Universal chimeric antigen receptor-expressing immune cells for allogeneic cell therapy

Abstract

This invention describes compositions and methods for treating diseases associated with the expression of group differentiation 33 (CD33) and / or group differentiation 5 (CD5). The compositions and methods relate to two chimeric antigen receptors (CARs) specific to CD33 and CD5, and T cells containing both CD33 and CD5 dual-CARs. A method for administering genetically modified T cells expressing dual-CARs is described. These genetically modified cells can be used for autologous and allogeneic therapy of T-cell malignancies.

Description

Technical Field

[0001] This invention relates to immune cells that express a universal chimeric antigen receptor for allogeneic cell therapy. Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 119,227, filed November 30, 2020. The entire teachings of the above application are incorporated herein by reference. Data in ASCII text files is incorporated by reference.

[0003] This application incorporates by reference a sequence list containing the following ASCII text files, which is filed concurrently with this application. a) File name: 58011002002_SEQUENCELISTING.txt; created on October 5, 2021, size is 139,353 bytes. Prior Technology

[0004] Graft-versus-host disease (GVHD) may be an obstacle to effective allologeneic cell therapy. Most T cells (>90%) in humans exhibit T cell receptor (TCR) heterodimers containing both α and β chains, and are referred to as αβ T cells. If αβT cells are used for allogeneic cell therapy, the endogenous TCRs on these allogeneic αβT cells can recognize the recipient's allogeneic antigens through the major histocompatibility complex (MHC)-dependent pathway, leading to GVHD. Furthermore, the expression of HLA on the surface of allogeneic T cells may cause rapid rejection by the host immune system (host-versus-graft disease, HVGD). Other types of immune cells (including γδT cells, NKT cells, and NK cells) can be used for allogeneic therapy, but each has significant drawbacks. Therefore, a simple and effective method for generating universal immune cells containing chimeric antigen receptors (CARs) for allogeneic therapy is the genetic modification of αβT cells. Early-generated universal CAR-T cells (universal CAR-T cells)... UCAR-T cells prevent GVHD by knocking out the TCR α and / or β chains, and by knocking out B2M or HLA-I molecules to avoid rejection by HVG. However, removing or reducing HLA-I expression may trigger potent NK cell cytotoxicity. To reduce NK cell cytotoxicity, HLA-E, HLA-F, or HLA-G have been ectopically expressed, but the resulting inhibition of NK cell cytotoxicity remains very limited.

[0005] Cellectis and Allogene designed an alternative strategy that uses a combination of αβT cells and a CD52 monoclonal antibody (alemtuzumab) to generate UCAR-T cells. This is achieved using a genetic editing tool—a transcription activator-like effector nuclease. The TALEN nuclease technique eliminates both TCR and CD52 in αβT cells to prevent GVHD and counteract the toxicity of aleizumab to UCAR-T cells. Preliminary results from a phase 1 study of UCAR-T in CD19-positive relapsed / refractory acute lymphoblastic leukemia (ALL) showed an 82% complete remission rate in adult and pediatric patients receiving a lymphocyte-clearing regimen consisting of fludarabine, cyclophosphamide, and aleizumab. However, even after allogeneic stem cell transplantation, more than 70% of these patients relapsed or died within one year. Summary of the Invention

[0006] One problem with previous processes was that they could take 3 to 4 weeks, which might be too long for some patients whose disease progresses very rapidly. In some cases, a patient's T cells may be of poor quality after several rounds of chemotherapy before T cell isolation. For patients with T-cell malignancies, it is difficult to isolate healthy T cells. Therefore, the medical need for "off-the-shelf" universal CAR-T (UCAR-T) cells from healthy donors for allogeneic therapy remains critically unmet.

[0007] This disclosure provides a solution to a pressing need in the field of allopathic off-the-shelf immunotherapy for cancer treatment. This disclosure relates to systems, components, and methods for expanding the use of engineered immune cells with receptors targeting two or more targets.

[0008] This article describes immune cells exhibiting universal chimeric antigen receptor (UCAR) expression (e.g., lymphocytes, such as T cells (UCAR-T cells)) and their use in treating diseases (e.g., cancer) and other physiological conditions. More specifically, this article describes UCAR-T cells and their use in treating diseases associated with CD33 and / or CD5 expression (e.g., acute myeloid leukemia (AML) and / or T-cell malignancies). UCAR-T cells contain one or more nucleic acid constructs encoding one or more chimeric antigen receptors targeting CD5 and CD33 and are referred to herein as "dual-CAR T cells".

[0009] The specific embodiment is a gene-transfected lymphocyte representing a CAR, which includes a signaling peptide, an extracellular domain including a variable light chain VL and a variable heavy chain VH domain that binds CD5 and a VL and VH domain that binds CD33, a hinge domain, a transmembrane domain, and a co-stimulatory domain.

[0010] In some specific embodiments, the messaging peptide has an N-terminus for the VL domain binding to CD5, an N-terminus for the VH domain binding to CD33, and an N-terminus for the VH domain binding to CD5. (This is repeated four times in the original text.) In some specific embodiments, the messaging peptide is N-terminus for the VH domain binding CD33, N-terminus for the VL domain binding CD5, N-terminus for the VH domain binding CD5, and N-terminus for the VL domain binding CD33. In some specific embodiments, the messaging peptide is a CD8α messaging peptide, a GM-CSF messaging peptide, a CD4 messaging peptide, a CD137 (4-1BB) messaging peptide, or a combination thereof. In some specific embodiments, one or more of the linker domains are (G4S)n, 218 linkers where n is 1 or 3, or a combination thereof.

[0011] In some specific embodiments, the hinge domain is a CD8α hinge domain, a CD28 hinge domain, a CD137 hinge domain, an IgG1 hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, or a combination thereof.

[0012] In some specific embodiments, the transmembrane structural domain is the CD8α transmembrane structural domain, CD28 transmembrane structural domain, CD3e transmembrane structural domain, CD45 transmembrane structural domain, CD4 transmembrane structural domain, CD5 transmembrane structural domain, CD9 transmembrane structural domain, CD16 transmembrane structural domain, CD22 transmembrane structural domain, CD33 transmembrane structural domain, CD37 transmembrane structural domain, CD64 transmembrane structural domain, CD80 transmembrane structural domain, CD86 transmembrane structural domain, CD134 transmembrane structural domain, CD137 transmembrane structural domain, transmembrane structural domain CD154, or a combination thereof.

[0013] In some specific embodiments, the costimulatory domains are 4-1BB costimulatory domains, CD28 costimulatory domains, OX40 costimulatory domains, CD2 costimulatory domains, CD7 costimulatory domains, CD27 costimulatory domains, CD28 costimulatory domains, CD30 costimulatory domains, CD40 costimulatory domains, CD70 costimulatory domains, CD134 costimulatory domains, PD1 costimulatory domains, ICOS costimulatory domains, NKG2D costimulatory domains, GITR costimulatory domains, TLR2 costimulatory domains, or combinations thereof.

[0014] The VH domain binding to CD5 includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:26 or SEQ ID NO:28. The VL domain binding to CD5 includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:27 or SEQ ID NO:29. The VH domain binding to CD33 includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:30. The VL domain binding to CD33 includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:31. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:14. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:15. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:16. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:17. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:18. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:19. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:20. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:21. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:22. In some embodiments, the CAR includes an amino acid sequence that is at least 95% identical to that of SEQ ID NO:23.

[0015] In some embodiments, the CAR includes an amino acid sequence selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

[0016] In some embodiments, the gene-transfected lymphocytes are T cells. The T cells express the T cell receptor α chain or T cell receptor β chain at a level that does not induce graft-versus-host disease (GVHD) when the gene-transfected lymphocytes are administered to a patient. In some embodiments, the gene-transfected lymphocytes are natural killer cells. In some specific embodiments, the gene-transfected lymphocytes express exogenous nucleic acids encoding interleukins or interleukin receptor genes. The encoded interleukin or interleukin receptor genes are interleukin-2, interleukin-7, interleukin-12, interleukin-15, or interleukin-21. In some specific embodiments, the lymphocytes express exogenous nucleic acids encoding suicide genes. In some embodiments, the pathological condition is a T-cell malignancy.

[0017] In one specific embodiment, the gene-transfected lymphocytes exhibit both CD5-binding CARs and CD33-binding CARs. The CD5-binding CAR includes a signaling peptide, an extracellular domain comprising CD5-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The CD33-binding CAR includes a signaling peptide, an extracellular domain comprising CD33-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain.

[0018] In some specific embodiments, for the CD33-binding CAR, the messaging peptide is N-terminus for the CD33-binding VL domain, N-terminus for the CD33-binding VH domain, and N-terminus for the hinge domain. In some specific embodiments, for the CD5-binding CAR, the messaging peptide is N-terminus for the CD5-binding VL domain, N-terminus for the CD5-binding VH domain, and N-terminus for the hinge domain.

[0019] In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:2. In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:3. In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:4. In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:5. In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:6. In some embodiments, the CD5-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:7. In some embodiments, the CD5-binding CAR includes an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0020] In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:8. In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:9. In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:10. In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:11. In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:12. In some embodiments, the CD33-binding CAR includes an amino acid sequence that is at least 95% identical to SEQ ID NO:13. In some embodiments, the CD33-binding CAR includes an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0021] In one specific embodiment, a method for producing gene-transfected lymphocytes expressing a chimeric antigen receptor binding to CD5 and CD33 includes introducing nucleic acid into the lymphocytes. The nucleic acid introduced into the lymphocytes encodes a message peptide, including extracellular domains, hinge domains, transmembrane domains, and co-stimulatory domains of the CD5-binding VL and VH domains and the CD33-binding VL and VH domains. In some specific embodiments, introducing the nucleic acid into the lymphocytes includes electroporation, transduction, or transduction. In some specific embodiments, the introduced nucleic acid is a viral vector, a non-viral vector, or naked DNA. The viral vector is a lentiviral vector or an adeno-associated viral vector. In some specific embodiments, the nucleic acid is integrated into the lymphocyte genome. The nucleic acid is randomly integrated into the lymphocyte genome. In some specific embodiments, the method is performed under conditions that allow CAR expression in gene-transfected lymphocytes. In some specific embodiments, cells express CAR after the introduction of nucleic acid.

[0022] In one specific embodiment, a method for manufacturing gene-transfected lymphocytes expressing a CD5-binding CAR and a CD33-binding CAR is disclosed, wherein the CD5-binding CAR and the CD33-binding CAR are linked by self-cleaving proteins, and wherein the method includes introducing nucleic acids into the lymphocytes. The nucleic acid encodes the CD5-binding CAR, which includes a signaling peptide, an extracellular domain including CD5-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The nucleic acid also encodes the CD33-binding CAR, which includes a signaling peptide, an extracellular domain including CD33-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain.

[0023] In some embodiments, the self-cleaving peptide is a 2A peptide. In some embodiments, the introduction of nucleic acid into lymphocytes includes electroporation, transduction, or transduction. In some embodiments, the nucleic acid is introduced into lymphocytes using a viral vector, a non-viral vector, or naked DNA. The viral vector is a lentiviral vector or an adeno-associated viral vector. In some embodiments, the nucleic acid is integrated into the lymphocyte genome. The nucleic acid is randomly integrated into the lymphocyte genome. In some embodiments, the method is performed under conditions that allow CAR expression in gene-transfected lymphocytes. In some embodiments, cells express CAR after the introduction of nucleic acid.

[0024] In one specific embodiment, the nucleic acid-encoded CAR includes a message peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes VL and VH domains that bind CD5, VL and VH domains that bind CD33, and a linker domain between adjacent VL and VH domains.

[0025] In one specific embodiment, the nucleic acid encodes a CD5-binding CAR and a CD33-binding CAR. The CD5-binding CAR includes a messaging peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes CD5-binding VL and VH domains and a connectant domain between adjacent VL and VH domains. The CD33-binding CAR includes a messaging peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes CD33-binding VL and VH domains and a connectant domain between adjacent VL and VH domains.

[0026] In one specific embodiment, a method of treating acute myeloid leukemia (AML) includes administering an effective amount of gene-transgenic lymphocytes to a patient in need. In some specific embodiments, the gene-transgenic lymphocytes are allogeneic. In some embodiments, the lymphocytes are autologous. In some specific embodiments, AML comprises leukemic cells expressing CD33 as a cell surface protein.

[0027] In one specific embodiment, a method of treating T-cell malignancies includes administering a therapeutically effective amount of gene-transfected lymphocytes to a patient in need. In some specific embodiments, the gene-transfected lymphocytes are allogeneic. In some specific embodiments, the gene-transfected lymphocytes are autologous. In some specific embodiments, the T-cell malignancies include T cells that express CD5 as a cell surface protein.

[0028] The gene-transfected lymphocytes described in this article target CD5, which is associated with the patient's T cells. By targeting the patient's T cells, the gene-transfected lymphocytes reduce host resistance to graft-versus-graft disease (HVGD). The combination of targeting the patient's T cells (CD5) and targeting cells associated with the disease state (CD33) provides a synergistic effect, as further described in this article. Simple Explanation of the Diagram

[0029] As illustrated in the accompanying drawings, the foregoing will become apparent from the following detailed description of exemplary embodiments, wherein the same reference numerals refer to the same parts in different views. The drawings are not necessarily drawn to scale, but rather focus on illustrating specific embodiments.

[0030] [Figure 1A] to [Figure 1B] illustrate the expression of CD5 and CD33 on the surface of T-ALL, AML, and genetically modified SK-Hep1 cell lines. T-ALL cell lines all expressed 100% CD5, while AML cell lines all expressed CD33 (Figure 1A). SK-Hep-1-CD5 and SK-Hep-1-CD33 cell lines were also generated by ectopic expression of CD5 and CD33 on SK-Hep-1 cells (Figure 1B).

[0031] [Figure 2] Illustration of TRAC gRNA screening to assess TRAC knockout efficiency. T cell lines were activated with CD3 / CD28 microbeads and electroporated with CRISPR Cas9 and indicated sgRNA (with or without enhancer). TRAC knockout efficiency was assessed by flow cytometry on day 5 after electroporation and following CD3 staining.

[0032] [Figures 3A] to [Figure 3B] illustrate the screening of CD5 gRNAs to assess CD5 knockout efficiency. T cell lines were activated with CD3 / CD28 microbeads and electroporated with CRISPR Cas9 and indicated sgRNAs. Five gRNAs were tested with and without electroporation enhancers. CD5 KO efficiency was assessed by flow cytometry on day 5 after electroporation and following CD5 staining.

[0033] [Figures 4A] to [Figure 4B] illustrate the CAR expression and purity of CD5 CAR on T cells. CD5 CAR expression was assessed using APC-labeled CD5 antigen (Figure 4A). T cell purity was assessed using anti-CD3 and anti-CD5 antibodies (Figure 4B).

[0034] [Figures 5A] to [5B] illustrate how CAR5 CARs were further evaluated and sequenced using short-term cytotoxicity assays by mixing fresh CD5 CAR-T cells with CSFE-labeled allogeneic pan-T cells (Figure 5A) or CCRF-CEM-luc (Figure 5B) at indicated effector cell to target cell ratios. NC represents untransduced T cells. CD19 CAR-T cells were used as a negative control.

[0035] [Figure 6A] to [Figure 6B] illustrate the different CD33 CAR (CAR33) manifestations on T cells (Figure 6A) and CAR33 screening using a luciferase-based assay to assess the cytotoxicity of CAR33 on MOLM13-luc cells (Figure 6B).

[0036] Figures 7A to 7F illustrate the structural configuration of the CD5 and CD33 bis-CAR constructs. Figures 7A to 7D are schematic diagrams of the two ring structures. Figures 7E to 7F are schematic illustrations of the peptide 2A (P2A) structure.

[0037] [Figure 8] illustrates the single-CAR and double-CAR performance in different indicated constructs 11 days after transduction with lentiviral vector constructs encoding CAR5, CAR33 or double CAR5-CAR33 with corresponding scFv.

[0038] Figures 9A to 9B illustrate how real-time cellular impedance monitoring (RTCA) was used to further evaluate the in vitro function of single-CAR and dual-CAR constructs by mixing fresh CAR-T cells with CD5-SK-Hep-1 (Figure 9A) or CD33-SK-Hep-1 (Figure 9B) cells.

[0039] [Figures 10A] to [Figures 10C] illustrate the evaluation of the function of the dual-CAR construct by using a luciferase-based assay to assess in vitro cytotoxicity against AML and T-ALL cell lines. CAR-T cells were co-cultured with MOLM13-luc (Figure 10A), MV-4-11-luc (Figure 10B), or CCRF-CEM-luc (Figure 10C) at a ratio of 2:1 or 1:1 (effect cells vs. target cells) for 18 hours.

[0040] [Figures 11A] to [Figures 11D] illustrate the cytotoxicity of dual-CAR CAR-T cells against autologous or allogeneic T cells. Fresh dual-CAR T cells were co-cultured with autologous T cells (Figures 11A to 11B) or allogeneic T cells (Figures 11C to 11D) at a ratio of 2:1 or 1:1. Cytotoxicity was assessed by CFSE-based flow cytometry after 18 or 44 hours of co-culture.

[0041] [Figures 12A] to [12F] illustrate the in vitro functional evaluation of CD5 / TCR knockout / IL15-armed dual CAR-T (CD5 / CD33) cells. Figures 12A to 12B illustrate the cytotoxic activity of CD5 / TCR knockout / IL15-armed dual CAR-T (CD5 / CD33) cells against MOLM13-luc (Figure 12A) or allogeneic T cells (Figure 12B) after 18 h of co-culture. Figures 12C to 12D illustrate the proliferation of CD5 / TCR knockout dual CAR-T (CD5 / CD33) cells when co-cultured with MOLM13-luc cell line (Figure 12C) or allogeneic T cells (Figure 12D). Figures 12E to 12F illustrate the release of intercytokines (IFN-γ) from CD5 / TCR knockout dual CAR-T (CD5 / CD33) cells after stimulation with CD33-positive cells (Figure 12E) or CD5-positive cells (Figure 12F).

[0042] [Figures 13A] through 13G illustrate the avoidance of allogeneic PBMC rejection by CD5 / TCR knockout / IL15-armed dual CAR-T (CD5 / CD33) cells (UCAR-T). UCAR-T cells (HLA-A2+) were co-cultured with allogeneic PBMCs (HLA-A2-), T-cell-depleted allogeneic PBMCs (PBMC-T), or NK-cell-depleted allogeneic PBMCs (PBMC-NK). UCAR-T and allogeneic T cells were gated by CD3 and HLA-A2 (Figures 13A to 13C). Figures 13D through 13G illustrate the quantification of HLA-A2+ levels on each indicator day relative to the number on day 0.

[0043] [Figures 14A] to [14C] illustrate the antitumor efficacy of CD5 / TCR knockout / IL15-armed dual CAR-T (CD5 / CD33) cells in MOLM13-luc and allogeneic T cell xenograft models. Figures 14A and 14B show bioluminescence imaging and signals of MOLM13-luc cancer growth. Figure 14C shows the Kaplan-Meier curves generated from animal survival in Figure 14A.

[0044] [Figures 15A] to [Figure 15C] illustrate flow cytometry analysis of cancer cells (HLA-A2- / human CD3-), allogeneic T cells (HLA-A2- / human CD3+), and CD5 / TCR knockout / IL15-armed dual CAR-T (CD5 / CD33) cells (HLA-A2 positive) in mice. Human cells were gated out using anti-mCD45 and anti-hCD45 (Figure 15A). T cells were gated out using anti-hCD3 and anti-hHLA-A2. UCAR-T cell lines were HLA-A2 positive while allogeneic T cell lines were HLA-A2 negative (Figure 15B). CD33 CAR performance was assessed by staining with CD33 antigen (Figure 15C).

[0045] In all the diagrams, "NC" stands for "No CAR-T". Implementation

[0046] Specific embodiments are illustrated below.

[0047] Allogeneic cell therapy strategies targeting both the patient's immune T cells and cancer cells have shown significant efficacy in reducing cancer levels in those patients. In short, this article describes the aforementioned dual-CAR system and T cells expressing dual-CARs, as well as methods for producing dual-CAR systems and T cells expressing dual-CARs. Dual-CAR systems eliminate both CD5+ and CD33+ cells, including CD5 / CD33 ectopic cell lines, tumor cell lines expressing CD5 or CD33, and pan-T cells from the same or different individuals. Therefore, these CAR-T cells eliminate CD5-expressing tumor cells (such as some T-cell malignancies) and also eliminate CD33-expressing tumor cells, as seen in, for example, AML. Because dual-CAR constructs eliminate allogeneic T cells, one use is to produce ready-made, universal CAR-T cells for allogeneic therapy of AML and other T-cell malignancies. By using gene editing technology to knock out TRAC, CAR-T cells will not cause graft-versus-host disease (GVHD) during allogeneic cell therapy. Because CAR5 eliminates endogenous T cells in the patient, CAR-T cells avoid rejection by the patient's immune system (HVG), resulting in prolonged survival and function. These dual-CAR modified T cells are expected to have potent tumor-eliminating efficacy against both AML and other T-cell malignancies in humans. The anti-tumor function of current systems can be further improved by combining dual-CAR modified T cells with cytokines including IL2, IL7, IL15, and IL21, whose signaling pathways have been shown to significantly increase the persistence of CAR-T cells in vivo.

[0048] In some specific embodiments, UCAR-T cells do not express the TCRα chain and / or β chain, or express low levels of the TCRα chain and / or β chain (e.g., to avoid inducing graft-versus-host disease (GVHD) during in vivo use). UCAR-T cells may also be engineered to not express CD5 or express low levels of CD5 (e.g., to limit cannibalism among UCAR-T cells in vivo). UCAR-T cells may also contain nucleic acids encoding intercytokines (e.g., IL2, IL7, IL12, IL15, or IL21) or intercytokine receptors (e.g., to increase the persistence and expansion of UCAR-T cells in vivo). UCAR-T cells may further contain nucleic acids containing suicide genes (e.g., to enable the elimination of UCAR-T cells in the event of severe side effects during treatment of patients). Acute myeloid leukemia

[0049] As used in this article, "Acute Myeloid Leukemia" or AML is a cell cancer originating in the bone marrow and often rapidly enters the bloodstream. AML infection involves white blood cells called "lymphocytes" (WBCs). Lymphocytes are mature white blood cells that develop from lymphoblasts in the bone marrow. Lymphocytes are the main cells that make up lymphoid tissue, a major part of the immune system, and are found in lymph nodes, the thymus, spleen, tonsils, and pinnatifida. In most cases of AML, cells express CD33 as a cell surface protein.

[0050] In order to treat AML patients and to avoid being bound by theory, it is believed that the dual-targeting CAR cells described in this article can avoid rejection by the recipient patient by targeting CD5 expressed on the surface of the patient's T cells, and can create space for the expansion of UCAR-T in vivo by targeting CD33 to kill CD33 expressed and CD33 expressed hematopoietic stem cells and other bone marrow cells in the recipient patient. T-cell malignant tumors

[0051] As used in this article, "T-cell malignancy" refers to lymphomas affecting T cells. T-cell malignancies are a heterogeneous group of diseases characterized by clonal growth and abnormal T-cell function, broadly classified into T-cell lymphomas (TCL) and T-cell leukemias, with mature and precursor subtypes. Despite advances in T-cell malignancies, new targeted therapies are needed to improve prognosis, particularly for relapsed and refractory patients. In some T-cell malignancies, T cells express CD5 as a cell surface protein (e.g., CD5-positive hematopoietic malignancies).

[0052] To treat T-cell malignancies and without being bound by theoretical limitations, the dual-targeting CAR cells described herein are believed to be able to kill CD5 and / or CD33-expressing T-cell tumor cells, normal T cells, and CD33-expressing hematopoietic stem cells. In some specific embodiments, patients may also receive allogeneic hematopoietic stem cell transplantation. Overview of chimeric antigen receptors

[0053] Chimeric antigen receptors (CARs) are proteins that bind to specific antigens. The chimeric antigen receptors described herein are designed to bind to one or more of group differentiation 5 (CD5) and group differentiation 33 (CD33).

[0054] In some specific embodiments, the gene-transfected lymphocytes exhibit both CD5-binding CARs and CD33-binding CARs. The CD5-binding CAR includes a signaling peptide, an extracellular domain comprising CD5-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The CD33-binding CAR includes a signaling peptide, an extracellular domain comprising CD33-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. Connector domains may be included between adjacent domains.

[0055] Lymphocytes can be genetically modified to express chimeric antigen receptors. These genetically modified lymphocytes, expressing CARs, can be administered to patients to treat diseases such as cancer. In this way, the lymphocytes are altered to bind to and attack disease-associated cells. Two types of lymphocytes that can be genetically modified to express chimeric antigen receptors are T cells and natural killer (NK) cells. These are respectively called CAR T cells and CAR NK cells.

[0056] CAR T cells can be produced by collecting blood from a patient and introducing the CAR gene into the patient's T cells. To engineer CAR T cells, blood is drawn from and collected from the patient. White blood cells (including T cells) are collected from the blood sample. The gene for the desired chimeric antigen receptor is transduced into the T cells in vitro. These CAR T cells are proliferated and then transferred to the patient via infusion. The CAR T cells are then able to bind to target antigens on the patient's cancer cells. Dual-CAR T cells

[0057] As used herein, the term "dual-CAR T cell" refers to a CAR T cell engineered to simultaneously express two tumor-associated antigen receptors on a single cell surface, reducing the likelihood of T cells attacking non-tumor cells. Figures 7A to 7F illustrate the tandem and loop configurations of multi-target CAR T cells. Tandem CARs within a single CAR molecule contain two distinct scFvs, which can be stacked in tandem or form loop structures.

[0058] Expressing two CAR molecules in a single viral plasmid may require codon optimization of the repetitive DNA to reduce the chance of DNA recombination. Another approach is to design a receptor that tandemly fuses two scFv molecules with different specificities to a single intracellular module. Tandem CARs offer the advantage of a smaller transgene size compared to bi-CARs, which is important when other transgenes (i.e., cytokines) are also incorporated into the CAR plasmid.

[0059] In some specific embodiments, the nucleic acid-encoded CAR includes a message peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes VL and VH domains that bind CD5, VL and VH domains that bind CD33, and a connectant domain between adjacent VL and VH domains. Connectant domains may be included between adjacent domains.

[0060] In some specific embodiments, the nucleic acid encodes a CD5-binding CAR and a CD33-binding CAR. The CD5-binding CAR includes a messaging peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes CD5-binding VL and VH domains and a connectant domain between adjacent VL and VH domains. The CD33-binding CAR includes a messaging peptide, an extracellular domain, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The extracellular domain includes CD33-binding VL and VH domains and a connectant domain between adjacent VL and VH domains.

[0061] In some specific embodiments, the CAR5 and CAR33 systems exhibit in the same construct and the two scFv systems are linked by a loop. In some specific embodiments, the CAR5 and CAR33 systems exhibit in the same construct and the two CAR systems are linked via a 2A self-cleaving peptide or IRES.

[0062] Dual-CARs can be constructed using single-chain variable fragments (scFv) and / or single-domain variable fragments (sdFv, V HH, or nanoantibodies) corresponding to CD5 and CD33 antigens in the same or different constructs. Messaging peptide domain

[0063] A "signal peptide" refers to the peptide sequence located at the N-terminus of the CAR (Cardiac Artery Activated Cell) that guides the protein to the endoplasmic reticulum and subsequently to the T cell surface. The signal peptide domain can be a full-length domain or a fragment thereof. In the CAR, the scFv (cell activator function) can fuse to the TCR membrane or an intracellular endodomain. A spacer may be included between the scFv and the TCR transmembrane to allow for variable orientation and antigen binding. The signal peptide guides the transport and localization of proteins within the cell, for example, directing proteins to a specific cellular organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0064] The message peptide domain is typically N-terminal to the extracellular domain of a CAR, and is generally located at the N-terminus of the CAR (i.e., the Nth-most domain). In some specific embodiments, the message peptide is the CD8α message peptide (SEQ ID NO:40), the granulocyte-macrophage colony-stimulating factor (GM-CSF) message peptide (SEQ ID NO:41), the CD4 message peptide (SEQ ID NO:42), the CD137 (4-1BB) message peptide (SEQ ID NO:43), or a combination thereof. In some specific embodiments, one or more of the linker domains are (G4S)n, 218 linkers (SEQ ID NO:44), or a combination thereof, where n is 1 or 3. extracellular domain

[0065] "Extracellular domain" refers to the antigen recognition domain of a chimeric antigen receptor exposed outside the cell. The extracellular domain can be a full-length domain or a fragment thereof. The extracellular domain interacts with its target antigen and is responsible for targeting CAR T cells to any cell expressing the matching molecule. The extracellular domain can be derived from the variable region of a monoclonal antibody and can be, for example, a single-chain variable fragment (scFv) of a monoclonal antibody. The scFv may include variable light chain (VL) and variable heavy chain (VH) regions, which may be capable of binding to the target antigen (e.g., CD33 or CD5). Other embodiments using the extracellular domain include single-domain antibodies (sdAbs), such as VHH sdAbs.

[0066] In some specific embodiments, the messaging peptide has an N-terminus for the VL domain binding to CD5, an N-terminus for the VH domain binding to CD33, and an N-terminus for the VH domain binding to CD5. (This is repeated four times in the original text.) In some specific embodiments, the messaging peptide has an N-terminus for the VH domain binding CD33, an N-terminus for the VL domain binding CD5, an N-terminus for the VH domain binding CD5, and an N-terminus for the VL domain binding CD33.

[0067] CARs are not limited to using scFvs as the target extracellular domain and other ligands and receptors can be used instead. For example, interleukin-specific CARs have been prepared by modifying interleukin molecules to form extracellular domains. Cytokines, innate immune receptors, tumor necrosis factor receptors, growth factors, and structural proteins can all be used as extracellular domains for CARs.

[0068] Suitable CD5 CAR scFvs include H65, hH65, and others that are commercially available and / or described in the literature. Humanized versions may also be used.

[0069] Suitable CD33 CAR scFvs include My9.6, humanized My9.6, M195, lintuzumab (HuM195), and others that are commercially available and / or described in the literature. Humanized versions may also be used.

[0070] In some specific embodiments, the extracellular domain of the CD5 CAR includes CD5-binding VL and VH domains and connecting subdomains between adjacent VL and VH domains. In some specific embodiments, the extracellular domain of the CD33 CAR includes CD33-binding VL and VH domains and connecting subdomains between adjacent VL and VH domains. Hinge structural domain

[0071] As used herein, the term "hinge domain" refers to the extracellular structural region of a CAR that separates the binding unit from the transmembrane domain. The hinge domain can be a full-length domain or a fragment thereof. Most CARs are designed with an immunoglobulin-like (Ig) domain hinge. The hinge domain is also referred to as a "spacer." These spacers typically provide stability for effective CAR expression and activity. The hinge domain provides flexibility in accessing the target antigen. The hinge domain can also influence the overall performance of CAR T cells.

[0072] In some specific embodiments, the hinge domain is a CD8α hinge domain, a CD28 hinge domain, a CD137 hinge domain, an IgG1 hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, an IgG4 hinge domain, or a combination thereof. Transmembrane domain

[0073] As used in this article, the term "transmembrane domain" ( A transmembrane domain refers to an amino acid sequence that crosses the cell membrane. A transmembrane domain can be a full-length domain or a fragment thereof. Generally, transmembrane domains are hydrophobic. Often, transmembrane domains are α-helices. Although the primary function of the transmembrane domain is to anchor CARs to the T cell membrane, the transmembrane domain may be involved in CAR T cell function.

[0074] In some specific embodiments, the transmembrane structural domain is the CD8α transmembrane structural domain, CD28 transmembrane structural domain, CD3e transmembrane structural domain, CD45 transmembrane structural domain, CD4 transmembrane structural domain, CD5 transmembrane structural domain, CD9 transmembrane structural domain, CD16 transmembrane structural domain, CD22 transmembrane structural domain, CD33 transmembrane structural domain, CD37 transmembrane structural domain, CD64 transmembrane structural domain, CD80 transmembrane structural domain, CD86 transmembrane structural domain, CD134 transmembrane structural domain, CD137 transmembrane structural domain, transmembrane structural domain CD154, or a combination thereof. Co-stimulatory domain

[0075] As used in this article, the term "co-stimulatory domain" ( The term "co-stimulatory domain" refers to a region that enhances the antigen-specific cytotoxicity and / or interferon production of CAR T cells. The co-stimulatory domain can be a full-length domain or a fragment thereof. Intracellular domains are typically derived from co-stimulatory molecules. Co-stimulatory signaling contributes to improving CAR T cell expansion, function, persistence, and antitumor activity. These can be provided by incorporating intracellular signaling domains from one or more T cell co-stimulatory molecules.

[0076] In some specific embodiments, the costimulatory domains are 4-1BB costimulatory domains, CD28 costimulatory domains, OX40 costimulatory domains, CD2 costimulatory domains, CD7 costimulatory domains, CD27 costimulatory domains, CD28 costimulatory domains, CD30 costimulatory domains, CD40 costimulatory domains, CD70 costimulatory domains, CD134 costimulatory domains, PD1 costimulatory domains, ICOS costimulatory domains, NKG2D costimulatory domains, GITR costimulatory domains, TLR2 costimulatory domains, or combinations thereof. Connecting substructures

[0077] A linker domain is a peptide chain segment within a protein that covalently links two adjacent domains. Linker domains can be full-length domains or fragments thereof. As shown in Figures 8A to 8F, linker domains connect target-specific extracellular domains and transmembrane domains. Linker domains can also connect adjacent VL and VH domains. Linker domains play a variety of structural and functional roles in naturally occurring proteins. For example, they play roles in modulating the biological activity of linked domains, in allosteric coupling, and in viral replication. Linker domains are also associated with protein engineering, such as altering the functionality of engineered antibodies.

[0078] In some specific embodiments, one or more of the connector domains are (G4S)n, 218 connectors (SEQ ID NO:44), or combinations thereof, wherein n is 1 or 3. Nucleic acid

[0079] "Nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). Nucleic acids include, for example, DNA (genetic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. Furthermore, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In some specific embodiments, nucleic acid molecules may be modified. In the case of double-stranded polymers, "nucleic acid" can refer to any one or both strands of the molecule.

[0080] "Nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as their non-natural derivatives and analogs. Therefore, nucleotides may include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0081] "Sequence identity" refers to the degree to which two nucleotide sequences, or two amino acid sequences, share the same residues at the same positions when aligned to achieve the maximum level of identity, expressed as a percentage. For sequence alignment and comparison, generally one sequence is designated as the reference sequence and compared with the test sequence. The sequence identity between the reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence, where the reference and test sequences share the same nucleotides or amino acids when aligned to achieve the maximum level of identity. As an example, when two sequences are considered to have 70% sequence identity after alignment to achieve the maximum level of identity, the test sequence has the same nucleotide or amino acid residues at 70% of the same positions across the entire length of the reference sequence.

[0082] Sequence alignments for comparison can be readily performed by those skilled in the art using appropriate alignment methods or algorithms to achieve the highest level of identity. In some examples, alignment may include introducing gaps to provide the highest level of identity. Examples include Smith & Waterman’s local homology algorithm (Adv. Appl. Math. 2:482 (1981)), Needleman & Wunsch’s homology alignment algorithm (J. Mol. Biol. 48:443 (1970)), and Pearson & Lipman’s similarity method exploration (Proc. Natl. Acad. Sci. USA 85:2444 (1988)), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Suite software, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection. (See Ausubel et al., Current Protocols in Molecular Biology for inspection).

[0083] When using sequence comparison algorithms, the test and reference sequences are input into the computer, and (if necessary) subsequent coordinates and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity (of one or more) test sequences relative to the reference sequence based on the specified program parameters. A commonly used tool for determining percentage sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP), which can be obtained from the National Center for Biotechnology Information at the National Library of Medicine, National Institutes of Health, USA (Altschul et al., J Mol Biol.215(3):403-10 (1990)).

[0084] In various specific embodiments, the two nucleotide sequences or the two amino acid sequences may have sequence identity of at least, for example, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. When determining the percentage sequence identity with one or more sequences described herein, the sequences described herein are reference sequences.

[0085] In some embodiments, the variable light chain domain has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO:4. In some embodiments, the variable heavy chain domain has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO:8. carrier

[0086] The terms "vector" and "vector structure" are used to describe the structure of a carrier. "Vector construct" and "expression vector" refer to the introduction of DNA or RNA sequences (e.g., foreign genes) into host cells to transform the host and promote the expression of the introduced sequence (e.g., transcription or translation). Vectors generally consist of DNA as a transmissible agent, into which foreign DNA encoding proteins is inserted using restriction enzyme technology. A common type of vector is the "plasmid," which is typically a self-contained molecule of double-stranded DNA that readily accepts additional (foreign) DNA and can be easily introduced into suitable host cells. Numerous vectors (including plasmids and fungal vectors) have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts.

[0087] The term "expression" refers to the permission or enabling of information in a gene or DNA sequence to be expressed, such as by activating cellular functions related to the transcription and translation of the corresponding gene or DNA sequence to produce proteins. DNA sequences are expressed in or through cellular processes to form "expression products" such as proteins. The expression product itself (e.g., the resulting protein) can also be referred to as "expressed" by the cell. For example, when polynucleotides or polypeptides are expressed or produced in a foreign host under the control of a foreign or natural promoter, or in a natural host cell under the control of a foreign promoter, the polynucleotide or polypeptide is recombinantly expressed. Gene delivery vectors typically include a transgenic gene (e.g., a nucleic acid encoding an enzyme) operatively linked to a promoter and other nucleic acid elements required for the expression of the transgenic gene in a host cell introduced into the vector. Suitable promoters and delivery constructs for gene expression are known in the art. Recombinant plasmids may also include inducible, regulated promoters for the expression of enzymes in cells.

[0088] Various gene delivery vectors are known in the art and include both viral and non-viral (e.g., naked DNA plasmids) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, for example, vectors derived from herpesviruses, baculovirus vectors, lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated viral vectors (AAV), and murine stem cell virus (MSCV). Viral vectors can be replicating or non-replicating. Such vectors can be introduced into many suitable host cells using the methods disclosed or cited herein or other methods known to those skilled in the art.

[0089] Non-viral vectors used for gene delivery include naked DNA, plasmids, transposons, and mRNA. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), and pMAL plasmids (New England Biolabs, Beverly, Mass.). Such vectors can be introduced into many suitable host cells using the methods disclosed or cited herein or other methods known to those skilled in the art.

[0090] In some embodiments, the vector includes an internal ribosome entry site (IRES). In some embodiments, the vector includes a selection marker, such as an ampicillin resistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein, such as green fluorescent protein (GFP). In some embodiments, the nucleic acid is suitable for secondary selection into pMSCV-IRES-GFP between EcoRI and XhoI. In some embodiments, the vector contains a multiple cloning site (MCS) for inserting the desired gene.

[0091] Although the genetic code is degenerate, since most amino acids are represented by multiple codons (called "synonyms"), it should be understood in the art that codon usage in a particular organism is non-random and biased towards specific codon triples. Therefore, in some specific embodiments, the vector includes a nucleotide sequence optimized (e.g., by codon optimization) for expression in a particular type of host cell. Codon optimization refers to the process of modifying a polynucleotide encoding a protein of interest to replace a specific codon in that polynucleotide with a codon encoding the same (one or more) amino acids but more commonly used / recognized in the host cell expressing the nucleic acid. In some cases, the polynucleotides described herein are codon-optimized for expression in T cells. Methods for creating gene-transplanted host cells

[0092] This document describes methods for creating gene-transfected host cells (such as gene-transfected T cells). For example, gene-transfected host cells can be created by introducing one or more of the vectors described in specific embodiments herein into host cells. Gene-transfected host cells can be constructed by collecting host cells from a host's blood and then modifying the host cells to express a transfected gene encoding a tumor-specific CAR. The transfected gene is introduced into the host cell genome using a vector carrying the transfected gene, as described herein. The gene-transfected host cells are then delivered to patients in need.

[0093] Dual-CARs can be constructed using single-chain variable fragments (scFv) and / or single-domain variable fragments (sdFv, VHH, or nanoantibodies) corresponding to CD5 and CD33 antigens in the same or different constructs.

[0094] The method includes introducing a vector into a host cell comprising nucleic acid encoding a message peptide, including extracellular domains, hinge domains, transmembrane domains, and co-stimulatory domains encoding CD5-binding VL and VH domains and CD33-binding VL and VH domains. In some embodiments, the introduction of the nucleic acid into lymphocytes includes electroporation, transduction, or transduction. In some embodiments, the introduced nucleic acid is a viral vector, a non-viral vector, or naked DNA. The viral vector is a lentiviral vector or an adeno-associated viral vector. In some embodiments, the nucleic acid is integrated into the lymphocyte genome. The nucleic acid is randomly integrated into the lymphocyte genome. In some embodiments, the method is performed under conditions that allow CAR to be expressed in gene-transfected lymphocytes. In some embodiments, cells express CAR after the introduction of the nucleic acid.

[0095] In one specific embodiment, the nucleic acid encodes a CD5-binding CAR and a CD33-binding CAR, wherein the CD5-binding CAR and the CD33-binding CAR are linked by a self-cleaving protein. The introduced nucleic acid encodes a CD5-binding CAR, which includes a signaling peptide, an extracellular domain including CD5-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. The introduced nucleic acid also encodes a CD33-binding CAR, which includes a signaling peptide, an extracellular domain including CD33-binding VL and VH domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain.

[0096] In some specific embodiments, the introduction of nucleic acids into lymphocytes includes electroporation, transduction, or transduction. In some specific embodiments, the nucleic acids are introduced into lymphocytes using viral vectors, non-viral vectors, or as naked DNA. Viral vectors are lentiviral vectors or adeno-associated viral vectors. In some specific embodiments, the nucleic acids are integrated into the lymphocyte genome. The nucleic acids are randomly integrated into the lymphocyte genome. In some specific embodiments, the method is performed under conditions that allow CAR expression in gene-transfected lymphocytes. In some specific embodiments, cells express CAR after the introduction of nucleic acids. αβT cell receptor knockout

[0097] "αβ T-cell receptor (TCR) knockout" refers to a method that can be used to prevent host-versus-graft disease (HVGD) in patients awaiting CAR T-cell therapy. Endogenous αβ TCRs on infused allogeneic T cells can recognize major and minor histocompatibility antigens in the recipient, leading to HVGD. To avoid HVGD during allogeneic CAR-T therapy using αβ T cells, αβ TCRs can be knocked out using CRISPR / Cas9 technology or other gene-editing techniques known to those skilled in the art. CD5 expression in UCAR-T cells can be deleted (e.g., knocked out) to avoid self-destruction of CD5 CARs. CRISPR / Cas9 knockout of endogenous αβ TCRs can increase the expression and functional activity of transduced CAR T cells. Methods of treating diseases

[0098] The dual-CAR T cells described in this article can be used in methods for treating individual diseases. Dual-CAR T cells are delivered to individuals in need (e.g., patients).

[0099] Diseases that can be treated by administering the dual-CAR T cells described herein include, but are not limited to, acute myeloid leukemia (AML) and T-cell malignancies. Generally, patients in need have cells expressing both CD33 and CD5 antigens. Dual-CAR T cells bind to cells expressing both CD33 and CD5 antigens. For patients in need, the CD5 CAR removes the patient's T cells, preventing rejection by the CAR-T cells, while the CD33 CAR kills the patient's CD33-expressing diseased cells. The CD33 CAR also kills CD33-expressing hematopoietic stem cells and other bone marrow cells, creating space for CAR-T cell expansion. For treating T-cell malignancies, dual-CARs kill CD5 and / or CD33-expressing T-cell tumors, followed by allogeneic hematopoietic stem cell transplantation.

[0100] In one specific embodiment, a method of treating AML includes administering an effective amount of gene-transgenic lymphocytes to a patient in need. In some specific embodiments, the gene-transgenic lymphocytes are allogeneic. In some embodiments, the lymphocytes are autologous. In some specific embodiments, AML includes leukemia cells that express CD33 as a cell surface protein.

[0101] In one specific embodiment, a method of treating T-cell malignancies includes administering a therapeutically effective amount of gene-transfected lymphocytes to a patient in need. In some specific embodiments, the gene-transfected lymphocytes are allogeneic. In some specific embodiments, the gene-transfected lymphocytes are autologous. In some specific embodiments, the T-cell malignancies include T cells that express CD5 as a cell surface protein.

[0102] In some specific embodiments, the dual-CAR T cell line is administered at approximately 1 million IU / m² or less (e.g., approximately 800,000 IU / m²; 600,000 IU / m²; 400,000 IU / m²; 200,000 IU / m²; 100,000 IU / m²; 80,000 IU / m²; 60,000 IU / m²; 40,000 IU / m²; 20,000 IU / m²; 10,000 IU / m²; 8,000 IU / m²; 6,000 IU / m²; 4,000 IU / m²; 2,000 IU / m²; 1,000 IU / m²; 800 IU / m²; 600 IU / m²; 400,000 IU / m²; 400,000 IU / m²; 8,000 IU / m²; 6,000 IU / m²; 400,000 IU / m²; 2,000 IU / m²; 1,000 IU / m²; 800 IU / m²; 600 IU / m²; 400,0 ... Administered at doses of 100 IU / m², 200 IU / m², or 100 IU / m². In some specific embodiments, the bi-CAR T cell line was administered at doses greater than about 1 million IU / m² (e.g., from about 1 million IU / m² to about 5 million IU / m²).

[0103] Methods for introducing dual-CAR T cells include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, local, oral, and intranasal administration. Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable mediators and solvents, water-based solutions, Ringer's solution, USP, and isotonic sodium chloride solutions can be used. Furthermore, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids (such as oleic acid) are used in the preparation of injectable formulations.

[0104] As used herein, the term "subject" means an animal, preferably a mammal, such as a human being, or a domestic or agricultural animal such as a dog, cat, horse, cow, pig, sheep, goat, and the like.

[0105] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" for the components described herein refer to an amount sufficient to produce beneficial and desired results (including effects at the cellular, tissue, or clinical level) when administered to individuals, including mammals (e.g., humans), and therefore, "effective amount" or its synonyms depend on the context in which they are applied. The amount of a given component described herein that corresponds to this amount will vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or condition, individual characteristics (e.g., age, sex, weight) or host to be treated, and the like, but can still be conventionally determined by someone of ordinary skill in the art.

[0106] As used in this article, "treatment / Treating" "Treatment" refers to medical management aimed at improving, stabilizing (i.e., preventing deterioration), preventing, or curing a disease, pathology, or symptom in an individual. This term includes active treatment (treatment aimed at improving a disease, pathology, or symptom), causal treatment (treatment targeting the cause of a related disease, pathology, or symptom), palliative treatment (treatment aimed at relieving symptoms), preventative treatment (treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathology, or symptom), and supportive treatment (treatment used to complement another therapy). Treatment also includes reducing the severity of a disease or symptom; preventing the spread of a disease or symptom; delaying or slowing the progression of a disease or symptom; improving or alleviating a disease or symptom; and remission (whether partial or complete), whether detectable or undetectable. "Improvement" or "palliating" of a disease or condition means a reduction in the severity and / or undesirable clinical signs and / or a slowing or prolonged progression of the disease, symptom, or condition compared to the extent or time course without treatment. "Treatment" can also mean extended survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from a condition or symptom, those at risk of developing a condition or symptom, or those requiring prevention of a condition or symptom. Values ​​and ranges

[0107] Unless otherwise indicated or apparent from the context and understanding of one of ordinary skill in the art, in various embodiments, values ​​expressed as ranges may be assumed to be any particular value or subrange within the stated range, unless the context explicitly specifies otherwise. The term "about" with respect to numerical values ​​generally refers to a value falling within ±8%, ±6% in some embodiments, ±4% in some embodiments, ±2% in some embodiments, ±1% in some embodiments, and ±0.5% in some embodiments, unless otherwise stated or apparent from the context. example

[0108] The following examples refer to the structures in Tables 1 and 2. [] []

[0109] The sequences of SEQ ID NO:2 to 23 include a message peptide domain, an extracellular domain comprising variable light chain (VL) and variable heavy chain (VH) domains, a hinge domain, a transmembrane domain, and a co-stimulatory domain. [pan] [-T] [Cell isolation, activation, and expansion]

[0110] Leukopak, sourced from healthy peripheral blood, was purchased from HemaCare. Pan-T cell lines were isolated using the EasySep Human T Cell Isolation Kit from Stemcell Technologies and frozen in CryoStor CS5 in a liquid N2 freezer. For use, cells were thawed, counted, and resuspended in X-VIVO 15 (Lonza) containing 5% FBS (Gibco) and 25 ng / ml IL2 (Peprotech). To activate pan-T cells, Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher) were used. Cells were cultured in a 37°C, 5% CO2 incubator. After 2–3 days, the Dynabeads were removed, and the activated T cells were further cultured in X-VIVO 15 + 5% FBS + 25 ng / ml IL2 for expansion. [Cell lines and cultures]

[0111] Cell line information is as follows: 293T cells (ATCC, #CRL3216); human hepatocellular carcinoma cell line SK-Hep-1 (ATCC, #HTB-52); acute T-lymphoblastic leukemia (T-ALL) Jurkat (ATCC, #TIB-152), and CCRF-CEM (ATCC, #CCL-119); acute myeloid leukemia (AML) MOLM13 (AddexBio, #C0003003); and MV-4-11 (ATCC, #CRL-9591). Subclones of SK-Hep-1 were generated by stable transduction using lentiviral vectors encoding CD5 or CD33. SK-Hep1-CD5 and SK-Hep1-CD33 cell lines were grown in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Secondary selection lines for CCRF-CEM, MOLM13, and MV-4-11 luciferase expression were generated by stable transduction of cells using a lentiviral vector (Biosettia, GlowCell-16) encoding firefly luciferase with GFP. CCRF-CEM-luciferase (luc), MOLM13-luc, and MV-4-11-luc cell lines were cultured in EMEM medium supplemented with 10% heat-inactivated FBS. All cells were transferred to RPMI 1640 medium with 10% FBS for growth during the cell cytolytic assay (toxicity assay). All cells were maintained in a humidified incubator at 37°C (with 5% CO2). [use] [CRISPR / Cas9 RNP] [Remove] [TRAC] [and] [CD5] [。]

[0112] αβ TCRs were knocked out using CRISPR / Cas9 technology. CD5 expression in UCAR-T cells was deleted to avoid cannibalism of CD5 CARs. Three gRNAs were designed for the human TRAC gene and five gRNAs for the human CD5 gene using online tools such as IDT and Synthego (high editing efficiency and low off-target editing possibility). The gRNAs and HiFi Cas9 were ordered from IDT. Cas9 was generated and gRNA ribonucleoproteins (RNPs) were formed by incubating gRNAs and Cas9 at a 2.5:1 molar ratio for 15 minutes at RT. To screen for effective gRNAs, pan-T cells were activated for 2 days using Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher) and RNPs were electroporated using Lonza 4D Nucleofector (Lonza Bioscience) program EO-115 in the presence of an electroporation enhancer (IDT). After CD3 and / or CD5 staining, knockout efficiency was assessed using an Attune NxT flow cytometer (Thermo Fisher). [Performance] [CAR] [Generation and transduction of recombinant lentiviral vectors] [] []

[0113] 293 T cells were seeded one day prior to transfection. When cell confluence reached 75%, the viral packaging vector and transfer vector were co-transfected using TransIT-VirusGEN® transfection reagent (Mirus) according to the recommended procedure. Viral supernatant was collected 48 hours after transfection and the virus was further used for transduction. Pan-T cells from a second healthy donor were used to generate chimeric antigen receptor (CAR) T cells. The pan-T cells were activated for two days with Dynabeads® Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) and supplemented with 25 ng / ml IL-2 two days before transduction. Magnetic beads were removed from the T cells, and the cells were electroporated using RNP and infected with lentiviral vectors encoding CAR constructs, including CD5 CAR (CAR5), CD33 CAR (CAR33), and CD19 CAR (CAR19). T cells were expanded in X-VIVO medium supplemented with 25 ng / ml IL-2, followed by CAR performance and function assays. Flow cytometry [] []

[0114] In the dark at RT, CD3 and CD5 were stained with CD3-FITC antibody (BD Biosciences, #555339) and CD5-APC antibody (BD Biosciences, #555355) for 30 minutes. CAR5 surface expression was determined by staining with Human CD5 Protein, HisTag (AcroBiosystems, Cat# CD5-H52H5) at RT for 1 hour, washing, and then incubating with His-APC antibody (R&D Systems, IC050A) in the dark at RT for 30 minutes. CAR33 expression was determined by staining with CD33 human protein-PE (Sino Biological, 12238-HCCH_P) at RT for 1 hour. All staining lines were performed at a 1:10 dilution. Dead cells were stained with DAPI (Thermo Scientific, Cat# BV-421, 1:5000) before running on an Attune NxT flow cytometer (Thermo Fisher Scientific). The data was further analyzed using Attune NxT flow software or FlowJo software. [In vitro cell cytotoxicity assay] [(] [Poisoning Test] [)] []

[0115] Different methods were used to assess the cytotoxic function of CAR T cells. For the CFSE-flow assay, pan-T cells, CCRF-CEM, MOLM13, and MV-4-11 cells were hydrated with carboxyfluorescein dibutylimide (CFSE-flow) before co-culturing with CAR T cells. Carboxyfluorescein succinimidyl ester (CFSE) (Biolegend, #423801) was used to label the cells. After a certain culture time (n=3), the number of viable target cells was analyzed by flow cytometry. For luciferase-based assays, CCRF-CEM-luc, MOLM13-luc, and MV-4-11-luc cells were co-cultured with CAR T cells at different E:T ratios for a certain period, and luminescence was read using a disk reader as an indicator of luciferase activity (n=3). For RTCA assays, SK-Hep-1-CD5 and SK-Hep-1-CD33 cells were seeded with different effector-to-target (E:T) ratios one day before the addition of CAR-T cells. Cell cytotoxicity was measured using the xCELLigence Real-Time Cell Analyzer (RTCA) (n=3). In all assays, the percentage of lysis was calculated using the average of the target-only wells as the no-lysis activity, and was calculated using the formula %lysis = 100 × (average of target-only values ​​- experimental group values) / (average of target-only values). [] [Cell line characteristics and generation] [] []

[0116] The expression of CD3, CD5, and CD33 in Jurkat, CCRF-CEM, MOLM13, and MV-4-11 cells was measured. As shown in Figure 1A, T-ALL cell lines (Jurkat and CCRF-CEM) showed 100% CD5 expression, while AML (MOLM13 and MV-4-11) showed CD33 expression. SK-Hep1-CD5 and SK-Hep1-CD33 cell lines were also generated by ectopic expression of CD5 and CD33 in SK-Hep-1 cells (Figure 1B). [TRAC] [Remove] [gRNA] [filter] [] []

[0117] To eliminate the T-cell receptor (TCR), three gRNAs targeting the TRAC locus were screened (Figure 2). Since the TCR and CD3 form a complex on the T-cell surface, CD3 expression is used to indicate TCR expression. Based on reduced CD3 expression, TRAC-gRNA1 showed the best knockout efficiency (>97%). In the presence of an electroporation enhancer, TRAC-gRNA3 achieved a ~95% knockout efficiency. TRAC-gRNA1 was selected for future experiments. [CD5] [Remove] [gRNA] [filter] [] []

[0118] To remove CD5, five gRNAs were screened (Figures 3A and 3B). In the presence of an electroporation enhancer, CD5-gRNAs 1, 4, and 5 showed knockout efficiencies exceeding 80% based on reduced CD5 expression, with CD5-gRNA 5 achieving a knockout efficiency of >90%. CD5-gRNA 5 was selected for future experiments. [] [CD5 CAR (CAR5)] [filter:] [CD5 CAR] [Expression and self] [T] [Cellular Poisoning] [] []

[0119] To screen for the optimal CAR5, six CAR5 constructs were screened (Figures 4A and 4B). T cells were activated for 2 days, and then electroporated with TRAC-RNP and CD5-RNP after Dynabead removal. Nuclearly stained cells were then infected with CAR virus particles. CAR5 expression exceeded 80% in CAR5-1, CAR5-2, CAR5-5, and CAR5-6, while CAR5 expression reached 70% in CAR5-3 and CAR5-4.

[0120] Almost all pan-T cells exhibited both CD3 and CD5. The knockout efficiency of TRAC and CD5 is shown in Figure 4B for CAR19, where the percentage of CD3-negative cells was ~98% and the percentage of CD5-negative cells was ~92%. After CD5 gene editing, ~7% of CD5-positive cells remained. In CAR5-1, CAR5-2, CAR5-5, and CAR5-6, CD5-positive cells disappeared, indicating that CAR5 had strong fratricide activity against CD5-positive T cells in those cases. In CAR5-3 and CAR5-4, CD5-positive cells were significantly reduced compared to CAR19. The data indicate that CAR5 in these constructs has cytotoxic activity against autologous T cells. [CD5 CAR (CAR5)] [filter:] [CAR5] [Regarding allogeneic species] [T] [Cells and] [T-ALL] [Cellular toxicity] [] []

[0121] To investigate whether CAR5 also possesses cytotoxic activity against allogeneic T cells, CAR5 T cells were co-cultured with CFSE-labeled allogeneic pan-T cells (from different donors) at different E:T ratios. After 20 hours of culture, the cells were stained with DAPI, and the number of viable allogeneic pan-T cells was counted by flow cytometry. As shown in Figure 5A, several CAR5 constructs exhibited strong cytotoxicity against allogeneic T cells. Next, CAR5 function was tested on the T-ALL cell line (CCRF-CEM), and several constructs showed significant cytotoxic activity against T-ALL cells (Figure 5B). In summary, several CAR5 constructs exhibited strong cytotoxic activity against CD5-expressing cells (including autologous and allogeneic T cells and T-cell malignant tumor cell lines). CAR5-1 and CAR5-5 were selected for the design of CD5 / 33 bi-CAR. [CD33 CAR (CAR33)] [filter] [] []

[0122] Six CAR33 constructs were designed, and cells were infected with CAR33 lentiviruses. The performance of CAR33 on T cells is shown in Figure 6A. In CAR33-1 and CAR33-2, CAR33 exhibited a single population, while in CAR33-3 through CAR33-6, CAR33 exhibited a smear-like population. To test CAR33 function, the AML cell line MOLM13 was used as the target cell line for assay using CFSE-based flow cytometry (Figure 6B). Compared to CAR19 and non-transduced T cells (NC), all six CAR33 constructs showed higher cytotoxic activity against MOLM13 cells. Due to their superior performance, CAR33-1 and CAR33-2 were selected for CD5 / 33 dual-CAR design and screening. [CD5 / 33] [pair] [-CAR] [design] [] []

[0123] CD5 / 33 dual-CARs can be expressed in T cells in various ways. In one specific embodiment, CAR5 and CAR33 lines are expressed on different vectors and transduced into lymphocytes. In another specific embodiment, CAR5 and CAR33 lines are expressed in the same construct with the two scFv lines linked in tandem (Figs. 7E-7F). In yet another specific embodiment, CAR5 and CAR33 lines are expressed in the same construct with the two scFv lines linked in a loop (Figs. 7A-7D). In yet another specific embodiment, CAR5 and CAR33 lines are expressed in the same construct with the two CAR lines linked via a 2A peptide or IRES. Here, the dual-CAR construct is designed with a loop structure (Figs. 7A-7D) and a tandem structure with 2A linkage (Figs. 7E-7F).

[0124] Figure 7A illustrates structures S036 and S040. Figure 7B illustrates structures S037 and S041. Figure 7C illustrates structures S038 and S042. Figure 7D illustrates structures S039 and S043. Figure 7E illustrates structure S048. Figure 7F illustrates structure S049. [CD5 / 33] [pair] [-CAR] [Performance] [] []

[0125] To verify CAR expression in pan-T cells after transduction with lentiviral vector constructs encoding single CAR5 (S005, S009), single CAR33 (S011, S012), or dual CAR5-CAR33 (S036, S037, S038, S039, S040, S041, S042, S043, S048, S049) corresponding to the scFv, CAR expression on T cells was measured by staining with CD5 (CAR5) and CD33 (CAR33) antigens 11 days post-transduction. CD19 CAR (S002) was used as a negative control. In both single and dual CAR constructs, almost all CAR-T samples showed over 70% CAR expression (CAR33) (Figure 8). [Extraterrestrial manifestations] [CD5] [or] [CD33] [on the cell line] [CD5 / 33] [pair] [-CAR] [Function] [] []

[0126] To detect the cytotoxic activity of the designed CAR constructs in vitro, SK-Hep-1 cells ectopically expressing CD5 and CD33 were used as the target cell line, and cell number was measured using real-time cell impedance monitoring (RTCA). The impedance-related cell index (CI) parameter, representing the number of cells up to 42 hours after co-culturing CAR-T cells with cancer cells, was measured. When co-cultured with SK-Hep-1-CD5 cells, the dual-CAR group and the single-CAR5 group showed significant cytotoxic effects compared to the target sample only (SK-Hep1-CD5 cells not co-cultured with CAR-T cells), the control CAR (CAR19), and the single-CAR33 group (Figure 9A). On the other hand, when co-cultured with SK-Hep-1-CD33 cells, the dual-CAR construct and the single-CAR33 group showed significant cytotoxic effects, but not in the CAR19 or single-CAR5 groups (Figure 9B). [CD5 / 33] [pair] [-CAR] [right] [CD33] [Positive] [AML] [and] [T-ALL] [In vitro cytotoxicity of cell lines] [] []

[0127] To evaluate the ability of CD5 / CD33 dual-CAR T cells to eliminate tumor cells in vitro, luciferase-based cytotoxicity assays were performed on two acute myeloid leukemia (AML) cell lines—MOLM13 (CD33+) and MV-4-11 (CD33+)—and the T-lymphocytic leukemia (ALL) cell line CCRF-CEM (CD5+) (Figs. 10A to 10C). After culturing CAR33 and CD5 / CD33 dual-CAR T cells with MOLM13 (Fig. 10A) and MV-4-11 cell lines (Fig. 10B) at two E:T ratios for 18 hours, 60–100% elimination was achieved. At a 1:1 E:T ratio, some dual-CAR constructs showed higher cytotoxicity compared to their single-CAR controls. After culturing CAR5 and CD5 / CD33 dual-CAR T cells with CCRF-CEM cells for 18 hours, almost 100% of cancer cells were eliminated (Fig. 10C). Data shows that the dual-CAR construct has a strong cytotoxic effect on AML and T-ALL and can completely eliminate these tumor cells. [CD5 / 33] [pair] [-CAR] [Can eliminate autologous and allogeneic substances] [T] [Cellular] [] []

[0128] For allogeneic cell therapy, the patient's T cell line was depleted using CAR5 in a dual-CAR construct. First, the ability of the dual-CAR construct to eliminate CD5-expressing autologous T cells was tested. Autologous pan-T cells were labeled with CFSE and co-cultured with CAR-T cells at two E:T ratios (2:1 and 1:1) for up to 42 hours. Some dual-CAR constructs showed 70–90% cytotoxic activity at 42 hours, similar to the activity of single-CAR5 constructs (Figure 12A). The dual-CAR constructs also showed strong cytotoxic activity against allogeneic T cells, with some eliminating approximately 70% of the allogeneic pan-T cells within two days (Figure 12B). [] [CD5 (CAR5)] [This can prevent rejection.] [] []

[0129] To determine whether CAR5 T cells could prevent T cell-based allogeneic HVGD rejection, CAR5-1 cells were transduced using an HLA-A2+ donor and co-cultured with HLA-A2-PBMCs alone, PBMCs depleted of NK cells by CD56 beads, or PBMCs depleted of T cells by CD3 beads for 0, 3, 5, or 7 days (Figs. 13A to 13C). Cells were further counted by flow cytometry and cell number was normalized using count beads. Figs. 13D to 13G show the fold change in cell number for the experiments in Figs. 13A to 13C, representing the number per day relative to day 0. Based on the results, CAR5 could completely deplete T cells (based on the number of CD3-positive cells), while more cells were maintained at day 3 (Figs. 13D to 13G). [Cytoin release and cell proliferation assay] [] []

[0130] CD5 / CD33 dual-CAR T cells were co-cultured with AML cell line (molm13), CCRF T cells, or allogeneic T cells. The supernatant was collected 24 hours after co-culture (Figs. 12E to 12F). Intercytokine release (INF-γ) was detected using an ELISA kit (Biolegend CAT# 430804). T cell counts were calculated 5 days after co-culture. [CD5 / 33] [pair] [-CAR] [Can slow tumor progression in the body] [] []

[0131] To determine the functional activity of the dual-CAR construct in vivo, five days prior to intravenous injection (iv) of CD5 / CD33 dual-CAR+ T cells (HLA-A2 positive) in immunodeficient NOD / SCID / IL-2Rγc null (NSG) mice, MOLM13-luc (luciferase) cell line and allogeneic T cells (HLA-A2 negative) were injected. Tumor growth was monitored weekly by bioluminescence imaging (Figs. 14A-14B). Survival curves of the animals were analyzed using the Kaplan-Meier method (Fig. 14C).

[0132] Blood samples from mice were collected for flow cytometry analysis to assess CAR-T cell persistence and expansion. Blood samples were stained with anti-mouse CD45, anti-human CD45, anti-human HLA-A2, anti-human CD3, and CD33 antigens for 30 min under RT, followed by elimination of red blood cells with blood lysis buffer. Samples were analyzed on a flow cytometer, and data were analyzed using FlowJo software v10.6.2 (Figures 15A to 15C).

[0133] Although exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of these embodiments as covered by the appended claims.

[0134] <![CDATA[ <110> Simcere Innovation, Inc. (USA) <![CDATA[ <120> Immune cells targeting universal chimeric antigen receptor expression in allogeneic cell therapy <![CDATA[ <130> 5801.1002002]]> <![CDATA[ <140> TW 110143992 <![CDATA[ <141> 2021-11-25 <![CDATA[ <150> US 63 / 119,227 <![CDATA[ <151> 2020 / 11 / 30 <![CDATA[ <160> 44]]> <![CDATA[ <170> FastSEQ Windows 4.0 version]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 491]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S002, CAR19]]> <![CDATA[ <400> 1]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 130 135 140 Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro 145 150 155 160 Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro 165 170 175 Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu 180 185 190 Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala 195 200 205 Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val 210 215 220 Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr 225 230 235 240 Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <![CDATA[<210> 2]]> <![CDATA[<211> 486]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[ <223> S005, CAR5-1]]> <![CDATA[ <400> 2]]> 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 Lys Met Thr Gln Ser Pro Ser Ser Met 20 25 30 Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln 35 40 45 Asp Ile Asn Ser Tyr Leu Ser Trp Phe His His Lys Pro Gly Lys Ser 50 55 60 Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Leu Asp Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr 100 105 110 Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn 130 135 140 Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 145 150 155 160 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 165 170 175 Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met Gly 180 185 190 Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 195 200 205 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu 210 215 220 Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Thr 225 230 235 240 Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 245 250 255 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[ <210> 3]]> <![CDATA[ <211> 486]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S006, CAR5-2]]> <![CDATA[ <400> 3]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asn Ile Gln Leu Val Gln Ser Gly Pro Glu Leu 20 25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys 50 55 60 Gly Leu Arg Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr 65 70 75 80 Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser 85 90 95 Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val 115 120 125 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Lys Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr 165 170 175 Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser Trp Phe His His 180 185 190 Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu 195 200 205 Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp 210 215 220 Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr Glu Asp Met Gly Ile Tyr 225 230 235 240 Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu Glu Met Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[<210> 4]]> <![CDATA[<211> 486]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S007, CAR5-3]]> <![CDATA[ <400> 4]]> 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 Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[ <210> 5]]> <![CDATA[ <211> 486]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S008, CAR5-4]]> <![CDATA[ <400> 5]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr 35 40 45 Ser Ile Thr Ser Gly Tyr Tyr Trp His Trp Ile Arg Gln Phe Pro Gly 50 55 60 Asn Lys Leu Gln Trp Met Gly Tyr Ile Ser Tyr Ser Gly Phe Thr Asn 65 70 75 80 Tyr Lys Thr Ser Leu Ile Asn Arg Ile Ser Ile Thr His Asp Thr Ser 85 90 95 Glu Asn Gln Phe Phe Leu Asn Leu Asn Ser Val Thr Thr Glu Asp Thr 100 105 110 Ala Thr Tyr Tyr Cys Ala Gly Asp Arg Thr Gly Ser Trp Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Val Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg Ile Ser Leu Thr 165 170 175 Cys Arg Thr Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Phe Gln Gln 180 185 190 Lys Pro Asp Gly Thr Phe Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu 195 200 205 Asp Ser Gly Val Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp 210 215 220 Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr 225 230 235 240 Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr 245 250 255 Lys Leu Glu Ile Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[<210> 6]]> <![CDATA[<211> 486]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S009, CAR5-5]]> <![CDATA[ <400> 6]]> 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 Arg Ala Ser Gln 35 40 45 Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Tyr Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr 100 105 110 Asp Glu Ser Pro Trp Thr Phe Gly Gly 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 Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 145 150 155 160 Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly 180 185 190 Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys 195 200 205 Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu 210 215 220 Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr 225 230 235 240 Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 245 250 255 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[ <210> 7]]> <![CDATA[ <211> 486]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S010, CAR5-6]]> <![CDATA[ <400> 7]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Lys Pro Gly Gly Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr 65 70 75 80 Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser 85 90 95 Lys Asn Thr Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu 195 200 205 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr Glu Asp Phe Gly Ile Tyr 225 230 235 240 Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu Glu Ile Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <![CDATA[<210> 8]]> <![CDATA[<211> 488]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S011, CAR33-1]]> <![CDATA[ <400> 8]]> 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 Arg Ala Ser Glu 35 40 45 Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Lys Glu Val 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 145 150 155 160 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 165 170 175 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 180 185 190 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 195 200 205 Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 210 215 220 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 225 230 235 240 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Ala 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 Lys Rec 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <![CDATA[ <210> 9]]> <![CDATA[ <211> 488]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S012, CAR33-2]]> <![CDATA[ <400> 9]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 65 70 75 80 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 85 90 95 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 165 170 175 Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe 180 185 190 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 195 200 205 Asn Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 210 215 220 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 225 230 235 240 Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln 245 250 255 Gly Thr Lys Val Glu Ile Lys Ala 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 Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <![CDATA[ <210> 10]]> <![CDATA[ <211> 492]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S013, CAR33-3]]> <![CDATA[ <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 Glu Ile Val Leu Thr Gln Ser Pro Gly Ser Leu 20 25 30 Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln 35 40 45 Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60 Gln Ile Pro Gly Gln Ser Pro Arg 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 Ile 100 105 110 Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr Phe Gly Gln 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 Val Gln Leu Gln Gln Pro Gly Ala Glu Val 145 150 155 160 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 180 185 190 Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 195 200 205 Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220 Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 225 230 235 240 Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 245 250 255 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <![CDATA[<210> 11]]> <![CDATA[<211> 492]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S014, CAR33-4]]> <![CDATA[<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 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 50 55 60 Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 65 70 75 80 Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln 145 150 155 160 Ser Pro Gly Ser Leu Ala Val Ser Pro Gly Glu Arg Val Thr Met Ser 165 170 175 Cys Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr 180 185 190 Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln Ser Pro Arg Leu Leu Ile 195 200 205 Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro 225 230 235 240 Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr 245 250 255 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <![CDATA[ <210> 12]]> <![CDATA[ <211> 492]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S015, CAR33-5]]> <![CDATA[ <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 Asn Ile Met Leu Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 35 40 45 Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60 Gln Ile 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 Ser Glu Asp Leu Ala Ile 100 105 110 Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg 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 Val Gln Leu Gln Gln Pro Gly Ala Glu Val 145 150 155 160 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 180 185 190 Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 195 200 205 Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 210 215 220 Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 225 230 235 240 Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 245 250 255 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​ <![CDATA[<223> S016, CAR33-6]]> <![CDATA[<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 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Ser Tyr Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln 50 55 60 Gly Leu Glu Trp Val Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser 65 70 75 80 Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val 115 120 125 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Met Leu Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser 165 170 175 Cys Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr 180 185 190 Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln Ser Pro Lys Leu Leu Ile 195 200 205 Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser 225 230 235 240 Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln Tyr Leu Ser Ser Arg Thr 245 250 255 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ser Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <![CDATA[<210> 14]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S036, Dual - CAR]]> <![CDATA[<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 Lys Met Thr Gln Ser Pro Ser Ser Met 20 25 30 Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln 35 40 45 Asp Ile Asn Ser Tyr Leu Ser Trp Phe His His Lys Pro Gly Lys Ser 50 55 60 Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Leu Asp Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr 100 105 110 Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 130 135 140 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 145 150 155 160 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 165 170 175 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 180 185 190 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 195 200 205 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly 245 250 255 Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr 260 265 270 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 275 280 285 Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met 290 295 300 Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 305 310 315 320 Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 325 330 335 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 340 345 350 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr 355 360 365 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Asn 370 375 380 Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 385 390 395 400 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 405 410 415 Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met Gly 420 425 430 Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 435 440 445 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu 450 455 460 Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Thr 465 470 475 480 Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 485 490 495 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 15]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S037, Dual-CAR]]> [[ID=1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val 115 120 125 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 145 150 155 160 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 165 170 175 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 180 185 190 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys 225 230 235 240 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly 245 250 255 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 260 265 270 Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 275 280 285 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 290 295 300 Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 305 310 315 320 Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys 325 330 335 Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala 340 345 350 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Met Thr Gln 385 390 395 400 Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg Val Thr Ile Thr 405 410 415 Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser Trp Phe His His 420 425 430 Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu 435 440 445 Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp 450 455 460 Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr Glu Asp Met Gly Ile Tyr 465 470 475 480 Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr 485 490 495 Lys Leu Glu Met Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 16]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S038, dual-CAR]]> <![CDATA[<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 Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu 35 40 45 Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Gly Gly Gly Gly Ser Asn Ile Gln Leu Val Gln Ser 130 135 140 Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln 165 170 175 Ala Pro Gly Lys Gly Leu Arg Trp Met Gly Trp Ile Asn Thr His Thr 180 185 190 Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser 195 200 205 Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys 210 215 220 Asn Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp 225 230 235 240 Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly 245 250 255 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 260 265 270 Gly Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu 275 280 285 Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser 290 295 300 Tyr Leu Ser Trp Phe His His Lys Pro Gly Lys Ser Pro Lys Thr Leu 305 310 315 320 Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser 325 330 335 Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp 340 345 350 Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro 355 360 365 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Gly Gly Gly Gly 370 375 380 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 385 390 395 400 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 405 410 415 Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 420 425 430 Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys 435 440 445 Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala 450 455 460 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 465 470 475 480 Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 485 490 495 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 17]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S039, bis-CAR]]> <![CDATA[<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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 65 70 75 80 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 85 90 95 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140 Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly Glu Arg 145 150 155 160 Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 165 170 175 Trp Phe His His Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Arg 180 185 190 Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr Glu Asp 210 215 220 Met Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Glu Met Lys Gly Ser Thr Ser Gly Ser Gly 245 250 255 Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asn Ile Gln Leu Val 260 265 270 Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser 275 280 285 Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val 290 295 300 Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met Gly Trp Ile Asn Thr 305 310 315 320 His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala 325 330 335 Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn 340 345 350 Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Arg Gly Tyr 355 360 365 Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser 370 375 380 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 385 390 395 400 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 405 410 415 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln 420 425 430 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln 435 440 445 Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 450 455 460 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr 465 470 475 480 Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr 485 490 495 Lys Val Glu Ile Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 18]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S040, dual-CAR]]> <![CDATA[<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 Arg Ala Ser Gln 35 40 45 Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Tyr Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr 100 105 110 Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 130 135 140 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 145 150 155 160 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 165 170 175 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 180 185 190 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 195 200 205 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly 245 250 255 Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Met Thr 260 265 270 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 275 280 285 Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met 290 295 300 Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 305 310 315 320 Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 325 330 335 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 340 345 350 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr 355 360 365 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu 370 375 380 Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser 385 390 395 400 Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 405 410 415 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly 420 425 430 Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys 435 440 445 Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu 450 455 460 Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr 465 470 475 480 Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 485 490 495 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 19]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S041, dual - CAR]]> <![CDATA[<400> 19]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu — 1 5 10 15 His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu 20 25 30 Val Lys Pro Gly Gly Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr — 35 40 45 Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr 65 70 75 80 Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser 85 90 95 Lys Asn Thr Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Ala Val Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 145 150 155 160 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 165 170 175 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 180 185 190 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys 225 230 235 240 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly 245 250 255 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 260 265 270 Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 275 280 285 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 290 295 300 Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 305 310 315 320 Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys 325 330 335 Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala 340 345 350 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 370 375 380 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 385 390 395 400 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 405 410 415 Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser Trp Phe Gln Gln 420 425 430 Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile Tyr Arg Ala Asn Arg Leu 435 440 445 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 450 455 460 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr Glu Asp Phe Gly Ile Tyr 465 470 475 480 Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe Gly Gly Gly Thr 485 490 495 Lys Leu Glu Ile Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 20]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S042, dual-CAR]]> <![CDATA[<400> 20]]> 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 Arg Ala Ser Glu 35 40 45 Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys 115 120 125 Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Ile Gln Leu Val Gln Ser 130 135 140 Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Val Arg Ile Ser Cys Ala 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gln 165 170 175 Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr His Thr 180 185 190 Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser 195 200 205 Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Ile Asn Ser Leu Arg 210 215 220 Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Arg Gly Tyr Asp Trp 225 230 235 240 Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 245 250 255 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 260 265 270 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 275 280 285 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser 290 295 300 Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu 305 310 315 320 Ile Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 325 330 335 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 340 345 350 Tyr Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro 355 360 365 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly 370 375 380 Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 385 390 395 400 Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 405 410 415 Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 420 425 430 Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys 435 440 445 Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala 450 455 460 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr 465 470 475 480 Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 485 490 495 Val Thr Val Ser Ser Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 21]]> <![CDATA[<211> 726]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S043, Dual-CAR]]> <![CDATA[<400> 21]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 <![CDATA[ ]] <![CDATA[ ]] Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 65 70 75 80 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 85 90 95 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 145 150 155 160 Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 165 170 175 Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile Tyr Arg 180 185 190 Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr Glu Asp 210 215 220 Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp Thr Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly 245 250 255 Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Ile Gln Leu Val 260 265 270 Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Val Arg Ile Ser 275 280 285 Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val 290 295 300 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr 305 310 315 320 His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 325 330 335 Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Ile Asn Ser 340 345 350 Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Arg Gly Tyr 355 360 365 Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 370 375 380 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 385 390 395 400 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 405 410 415 Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln 420 425 430 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln 435 440 445 Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 450 455 460 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr 465 470 475 480 Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr 485 490 495 Lys Val Glu Ile Lys Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 500 505 510 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 515 520 525 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 530 535 540 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 545 550 555 560 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 565 570 575 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 580 585 590 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 595 600 605 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 610 615 620 Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 625 630 635 640 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 645 650 655 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 660 665 670 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 675 680 685 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 690 695 700 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 705 710 715 720 Gln Ala Leu Pro Pro Arg 725 <![CDATA[<210> 22]]> <![CDATA[<211> 998]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S048, dual-CAR]]> [[ID=�0]] <![CDATA[<400> 22]]> 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 Arg Ala Ser Glu 35 40 45 Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Lys Glu Val 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 145 150 155 160 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 165 170 175 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 180 185 190 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 195 200 205 Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 210 215 220 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 225 230 235 240 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Ala 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 Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser 485 490 495 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala 500 505 510 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala 515 520 525 Ala Arg Pro Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala 530 535 540 Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile 545 550 555 560 Asn Ser Tyr Leu Ser Trp Phe His His Lys Pro Gly Lys Ser Pro Lys 565 570 575 Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg 580 585 590 Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser 595 600 605 Leu Asp Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu 610 615 620 Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Gly Gly 625 630 635 640 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Gln 645 650 655 Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys 660 665 670 Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 675 680 685 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met Gly Trp Ile 690 695 700 Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg 705 710 715 720 Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile 725 730 735 Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Thr Arg Arg 740 745 750 Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 755 760 765 Val Ser Ser Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 770 775 780 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 785 790 795 800 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 805 810 815 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 820 825 830 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 835 840 845 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 850 855 860 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 865 870 875 880 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 885 890 895 Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 900 905 910 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 915 920 925 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 930 935 940 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 945 950 955 960 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 965 970 975 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 980 985 990 Leu Pro Pro Arg Phe Glu 995 <![CDATA[<210> 23]]> <![CDATA[<211> 998]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> S049, dual-CAR]]> <![CDATA[<400> 23]]> 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 Arg Ala Ser Glu 35 40 45 Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys 50 55 60 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Ser Lys Glu Val 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 145 150 155 160 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 165 170 175 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 180 185 190 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 195 200 205 Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 210 215 220 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 225 230 235 240 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Ala 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 Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser 485 490 495 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala 500 505 510 Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala 515 520 525 Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 530 535 540 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile 545 550 555 560 Asn Ser Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys 565 570 575 Thr Leu Ile Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg 580 585 590 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser 595 600 605 Leu Gln Tyr Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu 610 615 620 Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly 625 630 635 640 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Gln 645 650 655 Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Val Arg 660 665 670 Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 675 680 685 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile 690 695 700 Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe Lys Gly Arg 705 710 715 720 Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Ile 725 730 735 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Thr Arg Arg 740 745 750 Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr 755 760 765 Val Ser Ser Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 770 775 780 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 785 790 795 800 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 805 810 815 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 820 825 830 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 835 840 845 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 850 855 860 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 865 870 875 880 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 885 890 895 Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 900 905 910 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 915 920 925 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 930 935 940 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 945 950 955 960 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 965 970 975 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 980 985 990 Leu Pro Pro Arg Phe Glu 995 <![CDATA[ <210> 24]]> <![CDATA[ <211> 758]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S026, CD5-GFP]]> <![CDATA[ <400> 24]]> Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Cys Leu Gly Arg Leu Ser Trp Tyr Asp Pro Asp 20 25 30 Phe Gln Ala Arg Leu Thr Arg Ser Asn Ser Lys Cys Gln Gly Gln Leu 35 40 45 Glu Val Tyr Leu Lys Asp Gly Trp His Met Val Cys Ser Gln Ser Trp 50 55 60 Gly Arg Ser Ser Lys Gln Trp Glu Asp Pro Ser Gln Ala Ser Lys Val 65 70 75 80 Cys Gln Arg Leu Asn Cys Gly Val Pro Leu Ser Leu Gly Pro Phe Leu 85 90 95 Val Thr Tyr Thr Pro Gln Ser Ser Ile Ile Cys Tyr Gly Gln Leu Gly 100 105 110 Ser Phe Ser Asn Cys Ser His Ser Arg Asn Asp Met Cys His Ser Leu 115 120 125 Gly Leu Thr Cys Leu Glu Pro Gln Lys Thr Thr Pro Pro Thr Thr Arg 130 135 140 Pro Pro Pro Thr Thr Thr Pro Glu Pro Thr Ala Pro Pro Arg Leu Gln 145 150 155 160 Leu Val Ala Gln Ser Gly Gly Gln His Cys Ala Gly Val Val Glu Phe 165 170 175 Tyr Ser Gly Ser Leu Gly Gly Thr Ile Ser Tyr Glu Ala Gln Asp Lys 180 185 190 Thr Gln Asp Leu Glu Asn Phe Leu Cys Asn Asn Leu Gln Cys Gly Ser 195 200 205 Phe Leu Lys His Leu Pro Glu Thr Glu Ala Gly Arg Ala Gln Asp Pro 210 215 220 Gly Glu Pro Arg Glu His Gln Pro Leu Pro Ile Gln Trp Lys Ile Gln 225 230 235 240 Asn Ser Ser Cys Thr Ser Leu Glu His Cys Phe Arg Lys Ile Lys Pro 245 250 255 Gln Lys Ser Gly Arg Val Leu Ala Leu Leu Cys Ser Gly Phe Gln Pro 260 265 270 Lys Val Gln Ser Arg Leu Val Gly Gly Ser Ser Ile Cys Glu Gly Thr 275 280 285 Val Glu Val Arg Gln Gly Ala Gln Trp Ala Ala Leu Cys Asp Ser Ser 290 295 300 Ser Ala Arg Ser Ser Leu Arg Trp Glu Glu Val Cys Arg Glu Gln Gln 305 310 315 320 Cys Gly Ser Val Asn Ser Tyr Arg Val Leu Asp Ala Gly Asp Pro Thr 325 330 335 Ser Arg Gly Leu Phe Cys Pro His Gln Lys Leu Ser Gln Cys His Glu 340 345 350 Leu Trp Glu Arg Asn Ser Tyr Cys Lys Lys Val Phe Val Thr Cys Gln 355 360 365 Asp Pro Asn Pro Ala Gly Leu Ala Ala Gly Thr Val Ala Ser Ile Ile 370 375 380 Leu Ala Leu Val Leu Leu Val Val Leu Leu Val Val Cys Gly Pro Leu 385 390 395 400 Ala Tyr Lys Lys Leu Val Lys Lys Phe Arg Gln Lys Lys Gln Arg Gln 405 410 415 Trp Ile Gly Pro Thr Gly Met Asn Gln Asn Met Ser Phe His Arg Asn 420 425 430 His Thr Ala Thr Val Arg Ser His Ala Glu Asn Pro Thr Ala Ser His 435 440 445 Val Asp Asn Glu Tyr Ser Gln Pro Pro Arg Asn Ser His Leu Ser Ala 450 455 460 Tyr Pro Ala Leu Glu Gly Ala Leu His Arg Ser Ser Met Gln Pro Asp 465 470 475 480 Asn Ser Ser Asp Ser Asp Tyr Asp Leu His Gly Ala Gln Arg Leu Phe 485 490 495 Glu Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 500 505 510 Val Glu Glu Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu Phe 515 520 525 Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly 530 535 540 His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly 545 550 555 560 Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro 565 570 575 Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser 580 585 590 Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met 595 600 605 Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly 610 615 620 Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val 625 630 635 640 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile 645 650 655 Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile 660 665 670 Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg 675 680 685 His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln 690 695 700 Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr 705 710 715 720 Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp 725 730 735 His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly 740 745 750 Met Asp Glu Leu Tyr Lys 755 <![CDATA[<210> 25]]> <![CDATA[ <211> 627]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> artificial sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> S027, CD33-GFP]]> <![CDATA[ <400> 25]]> Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 35 40 45 Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 50 55 60 Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 65 70 75 80 Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 85 90 95 Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 100 105 110 Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser 115 120 125 Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg 130 135 140 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn 145 150 155 160 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile 165 170 175 Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190 His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200 205 Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 210 215 220 Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 225 230 235 240 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly 245 250 255 Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala 260 265 270 Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys 275 280 285 Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly 290 295 300 Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr 305 310 315 320 Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu 325 330 335 Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys 340 345 350 Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln Phe Glu Gly Ser 355 360 365 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 370 375 380 Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 385 390 395 400 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 405 410 415 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 420 425 430 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 435 440 445 Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro 450 455 460 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 465 470 475 480 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 485 490 495 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 500 505 510 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 515 520 525 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 530 535 540 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 545 550 555 560 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 565 570 575 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 580 585 590 <00U5155>Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 595 6Q0 605 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 610 615 620 Leu Tyr Lys &25 <![CDATA[<210> 26]]> <![CDATA[<211> 118]]> ^ <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> It should be noted that there may be some errors in the original text such as "00U5155" which should probably be "0005155". This translation is based on the best understanding of the given content.<![CDATA[<223> CD5 ScFV V.sub.H]]> <![CDATA[<400> 26]]> Asn Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <![CDATA[<210> 27]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> CD5 ScFV V.sub.L]]> <![CDATA[<F00> 27]]> Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe His His Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Note: There seems to be an error in the original text where <400> is used instead of <220> in the CDATA section at line 35. I've corrected it in the translation. Also, <F00> at line 35 seems incorrect in the original, I've left it as <400> in the translation as it's not clear what it should be. If this is a known error in the original, you may want to correct it in the source text for a more accurate translation.Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 <![CDATA[<210> 28]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> CD5 ScFV V.sub.H]]> <![CDATA[<400> 28]]> Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Ser Phe 50 55 60 Lys Gly Arg Phe Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <![CDATA[<210> 29]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> CD5 ScFV V.sub.L]]> <![CDATA[<400> 29]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[<210> 30]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> CD33 ScFV V.sub.H]]> <![CDATA[<400> 30]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 31]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> CD33 ScFV V.sub.L]]> <![CDATA[<400> 31]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <![CDATA[ <210> 32]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> TRAC-gRNA1]]> <![CDATA[ <400> 32]]> ttcggaaccc aatcactgac 20 <![CDATA[ <210> 33]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> TRAC-gRNA2]]> <![CDATA[ <400> 33]]> aagttcctgt gatgtcaagc 20 <![CDATA[ <210> 34]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> TRAC-gRNA3]]> <![CDATA[ <400> 34]]> tcagggttct ggatatctgt 20 <![CDATA[ <210> 35]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD5-gRNA1]]> <![CDATA[ <400> 35]]> agcggttgca gagaccccat 20 <![CDATA[ <210> 36]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD5-gRNA2]]> <![CDATA[ <400> 36]]> tggccacctt gtacctgctg 20 <![CDATA[ <210> 37]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD5-gRNA3]]> <![CDATA[ <400> 37]]> ctggcacttc gagttggaac 20 <![CDATA[ <210> 38]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD5-gRNA4]]> <![CDATA[ <400> 38]]> gcttcaagaa ggagccacac 20 <![CDATA[ <210> 39]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD5-gRNA5]]> <![CDATA[ <400> 39]]> gagccttgcc tggaaatctg 20 <![CDATA[ <210> 40]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD8α signaling peptide]]> <![CDATA[ <400> 40]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <![CDATA[ <210> 41]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Granulocyte-macrophage community-stimulating factor (GM-CSF) Information Peptide <![CDATA[ <400> 41]]> Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser <![CDATA[ <210> 42]]> <![CDATA[ <211> 25]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD4 signaling peptide <![CDATA[ <400> 42]]> Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu 1 5 10 15 Ala Leu Leu Pro Ala Ala Thr Gln Gly 20 25 <![CDATA[ <210> 43]]> <![CDATA[ <211> 23]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> CD137 (4-1BB) Messenger Peptide <![CDATA[ <400> 43]]> Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser 20 <![CDATA[ <210> 44]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> 218 connectors]]> <![CDATA[ <400> 44]]> Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly

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Claims

1. A gene-transfected lymphocyte expressing a chimeric antigen receptor (CAR), the CAR comprising: a) a signaling peptide domain; b) an extracellular domain comprising variable light chain (VL) and variable heavy chain (VH) domains of binding group differentiation 5 ((CD5)), VL and VH domains of binding group differentiation 33 (CD33), and a connector domain between adjacent VL and VH domains; c) a hinge domain; d) a transmembrane domain; and e) a co-stimulatory domain; wherein the CAR comprises an amino acid sequence, wherein the amino acid sequence is any one of SEQ ID NO: 14 to 23.

2. The gene transfected into lymphocytes as requested in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

14.

3. The gene transfected into lymphocytes as requested in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

15.

4. The gene transfected into lymphocytes as requested in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

16.

5. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

17.

6. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

18.

7. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

19.

8. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

20.

9. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

21.

10. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

22.

11. The gene transfected into lymphocytes as claimed in claim 1, wherein the amino acid sequence of the CAR is SEQ ID NO:

23.

12. The gene-transfected lymphocytes of claim 1, wherein the gene-transfected lymphocytes are T cells.

13. The gene-transfected lymphocyte of claim 12, wherein the T cell expresses the T cell receptor α chain or the T cell receptor β chain at a level that does not elicit a graft-versus-host disease (GVHD) response when the gene-transfected lymphocyte is administered to a patient.

14. Gene-transfected lymphocytes as claimed in claim 1, wherein the gene-transfected lymphocytes are natural killer (NK) cells.

15. The gene-transfected lymphocytes of claim 1, wherein the gene-transfected lymphocytes express an exogenous nucleic acid encoding a cytokine or a cytokine receptor gene.

16. The gene transfected lymphocyte of claim 15, wherein the encoded interleukin or interleukin receptor gene is interleukin 2, interleukin 7, interleukin 12, interleukin 15, or interleukin 21.

17. The gene-transfected lymphocytes of claim 1, wherein the lymphocytes express exogenous nucleic acids encoding a suicide gene.

18. A method for manufacturing gene-transfected lymphocytes as claimed in claims 1 to 17, wherein the lymphocytes represent chimeric antigen receptors of group 5 (CD5) and group 33 (CD33), the method comprising introducing a nucleic acid into the lymphocyte encoding a chimeric antigen receptor (CAR) comprising: a) a signaling peptide domain; b) an extracellular domain comprising variable light chain (VL) and variable heavy chain (VH) domains of group 5 (CD5), VL and VH domains of group 33 (CD33), and a linker domain between adjacent VL and VH domains; c) a hinge domain; d) a transmembrane domain; and e) a co-stimulatory domain; wherein the CAR comprises an amino acid sequence, wherein the amino acid sequence is any one of SEQ ID NO: 14 to 23.

19. A nucleic acid encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: a) a message peptide domain; b) an extracellular domain comprising variable light chain (VL) and variable heavy chain (VH) domains of binding group differentiation 5 (CD5), VL and VH domains of binding group differentiation 33 (CD33), and a linker domain between adjacent VL and VH domains; c) a hinge domain; d) a transmembrane domain; and e) a co-stimulatory domain; wherein the CAR comprises an amino acid sequence, wherein the amino acid sequence is any one of SEQ ID NO: 14 to 23.

20. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

14.

21. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

15.

22. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

16.

23. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

17.

24. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

18.

25. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

19.

26. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

20.

27. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

21.

28. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

22.

29. The nucleic acid of claim 19, wherein the amino acid sequence of the CAR is SEQ ID NO:

23.

30. A nucleic acid encoding a chimeric antigen receptor (CAR) binding to group 5 (CD5) and a CAR binding to group 33 (CD33), wherein the CD5-binding CAR comprises: a) a signaling peptide domain; b) an extracellular domain comprising a variable light chain (VL) and a variable heavy chain (VH) domain binding to CD5 and a linker domain between adjacent VL and VH domains; c) a hinge domain; d) a transmembrane domain; and e) a costimulatory domain; wherein the CD5-binding CAR comprises an amino acid sequence of any one of SEQ ID NO: 2 to 7; and wherein the CD33-binding CAR comprises: f) a signaling peptide domain; g) an extracellular domain comprising a variable light chain (VL) and a variable heavy chain (VH) domain binding to CD33 and a linker domain between adjacent VL and VH domains; h) a hinge domain; i) a transmembrane domain; and j) a costimulatory domain; wherein the CD33-binding CAR comprises an amino acid sequence of any one of SEQ ID NO: 2 to 7; NO: The amino acid sequence of any one of 8 to 13.

31. Use of a gene-transfected lymphocyte of any one of claims 1 to 17 in the manufacture of a medicament for the treatment of acute myeloid leukemia (AML).

32. As requested in claim 31, wherein the transgenic lymphocytes are allogeneic or autologous.

33. As claimed in claim 31, wherein the AML comprises leukemia cells that express CD33 as a cell surface protein.

34. The use of a gene-transfected lymphocyte of any one of claims 1 to 17 in the manufacture of a medicament for the treatment of T-cell malignancies.

35. As requested in claim 34, wherein the transfected lymphocytes of the gene are allogeneic or autologous.

36. As claimed in claim 34, wherein the T-cell malignancy comprises T cells that express CD5 as a cell surface protein.

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