 10A upper panel and 10C). Similarly, analysis of HL-60 and CCRF-CEM cells alone confirmed high CD4 expression, 99.9% and 92.1% respectively ( Figure 10A middle panel and lower panel). Likewise, at an E:T ratio of 2:1, compared to the vector control, CD4CAR NK cells potently lysed 75% of HL-60 cells and 97% of CCRF-CEM cells ( Figure 10A and 10C ). These combined data show that CD4CAR NK cells specifically and potently target CD4+ cells in addition to retaining NK cell-intrinsic non-specific anti-tumor cell activity.

[0354] Co-culture studies were also performed using patient samples ( Figure 10Band 10C ). Patient 1 had aggressive forms of CD4+ cutaneous T cell lymphoma with Sézary syndrome and was unresponsive to standard chemotherapy. Sézary syndrome is a subset of PTCL. As assessed by flow cytometry, the leukemic cells of Patient 1 were 78.1% CD4+( Figure 10B ). Patient 2 had CD4+ pediatric T cell acute lymphoblastic leukemia (T-ALL). Similarly, as assessed by flow cytometry, the cells of Patient 2 were 43.7% CD4+( Figure 10B ). After co-culture at a low E:T ratio of 2:1 for 24 hours, CD4CAR NK cells lysed 58% of CD4+ Sézary syndrome cells in Patient 1 and 78% of CD4+ T-ALL cells in Patient 2 (N = 2). Additionally, at an increased E:T ratio of 5:1 (standard for CAR co-culture analysis), CD4CAR NK cells lysed 82% of Sézary syndrome cells in Patient 1 and 82% of T-ALL cells in Patient 2 (N = 2)( Figure 10C and Figure 14 ). These data strongly demonstrate dose-dependent responses and potent CD4CAR NK cell anti-tumor activity in both cell lines and patient sample settings for adult and pediatric CD4+ T cell leukemias and lymphomas.

[0355] CD4CAR NK cells specifically lysed CD4-expressing tumor cell lines in a dose-dependent manner.

[0356] CD4CAR NK cells specifically lysed the in vitro CD4+ Karpas 299 and CCRF-CEM leukemia cell lines in a dose-dependent manner at effector cell:target cell ratios of 1:4, 1:2, and 1:1( Figure 11 ). For each co-culture E:T ratio, CD4CAR NK effector cells or vector control NK effector cells were co-cultured with tumor cells composed of equal numbers of targeted CD4+ cells, CFSE-stained Karpas 299 or CFSE-stained CCRF-CEM, and CMTMR-stained "off-target" CD4−, CD5+ MOLT4 acute lymphoblastic leukemia cells.

[0357] MOLT4 cells were included to account for changes in initial cell numbers and spontaneous target cell death. After 24 hours, live cells were analyzed by flow cytometry. The percentage of target cell lysis was measured by comparing the survival of CD4+ target cells in CD4CAR NK co-cultures to the survival of CD4+ target cells in vector control NK co-cultures. Karpas 299 cells were eliminated at rates of 67%, 95%, and 100% at effector cell to target cell ratios of 1:4, 1:2, and 1:1, respectively( Figure 11)。And at the same E:T ratio, CCRFCEM cells were eliminated at rates of 39%, 58%, and 69% respectively ( Figure 11 )。As expected, CD4CAR NK cells did not lyse MOLT4 cells labeled with CMTMR, and flow cytometry analysis confirmed that it was <5% CD4+ ( Figure 16A )。Additional co-culture experiments confirmed that CD4CAR NK cells did not lyse MOLT4 cells at 0 h, 4 h, 8 h, and 24 h ( Figure 16B ), however, CD4CAR NK cells lysed Karpas 299 cells as early as 4 h, as detected by flow cytometry ( Figure 16C )。These combined data indicate that the anti-tumor cytotoxicity of CD4CAR NK cells is dose-dependent, rapid in onset, and highly specific for CD4+ cells.

[0358] Additional co-culture studies were performed using CD4+ T cells isolated from umbilical cord blood. In these experiments, CD4CAR NK cells completely depleted CD4+ T cells after 24-hour co-culture at a 2:1 effector cell:target cell ratio, and the remaining cells were 0.0% CD4+. As expected, after co-culture of CD4+ cord blood cells with corresponding vector control NK cells (CD56+, CD4-), the CD4+ population remained largely intact ( Figure 12A ), which further confirmed the specific and strong CD4CAR NK-mediated depletion of the CD4+ population on healthy tissues.

[0359] CD4CAR NK cells do not affect stem cell output in the hematopoietic compartment.

[0360] CFU (colony-forming unit) analysis showed that CD4CAR NK cells did not significantly affect the CD34+ cord blood stem cell output of the hematopoietic compartment. Hematopoietic compartment output was assessed by the presence of erythroid progenitors and granulocyte / macrophage progenitors on day 0, and determined by the number of erythroid burst-forming units (BFU-E) and granulocyte / monocyte colony-forming units (CFU GM) on day 16 ( Figure 12B )。This finding is consistent with the specific targeting of CD4 (a mature T cell marker) having a limited effect on hematopoietic stem cells and early progenitors, and the lack of evidence of lineage skewing (a measure of treatment safety).

[0361] CD4CAR NK cells showed significant in vivo anti-tumor activity

[0362] To evaluate the in vivo anti-tumor activity of CD4CAR NK cells, we developed a xenogeneic mouse model using NSG mice that were sub-lethally irradiated and injected intradermally with luciferase-expressing Karpas 299 cells to induce measurable tumor formation. On day 1, 24 hours after injection of Karpas 299 cells, and then every 5 days for a total of 6 courses, each administration involved intravenous injection of 5x10 6 CD4CAR NK cells or vector control NK control cells into the mice. On days 7, 14, and 21, the mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging to measure tumor burden ( Figure 13A ). The average light intensity for mice injected with CD4CAR NK was compared to the average light intensity for mice injected with vector control NK ( Figure 13B ). Before day 21, the mice injected with CD4CAR NK had significantly lower light intensity and thus less tumor burden than the vector control group (p<0.01). 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 13C ). Unpaired Student's T-test analysis showed that starting from day 17 (p<0.05) and continuing from days 19 to 25 (p<0.01), the average tumor size of mice injected with CD4CAR NK was significantly smaller than that of mice injected with vector control NK. Next, we compared the survival of mice across the two groups ( Figure 13D ). All mice injected with CD4CAR NK remained alive after day 30. However, the survival percentage of mice injected with vector control NK began to decrease at day 17 and none were alive before day 23. In summary, these in vivo data indicate that CD4CAR NK cells significantly reduce the tumor burden in NSG mice injected with Karpas 299 and extend the survival of these NSG mice.

[0363] Anti-CD5 chimeric antigen receptor (CD5CAR) T cells effectively target CD5-positive hematological malignancies

[0364] Examples

[0365] Results

[0366] Generation of the third-generation CD5CAR

[0367] The construct of CD5CAR and the anchored CD5 scFv antibody were designed to test the function and mechanism of CD5CAR T cells in targeting and lysing cells expressing CD5 and the ability of CD5CAR T cells to downregulate CD5 expression within their own CD5CAR T cell population ( Figure 17A) To confirm the CD5 CAR construct, the generated CD5 CAR lentivirus was transduced into HEK293 cells. After treatment with CD5 CAR or GFP-lentivirus for 48 h, the expression of CD5 CAR in HEK293 cells was verified by Western blot analysis using CD3ζ antibodies that recognize the C-terminus of the CD5 CAR protein (Figure 17B). The resulting band was the expected size of the CD5 CAR protein in CD5 CAR-transduced HEK293 cells, but GFP-transduced HEK293 cells did not show any specific bands by Western blot analysis. To evaluate the function of the CD5 CAR protein for subsequent experiments, the CD5 CAR lentivirus was transduced into activated human T cells. The expression of CD5 CAR on the T cell surface was evaluated by flow cytometry analysis using goat anti-mouse F(ab’) antibodies that recognize the scFv region of the CD5 CAR protein. Flow cytometry analysis showed approximately 20% CD5 CAR expression on CD5 CAR-transduced T cells compared to the isotype control group ( Figure 17C ). These results indicate that we successfully generated T cells expressing CD5 CAR for the following experiments.

[0368] Downregulation of CD5 expression for CAR therapy

[0369] Before CD5 CAR T cell co-culture and animal analysis, the expression of CD5 on the surface of CD5 CAR T cells was downregulated to avoid suicide within the CD5 CAR T population. Downregulation of CD5 will prevent the suicide of CAR T cells within the CAR T cell population, and downregulation of CD5 is associated with enhanced killing of the T cell population. CAR generated in T cells without CD5 expression can be a super-functional CAR, which is independent of the construct of the CAR itself. The steps for generating CD5 CAR T cells and the comparison of single transduction or double transduction using CD5 CAR lentivirus to downregulate CD5 are shown in Figure 18A and B. CD5 CAR T cells singly transduced with un-concentrated lent-CD5CAR virus did not show complete downregulation of cell surface CD5 protein before day 8, and the maximum CD5-negative population reached up to 46% on day 6 ( Figure 18C ). In the doubly transduced population, approximately 90% of the transduced T cells became CD5-negative after 4 days of culture. In contrast, the GFP T cell control group maintained a CD5+, CD3+ double-positive population of more than 95% from day 2 to day 8 ( Figure 18C ).

[0370] Downregulation of CD5 expression on T cells can be accomplished by transduction of the anchored CD5 CAR scFv lentivirus.

[0371] To further elucidate the mechanism by which CD5CAR downregulates CD5 expression on T cells, a novel construct (SEQ ID NO.7) titled "Anchored CD5 scFv" was created ( Figure 17A ). This construct contains an anti-CD5 scFv linked to a transmembrane domain via a hinge region that allows the CD5 scFv to be immobilized on the T cell surface. The anchored CD5 scFv polypeptide (SEQ ID NO.16) binds to the CD5 target without lysing the target cells, as observed with the functional CD5CAR. Single transduction and flow data analysis showed in Figure 19A and 19B , and partial downregulation of CD5 expression on T cells at day 7 of culture. This is consistent with the partial downregulation of CD5 expression seen in CD5CAR T cells after single transduction.

[0372] CD5CAR T cells effectively lyse T-cell ALL cell lines.

[0373] First, the cytotoxicity of CD5CAR T cells against the established T-cell ALL cell lines CCRFCEM and MOLT-4 and the anaplastic large cell leukemia cell line KARPAS 299 was tested, as shown in Figure 20A and 20B . When compared to the GFP control group, strong cytotoxicity against the two CD5+ cell lines was observed, and target cell lysis exceeded 75% for both cell lines. 0% lysis was observed in the anaplastic large cell line KARPAS 299, which is negative for CD5.

[0374] CD5CAR T cells effectively lyse T-cell ALL cells from human samples.

[0375] The ability of CD5CAR to lyse patient sample T-ALL cells was also evaluated using multiple patient samples, and CD5CAR cell co-culture is shown in Figure 21 and Figure 22 . Although high-efficiency cell killing similar to that seen when CD5CAR cells target T-cell ALL cell lines was noted for the leukemia cells of patient T-ALL 1, relatively weak lysis of target cells was shown for the leukemia cells of three other patients ( Figure 21A and Figure 21B ).

[0376] The ability of CD5CAR to kill patient leukemia cells is correlated with the intensity of CD5 expression, as shown in Figure 21A , 21B and 21D. As shown in Figure 21C and 21DAs shown, CD5 expression of T-ALL-1, T-ALL 3, T-ALL 6, and T-ALL 7 was observed by flow cytometry analysis. Except for the T-ALL-1 sample, the CD5 expression of these T-ALL patient samples was significantly lower.

[0377] CD5CAR T cells showed specific and potent killing of target cells.

[0378] As a control, the ability of CD5CAR T cells to eliminate CD5-negative leukemia T cells was also tested. The anaplastic large T-cell lymphoma cell line is a cell line that does not express CD5. Flow cytometry analysis showed that CD5CAR T cells were unable to lyse or eliminate KARPAS 299 cells, as Figure 21A shown in the lower panel.

[0379] Patient samples with a high degree of CD5 expression (T-ALL-8) were obtained from patients with minimal disease of T-ALL. Co-culture with CD5CAR and detailed analysis were performed, as Figure 22 shown. Three populations of cells were evaluated by flow cytometry after co-culture, including CD5+ normal T cells, CD5+CD34+ T-ALL cells, and CD5-CD34+ T-ALL cells. When compared to the GFP control group, CD5CAR showed specific and potent target cell lysis ability, and >93% of CD5-positive cell lysis for all CD5+ cell populations. CD5CAR killed leukemia cells as effectively as CD5 normal T cells. No killing was observed in the CD5-negative population.

[0380] CD5CAR T cells essentially eliminated the T cell population (CD5+CD34-).

[0381] CD5CAR T cells effectively eliminated normal T cells.

[0382] CD5CAR T cells demonstrated effective elimination of normal T cells in a dose-dependent manner at low ratios (effector cell:target cell) of 0.25:1, 0.5:1, and 1:1 in co-culture analysis (Figure 23). CD5CAR T cell or CD123CAR T (control) effector cells were co-cultured with GFP-labeled T cells. The percentage of target cell killing was measured by comparing the survival of GFP T cells in CD5CAR T co-culture relative to the survival of GFP T cells in the CD123CAR T control co-culture. CD5CAR T cells eliminated normal GFP T cells in a dose-responsive manner.

[0383] CD5 CAR T cells effectively eliminated all GFP T cells at a 1:1 effector-to-target cell ratio (Figure 23). Since CD5 CAR T cells effectively eliminated all normal T cells, the feasibility of CD5 CAR T therapy should depend on the ability to provide transient rather than permanent ablation. CD5 CAR T cells can be used as a novel conditioning regimen or "bridging therapy" for hematopoietic cell transplantation.

[0384] T cells continued to express CD5 when co-cultured with CD5 CAR or anchored CD5 scFv T cells.

[0385] One property of CD5 is internalization upon antibody binding. Thus, the target cell loses the target antigen, which can cause antigen escape. This phenomenon has been reported as a cause of failure in clinical studies using CAR T cell-based therapies.

[0386] We next used co-culture assays to investigate the question of whether CD5 CAR or anchored CD5 scFv T cells affect CD5 expression on CD5-positive T or leukemia cells. The procedures for generating CD5 CAR T cells or anchored CD5 scFv T cells and CD123 CAR T cells (control group) are shown in Figure 24A . After second transduction of T cells with lenti-CD5CAR or anchored CD5 scFv and CD123CAR viruses on day 3, CD5 expression of the transduced T cells was analyzed by flow cytometry. T cells transduced with CD5 CAR or anchored CD5 scFv lentiviruses showed a near-complete downregulation of surface CD5 protein ( Figure 24B ). In contrast, the control group of T cells transduced with CD123 CAR continued to express CD5.

[0387] We then co-cultured the transduced CD5 CAR or CD5-fixed scFv and CD123 CAR T cells with GFP-labeled T cells at a 1:1 (E:T) ratio for 2 or 4 days. As shown in Figure 25A and B, CD5 CAR T cells effectively eliminated all GFP-T cells. As expected, the transduced CD5-fixed scFv or CD123 CAR T cells were unable to lyse GFP T cells. In addition, GFP T cells continued to express CD5 when co-cultured with the transduced CD5-fixed scFv or CD123 CAR T cells. These studies indicate that CD5 antigen escape is unlikely to occur when using CD5 CAR for immunotherapy.

[0388] When T-ALL cells were transduced with lenti-CD5CAR or CD5-fixed scFv virus, CD5 expression in T-ALL cells was downregulated.

[0389] We next tested whether transduction of CD5CAR- or anchored CD5CAR lentivirus on T-ALL cells resulted in downregulation of CD5 expression. CCRF-CEM and MOLT-4 T-ALL cells were transduced with CD5CAR- or anchored CD5 scFv lentivirus. CD5CAR or anchored CD5 scFv significantly downregulated or reduced the amount of surface CD5 expression in these leukemic cells (Figure 25C). In contrast, these T cells continued to express CD5 when these cells were used to co-culture with transduced anchored CD5 scFv T cells( Figure 24A and B).

[0390] CD5CAR T cells showed profound in vivo anti-tumor activity

[0391] To evaluate the in vivo anti-tumor activity of CD5CAR T cells as a predictor of its efficacy in patients, we developed a xenograft mouse model that used NSG mice irradiated with a sublethal dose (2.0 Gy) and intravenously injected with 1.0 x 10 6 CCRF-CEM cells (CD5+) expressing firefly luciferase to induce measurable tumor formation. On the 3rd day after injection of CCRF-CEM-Luc+ cells, mice were intravenously injected with 5 x 10 6 CD5CAR T cells or vector control group T cells. These injections were repeated on the 4th, 6th, and 7th days, for a total of 20 x 10 6 T cells injected into each mouse. On the 5th, 8th, 10th, and 13th days, mice were subcutaneously injected with RediJect D-luciferin (Perkin-Elmer) and subjected to IVIS imaging (Caliper Life Sciences) to measure tumor burden( Figure 26A ). The average light intensity of mice injected with CD5CAR T cells was compared with the average light intensity of mice injected with vector control group T( Figure 26B ). Paired T-test analysis showed a highly significant difference between the two groups before the 13th day and the group injected with CD5CAR T had a smaller light intensity and thus a smaller tumor burden than the control group (p < 0.0012). Further analysis showed that before the 5th day, mice previously treated with CD5CAR T cells alone for 3 days had a 53% lower tumor burden compared to control group mice, and this percentage increased to 95% before the 8th day( Figure 26C)。On day 13, the tumor burden of the treated mice remained at near background levels. On day 15, a small amount of peripheral blood was drawn from each of the mice including 2 mice that did not receive injection of CCRF-CEM or T cells (to serve as a background control group), and the presence of transplanted CCRF-CEM cells (CD5+) was analyzed by flow cytometry. The results perfectly reflected this imaging as the percentage of tumor cells in the CD5 CAR T cell-treated mice that dropped to near background levels (<1%), while the mice given control group T cells had 28 to 43% CCRF-CEM tumor cells ( Figure 26D ). In summary, these in vivo data indicate that CD5 CAR T cells potently reduce the tumor burden in NSG mice injected with CCRF-CEM and prolong the survival of the NSG mice when compared to vector control T cells.

[0392] Anti-CD5 chimeric antigen receptor (CD5CAR) NK cells effectively eliminate CD5-positive hematological malignancies.

[0393] Examples

[0394] Results

[0395] Generation of CD5 NK-CAR

[0396] The anti-CD5 molecule is a molecular design composed of a single-chain variable fragment (scFv) that binds the CD28 and 4-1BB domains fused to the CD3ζ signaling domain to improve signal transduction, making it a third-generation CAR. The strong spleen focus-forming virus promoter (SFFV) is used for the efficient expression of the CD5CAR molecule on the surface of NK cells and the CD8 leader sequence is incorporated into the construct. The anti-CD5 scFv is linked to the intracellular signaling domain via a CD8-derived hinge (H) and transmembrane (TM) region. Then this CD5CAR construct is transduced into a lentiviral plasmid.

[0397] Generation of CD5CAR NK cells

[0398] The transduction efficiency of CD5CAR was determined by flow cytometry analysis. For enrichment of CD5CAR+ NK cells, the highest expressing NK cells were harvested using flow cytometry. After sorting, the CD5CAR high NK expression was amplified for in vitro and in vivo efficacy.

[0399] CD5CAR NK cells effectively eliminate human T cell acute lymphoblastic leukemia (TALL) cell lines The in vitro anti-T-ALL activity of CD5CAR NK cells was tested using the CCRF-CEM, MOLT-4, and Jurkat cell lines. All of these T-ALL cell lines highly express CD5.

[0400] During the co-culture experiments, CD5CAR NK cells showed profound killing of CCRF-CEM at low effector-to-target ratios (E:T) of 2:1 and 5:1. At these ratios, CD5CAR NK cells nearly eliminated CCRF-CEM cells ( Figure 27A ). CD5CAR NK cells lysed CCRF-CEM leukemia cells in vitro in a dose-dependent manner at effector cell:target cell ratios of 0.25:1, 0.5:1, 1:1, 2:1, and 5:1 ( Figure 27B and 27C ). Two additional T-ALL cell lines (MOLT-4 and Jurkat) were used to test the anti-leukemic activity of CD5NK cells. Co-culture studies of these two cell lines were performed with CD5CAR NK cells. CD5CAR NK cells substantially eliminated MOLT-4 and Jurkat cells at a low effector cell:target cell ratio of 2:1 ( Figure 28A and B).

[0401] CD5CAR NK cells effectively eliminated aggressive CD5+ T-ALL cells from human samples.

[0402] Co-culture experiments were also performed using patient samples ( Figure 29A , B). Both patient 1 and patient 2 had T-ALL and were refractory to standard chemotherapy. Patient 1 (T-ALL#1) had a small subset of CD5-positive T-ALL cells. The leukemia cells from this patient were co-cultured with CD5CAR NK cells. The gated target population and the patient's cells were labeled with the cell cytotracker dye (CMTMR) for quantification by flow cytometry. The target CD5+CD34+ cell population was gated against an isotype control. CD5CAR NK cells lysed approximately 60% of the CD34+CD5+ leukemia cells at an E:T ratio of 5:1. Importantly, CD5CAR NK cells did not show any activity against the CD5− cell population, indicating specificity and directed activity against the target antigen epitope (selectivity for the target antigen epitope). Patient 2 had an almost CD5-positive T-ALL population and was co-cultured with CD5CAR NK cells.

[0403] CD5CAR NK cells showed nearly complete lysis of the highly CD5-expressing target population with potent activity against the low (dim) CD5+CD34+ population ( Figure 29B ).

[0404] CD5CAR NK cells effectively eliminated aggressive CD5+ peripheral T-cell lymphoma (PTCL) cells from human samples.

[0405] Patient 3 has CD4+ PTCL (unclassified type) and patient 4 has Sézary syndrome (an aggressive form of PTCL that does not respond to standard chemotherapy). The lymphoma cells of patient 3 were co-cultured with CD5CAR NK cells for 24 hours. The leukemia cells were CD5+CD7 - positive and gated the CD5+CD7 - population and quantified by flow cytometry. The target CD5+CD7 - population was analyzed and showed cell viability relative to the transduced vector control NK cells. CD5CAR NK showed almost complete lysis of the leukemia CD5+CD7 - target population and complete lysis of the entire CD5+ population including normal T cells expressing CD5( Figure 29C ).

[0406] After 24 hours, the leukemia cells of patient #4 with Sézary syndrome were co-cultured with CD5CAR NK cells at E:T ratios of 2:1 and 5:1. CD5CAR NK cells showed potent anti-leukemic activity and more than 90% lysis of Sézary syndrome cells( Figure 29D ). Saturation was achieved at an E:T ratio of 2:1 where the leukemia cells were almost eliminated.

[0407] CD5CAR NK cells effectively depleted normal T cells.

[0408] T cells were isolated from umbilical cord blood and used for co-culture with CD5CAR NK cells. As Figure 30 shown, CD5CAR NK cells completely depleted T cells after 24 hours of co-culture at a low effector cell:target cell ratio of 2:1( Figure 30 ). The T cell population remained largely intact as compared to the T cells of the GFP control group.

[0409] CD5CAR NK cells effectively lysed CD5+ B-cell malignancies including mantle cell lymphoma (MCL) and chronic lymphocytic lymphoma (CLL).

[0410] Additional co-culture studies were performed on CD5+ Jeko lymphoma cell lines and lymphoma cells from patients with (MCL) and CLL. The JeKo-1 MCL cells were established from the peripheral blood mononuclear cells of a patient with a large cell variant of MCL. In co-culture studies at a low E:T of 2:1, CD5CAR NK cells effectively lysed approximately 80% of the Jeko cells (Figure 31A). Cells isolated from patient samples with MCL were also co-cultured with CD5CAR NK cells. The target population was gated and viable cells were quantified by flow cytometry. CD5CAR NK cells almost eliminated both populations of the CD5+CD19+ leukemia population and the CD5+CD19 - T cell population( Figure 31B)。Cells of patients with B-cell CLL were also co-cultured with CD5CAR NK cells. CD19 was used to gate the leukemia population by flow cytometry. CD5+CD19+CLL cells were almost eliminated by CD5CAR NK cells ( Figure 31C )。These studies strongly suggest that CD5CAR NK cells possess biological properties of profound anti-tumor activity in leukemia cell lines and patient leukemia samples ( Figure 32 ), including against T-ALL, PTCL, and B-cell lymphoma expressing CD5.

[0411] CD5CAR NK cells demonstrated potent anti-leukemia activity in vivo.

[0412] For a similar strategy with CD5CAR T cells, animal studies were employed to determine the in vivo anti-tumor activity of CD5CAR NK cells. NSG mice irradiated with a sublethal dose were intravenously injected with 1.0x10 6 CCRF-CEM cells expressing firefly luciferase to induce measurable tumor formation. Three days after the injection of CCRF-CEM-Luc+ cells, the mice were intravenously injected with 5x10 6 CD5CAR NK cells or vector control T cells. These injections were repeated on day 4, for a total of 10x10 6 T cells injected per mouse. On day 5, the mice were subcutaneously injected with RediJect D-luciferin and subjected to IVIS imaging to measure tumor burden ( Figure 33A ). The average light intensity of mice injected with CD5CAR NK cells was compared with that of mice injected with vector control NK cells ( Figure 33B ). On day 5 after tumor injection, the tumor burden in the treated mice was reduced by two-thirds. Paired t-test analysis showed a highly significant difference between the two groups (P = 0.0302). These in vivo data indicate that CD5CAR NK cells significantly reduce the tumor burden in NSG mice injected with CCRF-CEM in a rapid manner when compared to vector control NK cells.

[0413] Anti-CD3 chimeric antigen receptor (CD3CAR) NK cells efficiently lyse CD3-positive hematological malignancies

[0414] Examples

[0415] Results

[0416] Generation of CD3CAR

[0417] The anti-CD3 molecule is a molecular design that consists of a single-chain variable fragment (scFv) that binds the CD28 and 4-1BB domains fused to the CD3ζ signaling domain to improve signal transduction, making it a third-generation CAR. The strong spleen focus-forming virus promoter (SFFV) is used for the expression of the CD3CAR molecule on the surface of NK cells (NK-92), and the CD8 leader sequence is incorporated into the construct. The anti-CD3 scFv is linked to the intracellular signaling domain ( Figure 34A ) via a CD8-derived hinge (H) and transmembrane (TM) region. This CD3CAR construct is then transduced into a lentiviral plasmid. Western blot analysis was performed on HEK293-FT cells transfected with the CD3CAR lentiviral plasmid and the vector control plasmid to characterize the CD3CAR. Immunoblotting with an anti-CD3ζ monoclonal antibody showed a band of the expected size for the CD3CAR-CD3ζ fusion protein ( Figure 34B ), while there was no band for the vector control protein.

[0418] Generation of CD3CAR NK cells using NK-92 cells

[0419] The transduction efficiency of the CD3CAR was determined by flow cytometry analysis. For enrichment of CD3CAR NK cells, the highest-expressing NK cells were harvested using fluorescence-activated cell sorting (FAC). After sorting, NK cells with relatively high CD3CAR expression were obtained. The expression of CD3CAR after flow cytometry sorting was stable at approximately 30% CAR expression for subsequent NK cell expansion and cryopreservation.

[0420] CD3CAR NK cells effectively lyse human T-ALL cell lines To determine the efficacy of CD3CAR NK cells, we performed co-culture analysis using CD3+ T-ALL cell lines (Jurkat and CCRF-CEM). The CD3-positive cells in Jurkat and CCRF-CEM cells were approximately 80% and 10% positive for CD3, respectively. Then, the highly expressed CD3 cells in the CD3+ cells of the CCRF-CEM cell line were sorted, and the CD3 expression in the sorted CCRF-CEM cells was approximately 50%. During co-culture with Jurkat and CCRF-CEM cells, CD3CAR NK cells showed profound leukemia cell killing (Figure 35). At 6 hours of culture and at a low E:T ratio of 2:1, CD3CAR NK cells effectively lysed more than 60% of Jurkat cells ( Figure 35A)。We then compared the killing of sorted CD3 CCRF-CEM cells with relatively high expression with that of unsorted CCRF-CEM cells. These CD3 CAR NK cells showed more effective resistance against the population with high CD3 expression in sorted CCRF-CEM rather than unsorted CCRF-CEM with lower CD3 expression ( Figure 35B ) population.

[0421] CD3 CAR NK cells effectively eliminate CD3+ leukemia cells in human samples

[0422] The killing of CD3 CAR NK cells was also tested using patient samples. Flow cytometry analysis of two patient samples showed strong and uniform CD3 expression. As shown by flow cytometry analysis, co-culture of Sezary syndrome patient samples with CD3 CAR T cells effectively led to approximately 80% lysis of leukemia cells at a low E:T ratio of 2:1 ( Figure 36A ). Co-culture of patient samples, unsorted PTCL with CD3 CAR NK cells for 24 hours resulted in the actual elimination of CD3+ malignant cells ( Figure 36B ). CD3 CAR NK cells also affected a broad CD3+ population.

[0423] CD3 CAR NK cells can deplete normal T cells.

[0424] GFP-transduced normal T cells were used for co-culture with CD3 CAR NK cells. As Figure 37 shown, after 4 or 24 hours of culture, CD3 CAR NK cells depleted most normal T cells.

[0425] CD3 CAR NK cells show profound anti-leukemia activity in vivo

[0426] To determine the in vivo anti-tumor efficacy of CD3 CAR NK cells, sub-lethally irradiated NSG mice were intravenously injected with 1.0 x 10 6 CD3-positive (∼80%) Jurkat cells expressing firefly luciferase and measurable tumor formation was detected before day 3 or 4. Three days after Jurkat-Luc+ cell injection, 5 x 10 6 CD3 CAR NK cells or vector control NK cells were intravenously injected into each mouse (6 mice per group). These injections were repeated on days 3, 6, 7, and 10, for a total of 25 x 10 6 T cells injected per mouse. On days 4, 7, 9, and 13, the mice were subjected to IVIS imaging to measure tumor burden ( Figure 38A)。Two treated mice died due to the injection procedure on day 13. The mean fluorescence intensity of the mice injected with CD3CAR NK cells was compared with that of the mice injected with the vector control NK ( Figure 38B )。After the initial delay period, the tumor burden of the mice treated for 9 days then dropped to about less than two-thirds and only 13% on day 13 ( Figure 38C )。Paired t-test analysis showed a highly significant difference between the two groups (P = 0.0137). We conclude that these in vivo data confirm that CD3CAR NK cells significantly reduce the tumor burden in NSG mice injected with Jurkat and prolong the survival of these NSG mice when compared with vector control NK cells.

[0427] CRISPR / Cas nucleases target CD2, CD3, CD5, and CD7 expressed on T or NK cells.

[0428] T or NK cells show some surface antigens in common with leukemia or lymphoma such as CD2, CD3, CD5, and CD7. CD2, CD3, CD5, and CD7 can be good targets for T and NK cells because they are expressed in most T-cell leukemias / lymphomas.

[0429] Therefore, when one of the surface antigens CD2, CD3, CD5, and CD7 is selected as a target, if the T or NK cells used to generate the CAR share this antigen, this antigen needs to be deleted or downregulated in the T or NK cells used to generate the CAR to avoid suicide within the CAR T or NK cell population.

[0430] The steps for generating CAR T or NK cells targeting T-cell lymphoma or T-cell leukemia are described in Figure 39 . Three pairs of sgRNAs were designed using CHOPCHOP to target CD2, CD3, CD5, and CD7. Then the gene-specific sgRNA ( Figure 40 ) was transfected into a lentiviral vector (LentiU6-sgRNA-SFFV-Cas9-puro-wpre) expressing human Cas9 and puromycin resistance gene linked by an E2A self-cleaving linker. The U6-sgRNA cassette is located in front of the Cas9 element.

[0431] The expression of sgRNA and Cas9puro is driven by the U6 promoter and the SFFV promoter, respectively.

[0432] Examples

[0433] Results

[0434] CRISPR / Cas nucleases target CD5 on the T-cell line.

[0435] Lentiviruses carrying gene-specific sgRNAs were used to transduce CCRF-CEM and MOLT cells. Initially, loss of CD5 expression was observed in these T cell lines using both of two different CDISPR / Cas9 sgRNA sequences ( Figure 41A and 41C ). The most successful populations in terms of loss of CD5 expression were selected for each cell line, and these cells were sorted, normally amplified and found to have >99% purity of CD45+ and CD5- ( Figure 41B and 41D ).

[0436] The CRISPR / Cas nuclease targets CD7 on T cell lines and NK cells.

[0437] Lentiviruses carrying gene-specific sgRNAs were used to transduce CCRF-CEM, MOLT cells and NK cells (Figure 42). Flow cytometry analysis confirmed loss of CD7 expression in CCRF-CEM and NK-92 cells using the CRISPR / Cas9 method with two different sgRNAs ( Figure 42A and 42B ). The population (indicated by blue circles and arrows) was selected for sorting, amplification and analysis in Figure 42B . Loss of CD5 expression revealed by flow cytometry analysis was also seen in NK-92 cells using the above similar method targeting CD7 with CRISPR / Cas nuclease ( Figure 42C and 42D ). The sorted CD7-negative NK-92 cells were amplified ( Figure 42D ) and used to generate CD7CAR NK cells to eliminate CD7-positive leukemia cells.

[0438] CD7CAR NK 7- -92 cells have strong anti-leukemia activity

[0439] CD7 is expressed in both NK and T-ALL leukemia cells. To avoid autophagy within the CD7CAR NK-92 population, CD7 expression needs to be inactivated first. CD7-deficient NK-92 cells (NK 7- -92 cells) were generated and amplified as described in ( Figure 42D ). These amplified NK-92 cells were transduced with lentiviruses expressing CD7CAR. CD7CAR includes an anti-CD7 scFV that binds the CD28 and 4-BB domains fused to the CD3ζ signaling domain, making it a third-generation CAR. CD7CAR NK 7- -92 cells were used to test their ability to lyse leukemia cells expressing CD7. As shown in Figure 43, CD7CAR NK7- -92 cells exhibit potent anti-leukemia activity against the T-ALL cell line CCRF-CEM. As analyzed by flow cytometry, co-culture of CCRF-CEM cells at an E:T ratio of 5:1 effectively results in the lysis of approximately 50% of leukemia cells ( Figure 43A and 43B ).

[0440] The CD3 multimeric protein complex is described in Figure 44 . The complex includes the CD3δ chain, the CD3γ chain, and two CD3ε chains. These chains are associated with the T cell receptor (TCR) composed of αβ chains.

[0441] CD3CAR is used for graft-versus-host disease (GvHD).

[0442] CD3CAR is administered to a patient before or after stem cell transplantation. The patient is tested to have a high level of white blood cells.

[0443] CD3CAR is administered to a patient before or after bone marrow transplantation. The patient is tested to have a high level of white blood cells.

[0444] CD3CAR is administered to a patient before or after tissue transplantation. The patient is tested to have a high level of white blood cells.

[0445] Organ transplantation

[0446] CD3CAR is administered to an organ transplantation patient before organ transplantation surgery. The patient is tested to have organ rejection. The following histological signs are determined: (1) infiltrating T cells, in some cases accompanied by infiltration of eosinophils, plasma cells, and neutrophils, especially by counting ratios, (2) structural compromise of tissue anatomy, which varies with the type of transplanted tissue, and (3) damage to blood vessels.

[0447] CD3CAR is administered to an organ transplantation patient after organ transplantation surgery. The patient is tested to have organ rejection. The following histological signs are determined: (1) infiltrating T cells, in some cases accompanied by infiltration of eosinophils, plasma cells, and neutrophils, especially by counting ratios, (2) structural compromise of tissue anatomy, which varies with the type of transplanted tissue, and (3) damage to blood vessels. Sequence Listing <110> ICELL GENE THERAPEUTICS LLC MA, Yupo PINZ, Kevin Jiang, Xun Wada, Masayuki Chen, Kevin <120> Chimeric Antigen Receptors (CARs) Targeting Hematological Malignancies, Compositions Thereof, and Methods of Use <130> 2541-2 PCT <141> 2017-08-22 <150> 62 / 121,842 <151> 2015-02-27 <160> 38 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1593 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatta tgatgacaca gtcgccatca tctctggctg tgtctgcagg agaaaaggtc 120 actatgacct gtaagtccag tcaaagtgtt ttatacagtt caaatcagaa gaactacttg 180 gcctggtacc agcagaaacc agggcagtct cctaaactac tgatctactg ggcatccact 240 agggaatctg gtgtccctga tcgcttcaca ggcagtggat ctgggacaga ttttactctt 300 accatcagca gtgtgcaacc tgaagacctg gcagtttatt actgtcatca atacctctcc 360 tcgcacacgt tcggaggggg gaccaagctg gaaataaaac ggggtggcgg tggctcgggc 420 ggtggtgggt cgggtggcgg cggatctcaa ctgcagcagc ctggggctga gctggtgagg 480 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gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1380 gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1440 cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1500 ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1560 gcccttcaca tgcaggccct gccccctcgc taa 1593 <210> 2 <211> 1590 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 2 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacattg tgatgactca gtctccagcc accctgtctg tgactccagg agatagagtc 120 tctctttcct gcagggccag ccagagtatt agcgactact tacactggta tcaacaaaaa 180 tcacatgagt ctccaaggct tctcatcaaa tatgcttccc aatccatctc tgggatcccc 240 tccaggttca gtggcagtgg atcagggtca gatttcactc tcagtatcaa cagtgtggaa 300 cctgaagatg ttggagtgta ttactgtcaa aatggtcaca gctttccgct cacgttcggt 360 gctgggacca agctggagct gagacggggt ggcggtggct cgggcggtgg tgggtcgggt 420 ggcggcggat ctcaggtcca actgcagcag ccagggactg aactggtgag gcctgggtct 480 tcagtgaagc tgtcctgcaa ggcttctggc tacacgttca ccagctactg ggtgaactgg 540 gttaaacaga ggcctgacca aggccttgag tggattggaa ggattgatcc ttacgacagt 600 gaaactcact acaatcagaa gttcacggac aaggccatat cgactattga cacatcctcc 660 aacacagcct acatgcaact cagcaccctg acatctgatg cttctgcggt ctattactgt 720 tcaagatcac cccgagacag ctcgaccaac cttgctgact ggggccaagg gactctggtc 780 actgtctctt ctaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840 tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900 acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960 ggggtccttc tcctgtcact ggttatcacc ctttactgca ggagtaagag gagcaggctc 1020 ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1080 cagccctatg ccccaccacg cgacttcgca gcctatcgct ccaaacgggg cagaaagaaa 1140 ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1200 ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1260 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1320 aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1380 atggggggaa agccgcagag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1440 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1500 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1560 cttcacatgc aggccctgcc ccctcgctaa 1590 <210> 3 <211> 1578 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 3 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagagtg 120 accatcacct gcagcgccag cagcagcgtg agctacatga actggtacca gcagaccccc 180 ggcaaggccc ccaagagatg gatctacgac accagcaagc tggccagcgg cgtgcccagc 240 agattcagcg gcagcggcag cggcaccgac tacaccttca ccatcagcag cctgcagccc 300 gaggacatcg ccacctacta ctgccagcag tggagcagca accccttcac cttcggccag 360 ggcaccaagc tgcagatcgg cggcggcggc agcggcggcg gcggcagcgg cggcggcggc 420 agccaggtgc agctggtgca gagcggcggc ggcgtggtgc agcccggcag aagcctgaga 480 ctgagctgca aggccagcgg ctacaccttc accagataca ccatgcactg ggtgagacag 540 gcccccggca agggcctgga gtggatcggc tacatcaacc ccagcagagg ctacaccaac 600 tacaaccaga aggtgaagga cagattcacc atcagcagag acaacagcaa gaacaccgcc 660 ttcctgcaga tggacagcct gagacccgag gacaccggcg tgtacttctg cgccagatac 720 tacgacgacc actactgcct ggactactgg ggccagggca cccccgtgac cgtgagcagc 780 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 960 ctgtcactgg ttatcaccct ttactgcagg agtaagagga gcaggctcct gcacagtgac 1020 tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1080 ccaccacgcg acttcgcagc ctatcgctcc aaacggggca gaaagaaact cctgtatata 1140 ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1200 cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1260 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1320 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1380 ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1440 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1500 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1560 gccctgcccc ctcgctaa 1578 <210> 4 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 4 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcg tgatgaccca aagccccgac agcctggccg tgagcctggg cgagagggtg 120 accatgaact gcaaaagcag ccagtccctg ctgtactcca ccaaccagaa gaactacctg 180 gcttggtatc aacagaagcc cggacagagc cccaagctgc tgatctattg ggccagcact 240 agggaaagcg gcgtgcccga taggttcagc ggcagcggga gcggcacaga cttcactctg 300 accattagca gcgtgcaggc tgaggatgtg gccgtctact actgccagca gtactacagc 360 tacaggacct ttgggggcgg aactaagctg gagatcaagg gagggggggg atccggggga 420 ggaggctccg gcggaggcgg aagccaagtg caactgcagc agagcggccc agaggtggtc 480 aaacctgggg caagcgtgaa gatgagctgc aaggctagcg gctatacctt caccagctat 540 gtgatccact gggtgaggca gaaaccagga cagggcctgg actggatcgg ctacatcaac 600 ccctacaatg acggcaccga ttatgacgaa aaattcaagg ggaaggccac cctgaccagc 660 gacaccagca caagcaccgc ctacatggag ctgtccagcc tgaggtccga ggacaccgcc 720 gtgtattact gtgccaggga gaaggacaat tacgccaccg gcgcttggtt cgcctactgg 780 ggccagggca cactggtgac agtgagcagc accacgacgc cagcgccgcg accaccaaca 840 ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 900 gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 960 cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcagg 1020 agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080 cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1200 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1260 gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1320 cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1380 cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 1440 ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500 ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1560 aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaa 1608 <210> 5 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 5 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcg tgatgaccca gagccccgac agcctggccg tgagcctggg cgagagggcc 120 accatcaact gcagggccag caagagcgtg agcaccagcg gctacagcta catctactgg 180 taccagcaga agcccggcca gccccccaag ctgctgatct acctggccag catcctggag 240 agcggcgtgc ccgacaggtt cagcggcagc ggcagcggca ccgacttcac cctgaccatc 300 agcagcctgc aggccgagga cgtggccgtg tactactgcc agcacagcag ggagctgccc 360 tggaccttcg gccagggcac caaggtggag atcaagggcg gcggcggcag cggcggcggc 420 ggcagcggcg gcggcggcag cgaggagcag ctggtggaga gcggcggcgg cctggtgaag 480 cccggcggca gcctgaggct gagctgcgcc gccagcggct tcagcttcag cgactgcagg 540 atgtactggc tgaggcaggc ccccggcaag ggcctggagt ggatcggcgt gatcagcgtg 600 aagagcgaga actacggcgc caactacgcc gagagcgtga ggggcaggtt caccatcagc 660 agggacgaca gcaagaacac cgtgtacctg cagatgaaca gcctgaagac cgaggacacc 720 gccgtgtact actgcagcgc cagctactac aggtacgacg tgggcgcctg gttcgcctac 780 tggggccagg gcaccctggt gaccgtgagc agcaccacga cgccagcgcc gcgaccacca 840 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900 gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960 gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1080 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140 tccaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1200 caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1260 tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1320 aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1380 agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 1440 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1500 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1560 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 1611 <210> 6 <211> 1581 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 6 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc aggtgaccca gagccccagc agcctgagcg ccagcctggg cgagagaatc 120 agcctgacct gcagaaccag ccaggacatc agcaactacc tgaactggtt ccagcagaag 180 cccgacggca ccttcaagag actgatctac gccaccagca gcctggacag cggcgtgccc 240 aagagattca gcggcagcgg cagcggcagc gactacagcc tgaccatcag cagcctggag 300 agcgaggact tcgccgacta ctactgcctg cagtacgcca gctacccctt caccttcggc 360 agcggcacca agctggagat caagggaggg gggggatccg ggggaggagg ctccggcgga 420 ggcggaagcg aggtgcagct gcaggagagc ggccccggcc tggtgaagcc cagccagacc 480 ctgagcctga cctgcagcgt gaccggctac agcatcacca gcggctacta ctggcactgg 540 atcagacagt tccccggcaa caagctgcag tggatgggct acatcagcta cagcggcttc 600 accaactaca agaccagcct gatcaacaga atcagcatca cccacgacac cagcgagaac 660 cagttcttcc tgaacctgaa cagcgtgacc accgaggaca ccgccaccta ctactgcgcc 720 ggcgacagaa ccggcagctg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 780 gccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc aggagtaaga ggagcaggct cctgcacagt 1020 gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1080 gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa actcctgtat 1140 atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1200 tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1260 gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1320 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1380 aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1440 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1500 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1560 caggccctgc cccctcgctg a 1581 <210> 7 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 7 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc aggtgaccca gagccccagc agcctgagcg ccagcctggg cgagagaatc 120 agcctgacct gcagaaccag ccaggacatc agcaactacc tgaactggtt ccagcagaag 180 cccgacggca ccttcaagag actgatctac gccaccagca gcctggacag cggcgtgccc 240 aagagattca gcggcagcgg cagcggcagc gactacagcc tgaccatcag cagcctggag 300 agcgaggact tcgccgacta ctactgcctg cagtacgcca gctacccctt caccttcggc 360 agcggcacca agctggagat caagggaggg gggggatccg ggggaggagg ctccggcgga 420 ggcggaagcg aggtgcagct gcaggagagc ggccccggcc tggtgaagcc cagccagacc 480 ctgagcctga cctgcagcgt gaccggctac agcatcacca gcggctacta ctggcactgg 540 atcagacagt tccccggcaa caagctgcag tggatgggct acatcagcta cagcggcttc 600 accaactaca agaccagcct gatcaacaga atcagcatca cccacgacac cagcgagaac 660 cagttcttcc tgaacctgaa cagcgtgacc accgaggaca ccgccaccta ctactgcgcc 720 ggcgacagaa ccggcagctg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 780 gccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc tga 993 <210> 8 <211> 1623 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 8 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgggcgccc agcccgccat ggccgcctac aaggacatcc agatgaccca gaccaccagc 120 agcctgagcg ccagcctggg cgacagagtg accatcagct gcagcgccag ccagggcatc 180 agcaactacc tgaactggta ccagcagaag cccgacggca ccaacaagct gctgatctac 240 tacaccagca gcctgcacag cggcgtgccc agcagattca gcggcagcgg cagcggcacc 300 gactacagcc tgcacagcaa cctggagccc gaggacatcg ccacctacta ctgccagcag 360 tacagcaagc tgccctacac cttcggcggc ggcaccaagc tggagatcaa gagaggcggc 420 ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg cagcgaggtg 480 cagctggtgg agagcggcgg cggcctggtg aagcccggcg gcagcctgaa gctgagctgc 540 gccgccagcg gcctgacctt cagcagctac gccatgagct ggaacagaca gacccccgag 600 aagagactgg agtgggtggc cagcatcagc agcggcggct tcacctacta ccccgacagc 660 aacaagggca gattcaccat cagcagagac aacgccagaa acatcctgta cctgcagatg 720 agcagcctga gaagcgagga caccgccatg tactactgcg ccagagacga ggtgagaggc 780 tacctggacg tgtggggcgc cggcaccacc gtgaccgtga gcagcaccac gacgccagcg 840 ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 900 gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 960 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020 accctttact gcaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 1080 ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 1140 gcagcctatc gctccaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1200 atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1260 gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1320 cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1380 gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 1440 aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1500 gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1560 ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1620 taa 1623 <210> 9 <211> 1590 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 9 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagagtg 120 accatcacct gcaaggccag ccagaacatc gacaagtacc tgaactggta ccagcagaag 180 cccggcaagg cccccaagct gctgatctac aacaccaaca acctgcagac cggcgtgccc 240 agcagattca gcggcagcgg cagcggcacc gacttcacct tcaccatcag cagcctgcag 300 cccgaggaca tcgccaccta ctactgcctg cagcacatca gcagacccag aaccttcggc 360 cagggcacca aggtggagat caagggcggc ggcggcagcg gcggcggcgg cagcggcggc 420 ggcggcagcc aggtgcagct gcaggagagc ggccccggcc tggtgagacc cagccagacc 480 ctgagcctga cctgcaccgt gagcggcttc accttcaccg acttctacat gaactgggtg 540 agacagcccc ccggcagagg cctggagtgg atcggcttca tcagagacaa ggccaagggc 600 tacaccaccg agtacaaccc cagcgtgaag ggcagagtga ccatgctggt ggacaccagc 660 aagaaccagt tcagcctgag actgagcagc gtgaccgccg ccgacaccgc cgtgtactac 720 tgcgccagag agggccacac cgccgccccc ttcgactact ggggccaggg cagcctggtg 780 accgtgagca gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840 tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900 acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960 ggggtccttc tcctgtcact ggttatcacc ctttactgca ggagtaagag gagcaggctc 1020 ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1080 cagccctatg ccccaccacg cgacttcgca gcctatcgct ccaaacgggg cagaaagaaa 1140 ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1200 ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1260 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1320 aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1380 atggggggaa agccgcagag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1440 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1500 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1560 cttcacatgc aggccctgcc ccctcgctaa 1590 <210> 10 <211> 530 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 10 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Met Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ala Val Ser Ala Gly Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln 35 40 45 Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 65 70 75 80 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Leu Ala Val 100 105 110 Tyr Tyr Cys His Gln Tyr Leu Ser Ser His Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg 145 150 155 160 Pro Gly Ser Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 165 170 175 Thr Arg Tyr Trp Ile His Trp Val Lys Gln Arg Pro Ile Gln Gly Leu 180 185 190 Glu Trp Ile Gly Asn Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn 195 200 205 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Gly 210 215 220 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 225 230 235 240 Tyr Tyr Cys Ala Thr Glu Asp Leu Tyr Tyr Ala Met Glu Tyr Trp Gly 245 250 255 Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser 325 330 335 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 340 345 350 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 355 360 365 Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr 370 375 380 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 385 390 395 400 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 405 410 415 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 420 425 430 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 435 440 445 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 450 455 460 Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 465 470 475 480 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 485 490 495 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 500 505 510 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 515 520 525 Pro Arg 530 <210> 11 <211> 529 <212> PRT <213> Artificial Sequence <220> <223> Synthetic 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 Arg Pro Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu 20 25 30 Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln 35 40 45 Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser 50 55 60 Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile 85 90 95 Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly 100 105 110 His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Arg 115 120 125 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Arg Pro Gly Ser 145 150 155 160 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 165 170 175 Trp Val Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 180 185 190 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 195 200 205 Thr Asp Lys Ala Ile Ser Thr Ile Asp Thr Ser Ser Asn Thr Ala Tyr 210 215 220 Met Gln Leu Ser Thr Leu Thr Ser Asp Ala Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ser Arg Ser Pro Arg Asp Ser Ser Thr Asn Leu Ala Asp Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys 325 330 335 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 340 345 350 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 355 360 365 Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 370 375 380 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 385 390 395 400 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 405 410 415 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 420 425 430 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 435 440 445 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 450 455 460 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 465 470 475 480 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 485 490 495 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 500 505 510 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 515 520 525 Arg <210> 12 <211> 525 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 12 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser 35 40 45 Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro 50 55 60 Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 100 105 110 Ser Asn Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 130 135 140 Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg 145 150 155 160 Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 165 170 175 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile 180 185 190 Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val Lys Asp Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met 210 215 220 Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr 225 230 235 240 Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val 245 250 255 Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 260 265 270 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 275 280 285 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 290 295 300 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 305 310 315 320 Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu 325 330 335 Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 340 345 350 Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr 355 360 365 Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 370 375 380 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 385 390 395 400 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 405 410 415 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 420 425 430 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 435 440 445 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg 450 455 460 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 465 470 475 480 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 485 490 495 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 500 505 510 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 525 <210> 13 <211> 535 <212> PRT <213> Artificial sequence <220> <223> Synethic 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 Arg Pro Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 20 25 30 Ala Val Ser Leu Gly Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln 35 40 45 Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 65 70 75 80 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val 100 105 110 Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val 145 150 155 160 Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr 165 170 175 Phe Thr Ser Tyr Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly 180 185 190 Leu Asp Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr 195 200 205 Asp Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr 210 215 220 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 225 230 235 240 Val Tyr Tyr Cys Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp 245 250 255 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 340 345 350 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 355 360 365 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg 370 375 380 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 385 390 395 400 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 405 410 415 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 420 425 430 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 435 440 445 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 450 455 460 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 465 470 475 480 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 485 490 495 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 500 505 510 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 515 520 525 Met Gln Ala Leu Pro Pro Arg 530 535 <210> 14 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 20 25 30 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys 35 40 45 Ser Val Ser Thr Ser Gly Tyr Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Ile Leu Glu 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr 100 105 110 Cys Gln His Ser Arg Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Glu Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 145 150 155 160 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe 165 170 175 Ser Asp Cys Arg Met Tyr Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu 180 185 190 Glu Trp Ile Gly Val Ile Ser Val Lys Ser Glu Asn Tyr Gly Ala Asn 195 200 205 Tyr Ala Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 210 215 220 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Ser Ala Ser Tyr Tyr Arg Tyr Asp Val Gly Ala 245 250 255 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr 260 265 270 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 275 280 285 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 290 295 300 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 305 310 315 320 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 325 330 335 Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 340 345 350 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 355 360 365 Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly 370 375 380 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 385 390 395 400 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 405 410 415 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 420 425 430 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 435 440 445 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 450 455 460 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 465 470 475 480 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 485 490 495 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 500 505 510 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 515 520 525 His Met Gln Ala Leu Pro Pro Arg 530 535 <210> 15 <211> 526 <212> PRT <213> Artificial Sequence <220> <223> Synthetic 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 Arg Pro Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Glu Arg Ile Ser Leu Thr Cys Arg Thr Ser Gln 35 40 45 Asp Ile Ser Asn Tyr Leu Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr 50 55 60 Phe Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro 65 70 75 80 Lys Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr 100 105 110 Ala Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 145 150 155 160 Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr 165 170 175 Tyr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Gln Trp Met 180 185 190 Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Lys Thr Ser Leu Ile 195 200 205 Asn Arg Ile Ser Ile Thr His Asp Thr Ser Glu Asn Gln Phe Phe Leu 210 215 220 Asn Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 225 230 235 240 Gly Asp Arg Thr Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 245 250 255 Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg 325 330 335 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 340 345 350 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 355 360 365 Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 370 375 380 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 385 390 395 400 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 405 410 415 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 420 425 430 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 435 440 445 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg 450 455 460 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 465 470 475 480 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 485 490 495 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 500 505 510 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 525 <210> 16 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Glu Arg Ile Ser Leu Thr Cys Arg Thr Ser Gln 35 40 45 Asp Ile Ser Asn Tyr Leu Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr 50 55 60 Phe Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro 65 70 75 80 Lys Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr 100 105 110 Ala Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr 145 150 155 160 Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr 165 170 175 Tyr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Gln Trp Met 180 185 190 Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn Tyr Lys Thr Ser Leu Ile 195 200 205 Asn Arg Ile Ser Ile Thr His Asp Thr Ser Glu Asn Gln Phe Phe Leu 210 215 220 Asn Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 225 230 235 240 Gly Asp Arg Thr Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 245 250 255 Val Thr Val Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Asp Phe Ala Cys 290 295 300 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 305 310 315 320 Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 <210> 17 <211> 540 <212> PRT <213> Artificial Sequence <220> <223> Synthetic 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 Arg Pro Gly Ala Gln Pro Ala Met Ala Ala Tyr Lys Asp 20 25 30 Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45 Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu 50 55 60 Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Asn Lys Leu Leu Ile Tyr 65 70 75 80 Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95 Gly Ser Gly Thr Asp Tyr Ser Leu His Ser Asn Leu Glu Pro Glu Asp 100 105 110 Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr Thr Phe 115 120 125 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 165 170 175 Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr Ala Met 180 185 190 Ser Trp Asn Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser 195 200 205 Ile Ser Ser Gly Gly Phe Thr Tyr Tyr Pro Asp Ser Asn Lys Gly Arg 210 215 220 Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met 225 230 235 240 Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp 245 250 255 Glu Val Arg Gly Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr 260 265 270 Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 275 280 285 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 290 295 300 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 305 310 315 320 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 325 330 335 Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys Arg Ser Arg Leu Leu 340 345 350 His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 355 360 365 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 370 375 380 Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 385 390 395 400 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 405 410 415 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 420 425 430 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 435 440 445 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 450 455 460 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys 465 470 475 480 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 485 490 495 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 500 505 510 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 515 520 525 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 530 535 540 <210> 18 <211> 529 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence <400> 18 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln 35 40 45 Asn Ile Asp Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His 100 105 110 Ile Ser Arg Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr 145 150 155 160 Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe Tyr 165 170 175 Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly 180 185 190 Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro Ser 195 200 205 Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe 210 215 220 Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly Gln 245 250 255 Gly Ser Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Ser Lys 325 330 335 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 340 345 350 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 355 360 365 Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 370 375 380 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 385 390 395 400 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 405 410 415 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 420 425 430 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 435 440 445 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 450 455 460 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 465 470 475 480 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 485 490 495 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 500 505 510 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 515 520 525 Arg <210> 19 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 19 Lys Glu Ile Thr Asn Ala Leu Glu Thr Trp Gly Ala Leu Gly Gln Asp 1 5 10 15 Ile Asn Leu Asp Ile Pro Ser Phe Gln Met Ser Asp Asp Ile Asp Asp 20 25 30 Ile Lys Trp Glu Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln Phe Arg 35 40 45 Lys Glu Lys Glu Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu Phe Lys 50 55 60 Asn Gly Thr Leu Lys Ile Lys His Leu Lys Thr Asp Asp Gln Asp Ile 65 70 75 80 Tyr Lys Val Ser Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu Glu Lys 85 90 95 Ile Phe Asp Leu Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser 100 105 110 Trp Thr Cys Ile Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr 115 120 125 Asp Pro Glu Leu Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser 130 135 140 Gln Arg Val Ile Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys Phe 145 150 155 160 Lys Cys Thr Ala Gly Asn Lys Val Ser Lys Glu Ser Ser Val Glu Pro 165 170 175 Val Ser Cys Pro Glu Lys Gly Leu Asp 180 185 <210> 20 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 20 Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val 1 5 10 15 Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly 20 25 30 Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu 35 40 45 Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu 50 55 60 Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly 65 70 75 80 Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val 85 90 95 Cys Glu Asn Cys Met Glu Met Asp 100 <210> 21 <211> 371 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 21 Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys 1 5 10 15 Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His Trp Lys Asn Ser Asn 20 25 30 Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro 35 40 45 Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln 50 55 60 Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp 65 70 75 80 Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Leu 85 90 95 Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln 100 105 110 Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val 115 120 125 Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly Lys Thr Leu 130 135 140 Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly Thr Trp Thr Cys Thr 145 150 155 160 Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys Ile Asp Ile Val Val 165 170 175 Leu Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu 180 185 190 Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu Thr 195 200 205 Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys 210 215 220 Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys Arg 225 230 235 240 Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His 245 250 255 Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu 260 265 270 Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn 275 280 285 Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu 290 295 300 Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu 305 310 315 320 Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val Leu 325 330 335 Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly Gln 340 345 350 Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro 355 360 365 Val Gln Pro 370 <210> 22 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 22 Arg Leu Ser Trp Tyr Asp Pro Asp Phe Gln Ala Arg Leu Thr Arg Ser 1 5 10 15 Asn Ser Lys Cys Gln Gly Gln Leu Glu Val Tyr Leu Lys Asp Gly Trp 20 25 30 His Met Val Cys Ser Gln Ser Trp Gly Arg Ser Ser Lys Gln Trp Glu 35 40 45 Asp Pro Ser Gln Ala Ser Lys Val Cys Gln Arg Leu Asn Cys Gly Val 50 55 60 Pro Leu Ser Leu Gly Pro Phe Leu Val Thr Tyr Thr Pro Gln Ser Ser 65 70 75 80 Ile Ile Cys Tyr Gly Gln Leu Gly Ser Phe Ser Asn Cys Ser His Ser 85 90 95 Arg Asn Asp Met Cys His Ser Leu Gly Leu Thr Cys Leu Glu Pro Gln 100 105 110 Lys Thr Thr Pro Pro Thr Thr Arg Pro Pro Pro Thr Thr Thr Pro Glu 115 120 125 Pro Thr Ala Pro Pro Arg Leu Gln Leu Val Ala Gln Ser Gly Gly Gln 130 135 140 His Cys Ala Gly Val Val Glu Phe Tyr Ser Gly Ser Leu Gly Gly Thr 145 150 155 160 Ile Ser Tyr Glu Ala Gln Asp Lys Thr Gln Asp Leu Glu Asn Phe Leu 165 170 175 Cys Asn Asn Leu Gln Cys Gly Ser Phe Leu Lys His Leu Pro Glu Thr 180 185 190 Glu Ala Gly Arg Ala Gln Asp Pro Gly Glu Pro Arg Glu His Gln Pro 195 200 205 Leu Pro Ile Gln Trp Lys Ile Gln Asn Ser Ser Cys Thr Ser Leu Glu 210 215 220 His Cys Phe Arg Lys Ile Lys Pro Gln Lys Ser Gly Arg Val Leu Ala 225 230 235 240 Leu Leu Cys Ser Gly Phe Gln Pro Lys Val Gln Ser Arg Leu Val Gly 245 250 255 Gly Ser Ser Ile Cys Glu Gly Thr Val Glu Val Arg Gln Gly Ala Gln 260 265 270 Trp Ala Ala Leu Cys Asp Ser Ser Ser Ala Arg Ser Ser Leu Arg Trp 275 280 285 Glu Glu Val Cys Arg Glu Gln Gln Cys Gly Ser Val Asn Ser Tyr Arg 290 295 300 Val Leu Asp Ala Gly Asp Pro Thr Ser Arg Gly Leu Phe Cys Pro His 305 310 315 320 Gln Lys Leu Ser Gln Cys His Glu Leu Trp Glu Arg Asn Ser Tyr Cys 325 330 335 Lys Lys Val Phe Val Thr Cys Gln Asp Pro Asn Pro 340 345 <210> 23 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 23 Ala Gln Glu Val Gln Gln Ser Pro His Cys Thr Thr Val Pro Val Gly 1 5 10 15 Ala Ser Val Asn Ile Thr Cys Ser Thr Ser Gly Gly Leu Arg Gly Ile 20 25 30 Tyr Leu Arg Gln Leu Gly Pro Gln Pro Gln Asp Ile Ile Tyr Tyr Glu 35 40 45 Asp Gly Val Val Pro Thr Thr Asp Arg Arg Phe Arg Gly Arg Ile Asp 50 55 60 Phe Ser Gly Ser Gln Asp Asn Leu Thr Ile Thr Met His Arg Leu Gln 65 70 75 80 Leu Ser Asp Thr Gly Thr Tyr Thr Cys Gln Ala Ile Thr Glu Val Asn 85 90 95 Val Tyr Gly Ser Gly Thr Leu Val Leu Val Thr Glu Glu Gln Ser Gln 100 105 110 Gly Trp His Arg Cys Ser Asp Ala Pro Pro Arg Ala Ser Ala Leu Pro 115 120 125 Ala Pro Pro Thr Gly Ser Ala Leu Pro Asp Pro Gln Thr Ala Ser Ala 130 135 140 Leu Pro Asp Pro Pro Ala Ala Ser Ala Leu Pro 145 150 155 <210> 24 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 24 Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr Trp Asn Leu Gly Glu 1 5 10 15 Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser Asn Pro Thr Ser Gly 20 25 30 Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala Ala Ser Pro Thr Phe 35 40 45 Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala Ala Glu Gly Leu Asp 50 55 60 Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp Thr Phe Val Leu Thr 65 70 75 80 Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala 85 90 95 Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu 100 105 110 Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 115 120 125 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 130 135 140 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 145 150 155 160 Asp <210> 25 <211> 149 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 25 Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln Thr Asn Lys Met Val 1 5 10 15 Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser Asn Met Arg Ile Tyr 20 25 30 Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp Ser His His Glu Phe 35 40 45 Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile His Gly Glu Glu Val 50 55 60 Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala Ser Arg Phe Ile Leu 65 70 75 80 Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly Ile Tyr Phe Cys Met 85 90 95 Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys Gly Thr Gln Leu Ser 100 105 110 Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro Thr Lys Lys Ser Thr 115 120 125 Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro Glu Thr Gln Lys Gly 130 135 140 Pro Leu Cys Ser Pro 145 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 26 Gly Gln Asn Asp Thr Ser Gln Thr Ser Ser Pro Ser 1 5 10 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 27 gggtcatcac acacaag 17 <210> 28 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 28 gatgcccgcc acgcacc 17 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 29 gccacaaaga ccatcaag 18 <210> 30 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 30 ggagacttta tatgctg 17 <210> 31 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 31 ggcgtttggg ggcaaga 17 <210> 32 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 32 gtccactatg acaattg 17 <210> 33 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 33 gccggagctc caagcag 17 <210> 34 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 34 gggggccttg tcgttgg 17 <210> 35 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 35 gggtaccatc agctatg 17 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 36 gccagcgcca gaagcag 17 <210> 37 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 37 ggagactgct gcacctc 17 <210> 38 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 38 gccgcatcga cttctca 17

Claims

1. A modified cell, comprising a modified chimeric antigen receptor polynucleotide, which polynucleotide encodes a chimeric antigen receptor polypeptide, said chimeric antigen receptor polypeptide comprises: a signal peptide, a CD4 antigen recognition domain, a hinge region, a transmembrane region, at least one co-stimulatory domain, and a signaling domain; wherein the modified cell is a T cell or NK cell comprising a CD4 antigen recognition domain.

2. The modified cell according to claim 1, wherein the CD4 antigen recognition domain comprises a binding portion or variable region of a monoclonal antibody selective for CD4.

3. The modified cell according to claim 1, wherein the CD4 antigen recognition domain comprises CD4scFv.

4. The modified cell according to claim 1, wherein the CD4 antigen recognition domain comprises a polypeptide selective for SEQ ID NO.

21.

5. The modified cell according to any one of claims 1 to 4, wherein the hinge region comprises a hinge region of a human protein selected from the group consisting of: CD-8α, CD28, 4-1BB, OX40, CD3-ζ, its functional derivatives, and combinations thereof.

6. The modified cell according to any one of claims 1 to 4, wherein the transmembrane domain comprises a transmembrane region of a human protein selected from the group consisting of: CD-8α, CD28, 4-1BB, OX40, CD3-ζ, its functional derivatives, and combinations thereof.

7. The modified cell according to any one of claims 1 to 4, wherein the signaling domain comprises a signaling domain selected from the group consisting of: CD3ζ, FcRγ (FCER1G), FcγRlla, FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, DAP12, its active fragments, and combinations thereof.

8. The modified cell according to any one of claims 1 to 4, wherein the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of: OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, NKG2D, B7-H3, ligand binding to CD83, ICAM-1, LFA-1 (CDlla / CD18), ICOS, and 4-1BB (CD137), its active fragments, and combinations thereof.

9. The modified cell according to any one of claims 1 to 4, which comprises at least one of the following: SEQ ID NO.13 or SEQ ID NO.

14.

10. The modified cell according to any one of claims 1 to 4, wherein the modified chimeric antigen receptor polynucleotide comprises at least one of the following: SEQ ID NO. 4 and SEQ ID NO.

5.

11. The modified cell according to claim 10, wherein the polynucleotide is in a vector.

12. The modified cell according to claim 11, wherein the vector comprises a promoter.

13. The modified cell according to claim 1, wherein the cell is a CD8 T cell.

14. The engineered cell according to claim 1, wherein the cell is an NK-92 cell.

15. The engineered cell according to claim 1, wherein the cell is an NKT cell.

16. A method for generating an engineered cell according to any one of claims 1 to 15, the method comprising the following steps: i. providing peripheral blood cells or cord blood cells; ii. introducing a polynucleotide according to any one of claims 10 to 12 into the cells of step (i); iii. amplifying the cells of step (ii); and iv. isolating the cells of step (iii) to provide the engineered cell.

17. A method for reducing the number of CD4-positive T cell leukemia cells or CD4-positive T cell lymphoma cells in vitro, the method comprising the following steps: i. contacting the CD4-positive T cell leukemia cells or CD4-positive T cell lymphoma cells with an effective amount of an engineered cell comprising a polynucleotide encoding a chimeric antigen receptor polypeptide, the polynucleotide comprising: a signal peptide, an antigen recognition domain selected from CD4, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain; and ii. optionally analyzing and detecting CD4-positive T leukemia cells or CD4-positive lymphoma cells; wherein the engineered cell is a T cell comprising a CD4 antigen recognition domain.

18. The method according to claim 17, wherein the CD4-positive leukemia cells or CD4-positive lymphoma cells are in a patient.

19. The method according to claim 17, wherein the CD4-positive lymphoma / leukemia cell line or anaplastic large cell lymphoma or T-ALL cells.

20. The method according to claim 17, wherein the CD4-positive T cell leukemia or T cell lymphoma cells are Sezary cells or acute myeloid leukemia cells.

21. The method according to any one of claims 18-20, wherein the engineered cells comprise at least one of T cells and natural killer cells.

22. Use of an engineered cell according to any one of claims 1 to 16 in the preparation of a medicament for treating cell proliferative diseases, wherein the proliferative disease comprises a CD4-related proliferative disease selected from the group consisting of acute myeloid leukemia.

23. The use according to claim 22, wherein the engineered cells comprise T cells having a CD4 antigen recognition domain.

24. The use according to claim 22, wherein the acute myeloid leukemia is selected from the group consisting of: acute myeloid leukemia M4 and acute myeloid leukemia M5.

25. The use according to claim 22, wherein the cell proliferative disease is selected from the group consisting of T cell leukemia and T cell lymphoma.

26. The use according to claim 25, wherein the T cell leukemia comprises large granular lymphocyte leukemia, adult T cell leukemia, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia (T-ALL).

27. The use according to claim 26, wherein the T cell lymphoma includes peripheral T cell lymphoma, extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma.

28. The use according to any one of claims 22 to 27, wherein the modified cell line is deactivated.

29. The use according to any one of claims 22 to 27, wherein the modified cell further comprises a suicide system.

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