A chimeric antigen receptor and its application

Abstract

The present application provides a chimeric antigen receptor (CAR), wherein the CAR comprises a targeting portion, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the targeting portion specifically binds to the variable region Vα, Vβ and / or fragments thereof of a T cell receptor (TCR).

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a chimeric antigen receptor that specifically binds to the variable regions Vα, Vβ and / or fragments thereof of a T cell receptor (TCR) in a targeting portion. Background Art

[0002] T-cell tumors are common hematological malignancies. Compared with B-cell lymphoma or leukemia, T-cell lymphoma or leukemia has a poorer prognosis. Currently, chemotherapy is the main treatment for T-cell lymphoma or leukemia, and there is no specific targeted immunotherapy program.

[0003] Chimeric antigen receptor T cells (CAR-T cells) are artificially modified tumor-killing cells that combine the targeting recognition function of antibodies with the tumor-killing function of T cells, and are a major breakthrough in the field of tumor immunotherapy. However, CAR-T has the side effect of killing normal T cells, which poses a certain risk to the safety of tumor patients.

[0004] Most mature T cell tumors express TCRs or complexes, most of which are TCRs. The entire T cell population is very heterogeneous, with various Vα subtypes ranging from 0.85% to 8.3%. Therapies generally used to kill tumor cells (such as T cell tumor cells) will also affect normal T cells to a certain extent, thereby adversely affecting the patient's immune system, thereby hindering the treatment of tumors. Therefore, it is urgent to develop new CARs that have less impact on normal T cell populations. Summary of the invention

[0005] The present application provides a chimeric antigen receptor (CAR) and its application, wherein the targeting portion of the CAR specifically binds to the variable region Vα, Vβ and / or their fragments of the T cell receptor (TCR). The CAR provided in the present application has one or more of the following properties: 1) can specifically bind to the variable region Vα, Vβ and / or their fragments of the T cell receptor (TCR); 2) can selectively kill tumor cells derived from T cells; 3) has little effect on the normal T cell population; 4) can be used to treat T cell lymphoma.

[0006] On the one hand, the present application provides a chimeric antigen receptor (CAR), wherein the CAR comprises a targeting portion, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the targeting portion specifically binds to the variable region Vα, Vβ and / or fragments thereof of the T cell receptor (TCR).

[0007] In certain embodiments, the targeting moiety specifically binds to the variable region Vα or a fragment thereof of a TCR. In certain embodiments, the Vα or a fragment thereof comprises Vα24 or a fragment thereof. In certain embodiments, the Vα or a fragment thereof further comprises Jα or a fragment thereof. In certain embodiments, the Jα or a fragment thereof comprises Jα18 or a fragment thereof.

[0008] In certain embodiments, the targeting moiety specifically binds to the variable region Vβ or a fragment thereof of a TCR. In certain embodiments, the Vβ or a fragment thereof comprises Vβ8 or a fragment thereof. In certain embodiments, the fragment of the TCR variable region comprises CDR3 of the TCR variable region.

[0009] In certain embodiments, the variable region Vα, Vβ and / or fragments thereof of the TCR comprises the amino acid sequence shown in any one of SEQ ID NOs: 58-61.

[0010] In certain embodiments, the targeting moiety comprises an antibody or fragment thereof.

[0011] In certain embodiments, the antibody competes with a reference antibody for binding to the variable region Vα, Vβ and / or fragments thereof of the TCR, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:64 and SEQ ID NO:21; the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:65 and SEQ ID NO:22; the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:66 and SEQ ID NO:23; the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:67 and SEQ ID NO:29; the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:68 and SEQ ID NO: The HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 69 and SEQ ID NO: 31.

[0012] In certain embodiments, the light chain variable region of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70 and SEQ ID NO: 27; and the heavy chain variable region of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71 and SEQ ID NO: 35.

[0013] In certain embodiments, the reference antibody comprises Vα24Jα18 antibody.

[0014] In certain embodiments, the antibody comprises a heavy chain complementary determining region 1 (HCDR1), wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 67, and SEQ ID NO: 29. In certain embodiments, the antibody further comprises a heavy chain complementary determining region 2 (HCDR2), wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 68, and SEQ ID NO: 30. In certain embodiments, the antibody further comprises a heavy chain complementary determining region 3 (HCDR3), wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 69, and SEQ ID NO: 31.

[0015] In certain embodiments, the antibody comprises a light chain complementary determining region 1 (LCDR1), wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 64, and SEQ ID NO: 21. In certain embodiments, the antibody further comprises a light chain complementary determining region 2 (LCDR2), wherein the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 65, and SEQ ID NO: 22. In certain embodiments, the antibody further comprises a light chain complementary determining region 3 (LCDR3), wherein the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 66, and SEQ ID NO: 23.

[0016] In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71, and SEQ ID NO: 35. In certain embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70, and SEQ ID NO: 27.

[0017] In certain embodiments, the antibody is a single chain antibody.

[0018] In certain embodiments, the antibody comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:62 and SEQ ID NO:19.

[0019] In certain embodiments, the transmembrane domain comprises a polypeptide derived from a protein selected from the group consisting of CD8, CD28, CD137 and / or CD3. In certain embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:40.

[0020] In some embodiments, the costimulatory domain comprises a polypeptide derived from a protein selected from the group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 and / or CD226. In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 42.

[0021] In certain embodiments, the intracellular signaling domain comprises a polypeptide from CD3ζ. In certain embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:44.

[0022] In some embodiments, the CAR further comprises a hinge region, wherein the hinge region connects the antibody and the transmembrane domain. In some embodiments, the hinge region comprises a polypeptide selected from the following proteins: CD8a and / or IgG. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 38.

[0023] In certain embodiments, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO:46, SEQ ID NO:72 and SEQ ID NO:50.

[0024] On the other hand, the present application provides an isolated nucleic acid molecule encoding the CAR described in the present application.

[0025] On the other hand, the present application provides an isolated nucleic acid molecule encoding CAR, which comprises the nucleic acid sequence shown in SEQ ID NO: 47, SEQ ID NO: 73 or SEQ ID NO: 51.

[0026] On the other hand, the present application provides a vector comprising the nucleic acid molecule described in the present application. In certain embodiments, the vector described in the present application comprises an mRNA vector, a plasmid vector, a retroviral vector and / or a lentiviral vector.

[0027] On the other hand, the present application provides an immune effector cell, which comprises and / or can express CAR described in the present application, nucleic acid molecules described in the present application, or vectors described in the present application. In some embodiments, the immune effector cell is selected from T lymphocytes and natural killer (NK) cells. In some embodiments, the T lymphocytes and / or natural killer cells are natural sources, or are induced by stem cells.

[0028] On the other hand, the present application provides a method for preparing immune effector cells, which comprises introducing the vector described in the present application into the immune effector cells.

[0029] On the other hand, the present application provides a composition comprising the immune effector cells described in the present application.

[0030] On the other hand, the present application provides the use of the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, or the immune effector cell described in the present application for preparing a drug, wherein the drug is used to treat T cell lymphoma or leukemia.

[0031] In certain embodiments, the T-cell lymphoma or leukemia comprises one or more selected from the following groups: peripheral T-cell lymphoma, unspecified peripheral T-cell lymphoma, angioimmunoblastic lymphoma, extranodal NKT-cell lymphoma, anaplastic T-cell lymphoma, ALK-positive anaplastic cell lymphoma, ALK-negative anaplastic cell lymphoma, T-lymphoblastic lymphoma, cytotoxic T-cell lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), T-prolymphocytic leukemia (T-PLL), T-large granular lymphocyte (T-LGL) leukemia, hepatosplenic T-cell lymphoma (HSTL), Sezary syndrome (SS), subcutaneous tonsillar T-cell lymphoma, and unclassified T-cell lymphoma.

[0032] Those skilled in the art can easily perceive other aspects and advantages of the present disclosure from the detailed description below. In the detailed description below, only exemplary embodiments of the present disclosure are shown and described. As will be appreciated by those skilled in the art, the content of the present disclosure enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the descriptions in the drawings and specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0034] Figure 1 Shown is a schematic diagram of the CAR structure targeting TCR Vα or Vβ subtype described in the present application.

[0035] Figure 2 Shown are the titer determination results of the T120BBZ lentivirus described in this application.

[0036] Figure 3 Shown are the CAR expression analysis test results of the T120BBZ CAR-T described in this application.

[0037] Figure 4 The T120BBZ CAR-T cell line against Vα24Jα18 described in this application is shown. + The detection results of tumor cell killing.

[0038] Figure 5 The Vα24Jα18 + The test results show that tumor cells can specifically induce T120BBZ CAR-T to secrete TNF.

[0039] Figure 6A-6B The F231BBZ CAR-T described in this application can specifically kill Vβ8 + Tumor cells and release IFN.

[0040] Figures 7A-7C Shown is the in vivo anti-tumor activity analysis of T120BBZ CAR-T described in this application.

[0041] Figures 8A-8C Shown is the TCR expression analysis of T120BBZ CAR-T described in this application.

[0042] Fig. 9 Shown are the titer analysis results of the 1168BBZ lentivirus described in this application.

[0043] Fig.10 Shown are the CAR expression analysis test results of the 1168BBZ CAR-T described in this application.

[0044] Fig.11 The 1168BBZ CAR-T cell line against Vβ8 + The detection results of tumor cell killing. DETAILED DESCRIPTION

[0045] The following specific embodiments illustrate the implementation of the present invention, and those familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present application provides a CAR that can specifically bind to the variable regions Vα, Vβ and / or fragments of T cell receptors, which can selectively kill tumor cells derived from T cells and have little effect on normal T cell populations.

[0046] The present invention is further described below: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and related terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0047] In the present application, the "chimeric antigen receptor" (Chimeric Antigen Receptor, CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which may include a tumor-associated antigen (TAA) binding region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. In some embodiments, the intracellular domain may be derived from a different protein than the transmembrane domain.

[0048] In the present application, the "targeting moiety" generally refers to any oligopeptide or polypeptide in a chimeric antigen receptor that can specifically bind to a target protein or a fragment thereof. In the present application, the targeting moiety may be an antigen binding region, for example, it may be an antigen binding portion of an antibody. In certain embodiments, the targeting moiety may include a single-chain antibody (scFv) or a portion thereof that specifically recognizes a target protein (e.g., an antigen). In the present application, the targeting moiety may be a tumor-associated antigen binding region. In the present application, the tumor-associated antigen may refer to a biological molecule that is antigenic and whose expression is associated with a tumor. For example, the tumor-associated antigen may include a tumor-specific antigen that is present only in tumor cells but not in other normal cells; it may also include antigens that are present in tumor cells, in other organs and tissues, or in heterogeneous and allogeneic normal cells, or antigens expressed during development and differentiation.

[0049] In the present application, the "transmembrane domain" generally refers to a domain that spans the cell membrane. For example, the transmembrane domain may be a domain that spans the cell membrane of the chimeric antigen receptor. In the present application, the transmembrane domain may be derived from a natural polypeptide, or may be obtained by artificial design. For example, the transmembrane domain may be a transmembrane domain derived from a protein selected from the group consisting of: T cell receptor α or β chain, 3ζ chain, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 or GITR.

[0050] In the present application, the "costimulatory domain" generally refers to an intracellular domain that can provide an immune co-stimulatory molecule. The co-stimulatory molecule can be a molecule required for lymphocytes to effectively respond to antigens. The co-stimulatory domain may include a co-stimulatory domain of CD28. The co-stimulatory domain may include a co-stimulatory domain of the TNF receptor family, such as a co-stimulatory domain of OX40 and 4-1BB.

[0051] In the present application, the "intracellular signaling domain" generally refers to a domain located inside a cell that can transduce a signal. In the present application, the intracellular signaling domain can conduct a signal into the cell. For example, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor. For example, the intracellular signaling domain can be derived from an intracellular domain selected from CD3ζ, CD28, 4-1BB or OX40. For another example, the intracellular signaling domain can be an intracellular domain derived from GITR.

[0052] In the present application, the "T cell receptor (TCR)" generally refers to a specific receptor on the surface of a T cell. The T cell receptor is a heterodimer and can be composed of two different subunits. Most T cell receptors (e.g., 95% and above, 96% and above, 97% and above, etc.) are composed of α subunits and β subunits, and each peptide chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. In certain embodiments, the T cell receptor may be composed of a γ subunit and a δ subunit, and the ratio of these two subunits may vary with individual development or disease.

[0053] In the present application, the "TCR variable region" generally refers to the V region of the α and β peptide chains of the TCR, namely Vα and Vβ. The Vα and Vβ may have antigen specificity. The Vα and Vβ may each have three hypervariable regions CDR1, CDR2 and CDR3. Among them, the CDR3 of the TCR variable region has the greatest degree of variation, and the CDR3 of the TCR variable region may determine the antigen binding specificity of the TCR.

[0054] In the present application, the "Vα" generally refers to the variable region of the α chain of TCR. The term may also include the full-length sequence of Vα and / or the functional fragment of Vα, as long as it can be recognized by the antibody targeting it. For example, the Vα described in the present application may be a Vα with the registration number of Gene ID 6955 (human source) and the Vα with Gene ID 21473 (mouse source) in GenBank. In the present application, the "Vα24" generally refers to a variant of the Vα fragment. The Vα24 may be derived from different strains such as humans, macaques, mice or rats. For example, the Vα24 may be of macaque origin, and its registration number in GenBank is ABG76794.1; it may also be of human origin, and its PDB number is 4EN3_A.

[0055] In the present application, the "Jα" generally refers to the Jα fragment of the variable region of the α chain of TCR, which may have multiple variants. The Jα may be Jα18. In the present application, the "Jα18" generally refers to a variant of the Jα fragment. The Jα18 may be derived from different germline species such as humans, macaques, mice or rats. For example, the Jα18 described in the present application may be human, and its sequence may be shown as SEQ ID NO: 58.

[0056] In the present application, the "Vα24Jα18" generally refers to a variant obtained by recombination of a Vα variant Vα24 and a Jα fragment variant Jα18. For example, the sequence of Vα24Jα18 described in the present application may be shown as SEQ ID NO:59.

[0057] In the present application, the "Vβ" generally refers to the variable region of the β chain of TCR. The term may also include the full-length sequence of Vβ and / or the functional fragment of Vβ, as long as it can be recognized by the antibody targeting it. For example, the Vβ described in the present application may be the Vβ with accession number Gene ID 6957 (human source) and Gene ID 21577 (mouse source) in GenBank.

[0058] In the present application, the "Vβ8" generally refers to a variant of the variable region of the β chain of TCR. For example, the Vβ8 described in the present application can be derived from different germ lines such as human, macaque, mouse or rat. For example, the Vβ8 described in the present application can be human, and its sequence is shown in SEQ ID NO: 61; it can also be mouse, and its sequence is shown in SEQ ID NO: 60.

[0059] In the present application, the "variant" generally refers to a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a TCR variable region). For example, the functional variant may include a protein or polypeptide that has been substituted, deleted and / or inserted with at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acids. The functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0060] In the present application, the "homolog" generally refers to a protein or polypeptide having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to a TCR variable region).

[0061] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology percentage ratio " in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number of position to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio, can realize by multiple ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and DJ. Lipman, "Improved tools for biological sequence comparisons," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ. Lipman and W. R. Earson, "Rapid and sensitive protein similarity searches," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "A basic local alignment search tool," Journal of Molecular Biology, 215: 403-410, 1990.

[0062] In the present application, the "CDR3 of the TCR variable region" generally refers to the complementary determining region 3 of the TCR β chain. The CDR3 of the TCR variable region can be a site that directly binds to the antigen peptide. The gene of the CDR3 of the TCR variable region can be composed of the V end, the D segment, the J front end, and the insertion sequence (VnDnJ) between the rearranged V~D and D~J.

[0063] In the present application, the "Vα24Jα18 antibody" generally refers to an antibody that can specifically bind to Vα24Jα18. For example, in the present application, the Vα24Jα18 antibody can be 6B11 (Invitrogen Product#25-5806-41). For example, in the present application, the Vα24Jα18 antibody can also be scFv T120 (whose sequence can be shown as SEQ ID NO: 1).

[0064] In the present application, the "Vβ8 antibody" generally refers to an antibody that can specifically bind to Vβ8. For example, in the application, the Vβ antibody can be scFv F231 (whose sequence can be shown as SEQ ID NO: 19). For another example, in the present application, the Vβ antibody can be scFv 1168 (whose amino acid sequence can be shown as SEQ ID NO: 62, and its nucleic acid sequence can be shown as SEQ ID NO: 63).

[0065] In the present application, the "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), and antibody fragments that bind to antigens (e.g., Fab, Fab' and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain may be composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0066] In the present application, the "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide or ribonucleotide or its analog in an isolated form of any length separated from its natural environment or artificially synthesized. The nucleic acid molecule described in the present application can be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In the present application, nucleic acids encoding the antibodies or antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific operations, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0067] In the present application, the "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which is used to transfer the inserted nucleic acid molecule into a host cell and / or between host cells. The vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product. In the present application, one or more of the nucleic acid molecules may be included in the vector. In addition, other genes may also be included in the vector, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art, and for example, may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes a 5' non-transcribed sequence and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, a 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence functionally linked to a nucleic acid for transcriptional control.

[0068] In the present application, the "mRNA vector" generally refers to an mRNA fragment that can express an exogenous gene or an endogenous gene in a host cell. For example, in the present application, the mRNA vector may include F231mBBZ mRNA and 20mBBZ mRNA.

[0069] In this application, the "plasmid vector" generally refers to a plasmid artificially constructed on the basis of a natural plasmid to adapt to laboratory operations. Compared with natural plasmids, plasmid vectors usually carry one or more selectable marker genes (such as antibiotic resistance genes) and an artificially synthesized multiple cloning site sequence containing multiple restriction endonuclease recognition sites, and most of the non-essential sequences are removed to reduce the molecular weight as much as possible to facilitate genetic engineering operations.

[0070] In this application, the "retroviral vector" generally refers to a type of RNA virus whose genetic information is stored in ribonucleic acid, and most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains genes for proteins that make up the center and structure of the virus; pol, which contains genes for reverse transcriptase and env, which contains genes that make up the viral shell. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the exogenous genes it carries into the host cell genome, for example, the CAR molecule can be integrated into the host cell.

[0071] In the present application, the "lentiviral vector" generally refers to a diploid RNA viral vector belonging to a retrovirus. The lentiviral vector is based on the genome of the lentivirus, and multiple sequence structures related to viral activity are removed to make it biologically safe, and then the sequence and expression structure of the target gene required for the experiment are introduced into this genome skeleton to prepare the vector. Compared with other retroviruses, lentiviral vectors have a wider range of hosts and have the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc., it can greatly improve the transduction efficiency of the target gene (see Chen Chen and Wan Haisu, "Lentiviral Vectors and Research Progress, Chinese Journal of Lung Cancer 17.12 (2014): 870-876.PMC"). Through lentiviral vector transfection, the retroviral vector can randomly and stably integrate its own genome and the exogenous genes it carries into the host cell genome, for example, the CAR molecule can be integrated into the host cell.

[0072] In the present application, the "immune effector cells" generally refer to immune cells that participate in the clearance of foreign antigens and perform effector functions in immune responses, such as plasma cells, cytotoxic T cells, NK cells, APSC multipotent cells, mast cells, etc.

[0073] In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.

[0074] In this application, "T cell lymphoma" generally refers to a lymphoma that affects T cells. The T cell lymphoma may include the following types: angiocentric lymphoma, cutaneous T cell lymphoma, anaplastic large-cell lymphoma, and angioimmunoblastic T cell lymphoma. T cell lymphoma can develop in lymphoid tissues such as lymph nodes and spleen or outside of lymphoid tissues (i.e., gastrointestinal tract, liver, nasal cavity, skin, etc.).

[0075] In the present application, "leukemia" is generally a broad definition of blood cell cancer. For example, the leukemia can be a disease caused by the massive proliferation and accumulation of leukemic cells. The leukemia usually starts in the bone marrow. In the present application, the type of the leukemia can depend on the type of blood cell that becomes cancer and / or the growth rate of the blood cell. For example, the leukemia can include subtypes such as adult T cell leukemia / lymphoma (ATLL), T prolymphocytic leukemia (T-PLL), T large granular lymphocyte (T-LGL) leukemia.

[0076] In the present application, the "natural source" generally refers to a method of obtaining a wild type that has not been artificially processed. In the present application, the natural source may be a method of obtaining cells collected directly from a donor. For example, it may be a method of obtaining cells that have not been artificially gene edited or modified. In the present application, the donor may be a healthy or diseased mammal. For example, humans, mice, monkeys, etc. In the present application, the disease may refer to cancer. For example, the disease may be a leukemia or lymphoma of T cell origin (e.g., T cell lymphoma).

[0077] In the present application, the "induction by stem cells" generally refers to the process of differentiating stem cells by artificial induction in vitro. In the present application, the in vitro induction method is generally to add induction factors to adult stem cells cultured in vitro, and the induction factors may include various substances that can affect cell differentiation, such as serum, sugars, vitamins, and various protein factors. In the present application, the induction by stem cells may be induced pluripotent stem cells (iPS cells). The induced pluripotent stem cells can be obtained by transferring a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells using a viral vector. In the present application, the iPS cells may be a cell type similar to embryonic stem cells and embryonic APSC pluripotent cells.

[0078] In the present application, the term "and / or" should be understood to mean either one of the alternatives or both of the alternatives.

[0079] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0080] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "for", "consisting of...".

[0081] 1. Chimeric Antigen Receptor

[0082] In the present application, the chimeric antigen receptor (CAR) may comprise a targeting portion, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the targeting portion can specifically bind to the variable region Vα, Vβ and / or fragments thereof of the T cell receptor (TCR).

[0083] a. Targeting moiety

[0084] In certain embodiments, the targeting moiety may specifically bind to the variable region Vα or a fragment thereof of the TCR. In certain embodiments, the Vα or a fragment thereof may include Vα24 or a fragment thereof. In certain embodiments, the Vα or a fragment thereof may also include Jα or a fragment thereof. In certain embodiments, the Jα or a fragment thereof may include Jα18 or a fragment thereof. In certain embodiments, the targeting moiety may include a portion that targets Vα24Jα18. In the present application, the portion that targets Vα24Jα18 includes scFv T120, the sequence of which is shown in SEQ ID NO: 1.

[0085] In certain embodiments, the targeting moiety specifically binds to the variable region Vβ or a fragment thereof of the TCR. In certain embodiments, the Vβ or a fragment thereof may include Vβ8 or a fragment thereof. In certain embodiments, the fragment of the TCR variable region may include the CDR3 of the TCR variable region. In certain embodiments, the targeting moiety may include a portion targeting Vβ8. In the present application, the portion targeting Vβ8 may include scFv F231, the sequence of which is shown in SEQ ID NO: 19. For another example, in the present application, the portion targeting Vβ8 may also include scFv 1168, the sequence of which is shown in SEQ ID NO: 62.

[0086] In the present application, the variable region Vα, Vβ or a fragment thereof of the TCR may comprise an amino acid sequence shown in any one of SEQ ID NOs: 58-61, Gene ID 21473, Gene ID 6955, Gene ID 21577 and Gene ID 6957.

[0087] In the present application, the targeting moiety may include an antibody or a fragment thereof.

[0088] In the present application, the antibody can compete with the reference antibody for binding to the variable region Vα, Vβ and / or fragments thereof of the TCR, wherein the reference antibody can comprise a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody can comprise LCDR1, LCDR2 and LCDR3, the LCDR1 can comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 64 and SEQ ID NO: 21; the LCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 22; the LCDR3 can comprise the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 66 and SEQ ID NO: 23; the heavy chain variable region of the reference antibody can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 67 and SEQ ID NO: 29; the HCDR2 can comprise the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 68 and SEQ ID NO: The HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 69 and SEQ ID NO: 31.

[0089] In the present application, the light chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70 and SEQ ID NO: 27; and the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71 and SEQ ID NO: 35.

[0090] In the present application, the reference antibody may include Vα24Jα18 antibody. For example, the Vα24Jα18 antibody may be scFv T120, the sequence of which is shown in SEQ ID NO:1.

[0091] In the present application, the antibody may comprise a heavy chain complementary determining region 1 (HCDR1), and the HCDR1 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 11, SEQ ID NO: 67 and SEQ ID NO: 29. In the present application, the antibody may further comprise a heavy chain complementary determining region 2 (HCDR2), and the HCDR2 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 12, SEQ ID NO: 68 and SEQ ID NO: 30. In the present application, the antibody may further comprise a heavy chain complementary determining region 3 (HCDR3), and the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NO: 13, SEQ ID NO: 69 and SEQ ID NO: 31.

[0092] In the present application, the antibody may comprise a light chain complementary determining region 1 (LCDR1), wherein the LCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, SEQ ID NO: 64 and SEQ ID NO: 21. In the present application, the antibody may further comprise a light chain complementary determining region 2 (LCDR2), wherein the LCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 22. In the present application, the antibody may further comprise a light chain complementary determining region 3 (LCDR3), wherein the LCDR3 comprises an amino acid sequence as shown in any one of SEQ ID NO: 5, SEQ ID NO: 66 and SEQ ID NO: 23.

[0093] In the present application, the antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 71 and SEQ ID NO: 35. In the present application, the antibody comprises a light chain variable region, and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 70 and SEQ ID NO: 27.

[0094] In the present application, the antibody may be a single-chain antibody (scFv). In the present application, the single-chain antibody may be formed by connecting one or more scFv. For example, the single-chain antibody may include one scFv, and the single-chain antibody may include more than one scFv (e.g., 2, 3, 4, 5, etc.). In some embodiments, the multiple scFvs may be directly connected. In some embodiments, the multiple scFvs may be connected by a connecting peptide.

[0095] For example, the single-chain antibody may include scFv T120, whose sequence is shown in SEQ ID NO: 1; it may include a light chain variable region, whose sequence is shown in SEQ ID NO: 9; it may include LCDR1-3, whose sequences are shown in SEQ ID NO: 3-5, respectively; it may include a heavy chain variable region, whose sequence is shown in SEQ ID NO: 17; it may include HCDR1-3, whose sequences are shown in SEQ ID NO: 11-13, respectively; it may include a connecting peptide between VL and VH, and its connecting peptide sequence is shown in SEQ ID NO: 52.

[0096] For example, the single-chain antibody may include scFv F231, whose sequence is shown in SEQ ID NO: 19; it may include a light chain variable region, whose sequence is shown in SEQ ID NO: 27; it may include LCDR1-3, whose sequences are shown in SEQ ID NO: 21-23, respectively; it may include a heavy chain variable region, whose sequence is shown in SEQ ID NO: 35; it may include HCDR1-3, whose sequences are shown in SEQ ID NO: 29-31, respectively; it may include a connecting peptide between VL and VH, and its connecting peptide sequence is shown in SEQ ID NO: 52.

[0097] For example, the single-chain antibody may include scFv 1168, whose sequence is shown in SEQ ID NO: 62; it may include a light chain variable region, whose sequence is shown in SEQ ID NO: 70; it may include LCDR1-3, whose sequences are shown in SEQ ID NO: 64-66, respectively; it may include a heavy chain variable region, whose sequence is shown in SEQ ID NO: 71; it may include HCDR1-3, whose sequences are shown in SEQ ID NO: 67-68, respectively; it may include a connecting peptide between VL and VH, whose connecting peptide sequence is shown in SEQ ID NO: 52.

[0098] b. Transmembrane domain

[0099] In the present application, the transmembrane domain may include a transmembrane domain from a protein selected from the group consisting of CD8, CD28, CD137 and / or CD3. In the present application, the transmembrane domain may include an amino acid sequence as shown in SEQ ID NO: 40. For example, in the present application, the transmembrane domain may be a CD8 transmembrane region, the sequence of which is shown in SEQ ID NO: 40. In the present application, CAR T120BBZ and CAR 1168BBZ may include a transmembrane domain of CD8.

[0100] c. Co-stimulatory domain

[0101] In the present application, the costimulatory domain may include the costimulatory domain of the following proteins: ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 and / or CD226. In the present application, the costimulatory domain includes the amino acid sequence shown in SEQ ID NO: 42. For example, in the present application, the costimulatory domain may be 4-1BB, and its sequence is shown in SEQ ID NO: 42. In the present application, CAR T120BBZ, CAR1168BBZ and CAR F231mBBZ may include the costimulatory domain of 4-1BB.

[0102] d. Signal transduction domain

[0103] In the present application, the intracellular signaling domain may include a signaling domain from CD3ζ. In the present application, the intracellular signaling domain may include an amino acid sequence as shown in SEQ ID NO: 44. For example, in the present application, the signaling domain may be CD3ζ, and its sequence is as shown in SEQ ID NO: 44. In the present application, CART120BBZ, CAR 1168BBZ and CAR F231mBBZ may include a signaling domain of CD3ζ.

[0104] e. Hinge region

[0105] In the present application, the CAR may further include a hinge region, which may connect the antibody and the transmembrane domain. In the present application, the hinge region may include a hinge region from a protein selected from the group consisting of CD8a and / or IgG. In the present application, the hinge region is CD8a, which may include an amino acid sequence as shown in SEQ ID NO: 38. For example, in the present application, the hinge region may be CD8a, whose sequence is shown in SEQ ID NO: 38. In the present application, CART120BBZ, CAR 1168BBZ, and CAR F231mBBZ may include a hinge region of CD8a.

[0106] f.CAR

[0107] The CAR described in the present application may comprise an amino acid sequence as shown in any one of SEQ ID NO: 46, SEQ ID NO: 64 and SEQ ID NO: 50. For example, the CAR may be selected from CAR T120BBZ, whose sequence is shown in SEQ ID NO: 46. For another example, the CAR may be selected from CARF231mBBZ, whose sequence is shown in SEQ ID NO: 50. For another example, the CAR may be selected from CAR 1168BBZ, whose sequence is shown in SEQ ID NO: 72.

[0108] In certain embodiments, the CAR described herein may include, from the N-terminus, a targeting portion (the targeting portion sequence is as shown in SEQ ID NO: 1 and SEQ ID NO: 19), a transmembrane domain (the transmembrane domain sequence is as shown in SEQ ID NO: 40), a co-stimulatory domain (the co-stimulatory domain sequence is as shown in SEQ ID NO: 42) and an intracellular signaling domain (the intracellular signaling domain sequence is as shown in SEQ ID NO: 44).

[0109] For example, the CAR described in the present application may be CAR T120BBZ, whose targeting portion is scFv T120, and the amino acid sequence of scFv T120 is shown in SEQ ID NO: 46. Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40. The costimulatory domain may be 4-1BB, and its amino acid sequence is shown in SEQ ID NO: 42. The intracellular signaling domain may be CD3ζ, and its amino acid sequence is shown in SEQ ID NO: 44. The amino acid sequence of CAR T120BBZ is shown in SEQ ID NO: 46.

[0110] For example, the CAR described in the present application may be CARF231mBBZ, whose targeting portion is scFv F231, and the amino acid sequence of scFv F231 is shown in SEQ ID NO: 19. Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40. The costimulatory domain may be 4-1BB, and its amino acid sequence is shown in SEQ ID NO: 42. The intracellular signaling domain may be CD3ζ, and its amino acid sequence is shown in SEQ ID NO: 44. The amino acid sequence of CAR F231mBBZ is shown in SEQ ID NO: 50.

[0111] For example, the CAR described in the present application may be CAR 1168BBZ, whose targeting portion is scFv 1168, and the amino acid sequence of scFv 1168 is shown in SEQ ID NO: 61. Its transmembrane domain is CD8a, and its amino acid sequence is shown in SEQ ID NO: 40. The costimulatory domain may be 4-1BB, and its amino acid sequence is shown in SEQ ID NO: 42. The intracellular signaling domain may be CD3ζ, and its amino acid sequence is shown in SEQ ID NO: 44. The amino acid sequence of CAR 1168mBBZ is shown in SEQ ID NO: 72.

[0112] The proteins, polypeptides, amino acid sequences and / or nucleotide sequences involved in the present application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the proteins, polypeptides, or the proteins, polypeptides encoded by the nucleotide sequences.

[0113] Nucleic acid, vector, cell, composition

[0114] On the other hand, the present application provides an isolated nucleic acid molecule encoding the CAR described in the present application. In the present application, the isolated nucleic acid molecule encoding CAR may include a nucleic acid sequence shown in SEQ ID NO: 47 or SEQ ID NO: 51. The nucleic acid molecule described in the present application may be isolated. For example, it may be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0115] On the other hand, the present application provides a vector, which may include the nucleic acid molecule described in the present application. In the present application, the vector may include an mRNA vector, a plasmid vector, a retroviral vector and / or a lentiviral vector. For example, the vector described in the present application may be a lentiviral vector, which may include the nucleic acid sequence shown in SEQ ID NO: 47 or SEQ ID NO: 51. In addition, the vector may also include other genes, such as a marker gene that allows the vector to be selected in an appropriate host cell and under appropriate conditions. In addition, the vector may also include expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that are involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence for transcriptional control functionally connected to a nucleic acid. One or more nucleic acid molecules described herein may be operably connected to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage, an mRNA vector, or other vectors commonly used in, for example, genetic engineering.

[0116] In some embodiments, the vector may be a plasmid vector, for example, it may include a pCDH-MSCVEF vector. In some embodiments, the vector may be a lentiviral vector, for example, the lentiviral vector may be VSV-g, pMD Gag / Pol or RSV-REV, which may be co-transfected with a plasmid vector to obtain a lentiviral vector loaded with a plasmid after a host cell (e.g., 293X cell) is co-transfected. In the present application, a lentiviral vector loaded with a plasmid may include a T120BBZ virus and may also include a 20BBZ virus. In some embodiments, the vector may be an mRNA vector, for example, the mRNA vector may be an ARCA-capped RNA (AM1345, ThermorFisher), which may be used to produce an mRNA vector loaded with a plasmid. In the present application, an mRNA vector loaded with a plasmid may include F231mBBZ mRNA and may also include 20BBZ mRNA.

[0117] On the other hand, the present application provides immune effector cells, which include and / or can express CAR described in the present application, nucleic acid molecules described in the present application, or vectors described in the present application. In the present application, the immune effector cells can be selected from T lymphocytes and natural killer (NK) cells. In the present application, the T lymphocytes and / or natural killer cells can be natural sources, or induced by stem cells.

[0118] On the other hand, the present application provides a method for preparing immune effector cells, which includes introducing the vector described in the present application into the immune effector cells. In some embodiments, each or each cell may include one or a vector described in the present application. In some embodiments, each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) vectors described in the present application. For example, the vector described in the present application can be introduced into the cell, such as a eukaryotic cell (e.g., a mammalian cell). For example, the mammalian cell can be a 293T cell or a 293X cell. The vector described in the present application can be introduced into the cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0119] On the other hand, the present application provides a composition, which may include the immune effector cells described in the present application. In certain embodiments, it may include the immune effector cells and a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex and / or a nonionic surfactant, etc. In the present application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via a subcutaneous reservoir.

[0120] Pharmaceutical use

[0121] On the other hand, the present application provides the use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell for preparing a drug, wherein the drug is used to treat T cell lymphoma or leukemia.

[0122] On the other hand, the present application provides the use of the CAR, the nucleic acid molecule, the vector, or the immune effector cell in the preparation of a drug for treating T-cell lymphoma or leukemia.

[0123] On the other hand, the present application provides a method for treating T-cell lymphoma or leukemia, comprising administering the CAR, the nucleic acid molecule, the vector, or the immune effector cell to the patient.

[0124] In the present application, the T-cell lymphoma or leukemia may include one or more selected from the following groups: peripheral T-cell lymphoma, unspecified peripheral T-cell lymphoma, angioimmunoblastic lymphoma, extranodal NKT-cell lymphoma, anaplastic T-cell lymphoma, ALK-positive anaplastic cell lymphoma, ALK-negative anaplastic cell lymphoma, T-lymphoblastic lymphoma, cytotoxic T-cell lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), T-prolymphocytic leukemia (T-PLL), T-large granular lymphocyte (T-LGL) leukemia, hepatosplenic T-cell lymphoma (HSTL), Sezary syndrome (SS), subcutaneous tonsillar T-cell lymphoma and unclassified T-cell lymphoma.

[0125] In certain embodiments, the CAR, the nucleic acid molecule, the vector, or the immune effector cell can significantly improve the in vivo killing effect on tumor cells. For example, the tumor volume can be reduced by more than 50% (for example, more than 60%, more than 70%, more than 80%, more than 90%, etc.).

[0126] In certain embodiments, the CAR, the nucleic acid molecule, the vector, or the immune effector cell can significantly improve the in vitro killing effect on tumor cells. For example, the tumor volume can be reduced by more than 50% (for example, more than 60%, more than 70%, more than 80%, more than 90%, etc.).

[0127] In certain embodiments, the CAR, the nucleic acid molecule, the vector, or the immune effector cell can selectively kill tumor cells with a specific antigen, while substantially not killing normal T cells (e.g., the normal T cells can be normal, healthy T cells that do not belong to T cell lymphoma). For example, the CAR, the nucleic acid molecule, the vector, or the immune effector cell can selectively kill tumor cells with a specific antigen (e.g., Vα24Jα18 + ) The tumor cell killing effect is improved by more than 50% (for example, more than 60%, more than 70%, more than 80%, more than 90%, etc.), while there is basically no damage to normal T cells (for example, the number of T cells is reduced by less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, etc.).

[0128] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate the working modes of the apparatus, method and system of the present application, and are not intended to limit the scope of the invention of the present application.

[0129] Example

[0130] In this application, the corresponding relationship between the binding target, targeting part (scFv), CAR, vector, and cell name is shown in Table 1 below:

[0131] Table 1

[0132]

[0133] Example 1 Preparation of anti-Vα24Jα18 CAR-T cells (T120BBZ CAR-T cells)

[0134] 1.1 Construction of lentiviral vector pCDH-MSCVEF-T120BBZ and virus production

[0135] To construct the CAR described in this application (see Figure 1 ). First, a CAR targeting Vα24Jα18 was prepared, and the following sequences were artificially synthesized: scFv T120 (SEQ ID NO: 1), hinge region (SEQ ID NO: 38), transmembrane region (SEQ ID NO: 40), 4-1BB co-stimulatory factor (SEQ ID NO: 42), CD3ζ intracellular signaling domain (SEQ ID NO: 44). Among them, BBZ can be obtained by connecting the 4-1BB co-stimulatory factor and the CD3ζ intracellular signaling domain end to end, and its sequence is shown in SEQ ID NO: 54.

[0136] Meanwhile, scFv 20 was constructed as a control, and its sequence is shown in SEQ ID NO:37.

[0137] The scFv T120 (amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) and BBZ (SEQ ID NO: 54) that can specifically bind to TCR Vα24Jα18 were cloned into the pCDH-MSCVEF vector by overlap PCR, and EcoRI and BamHI restriction sites were added at both ends. PCR amplification was performed, and the EcoRI restriction site (including protective bases), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3ζ intracellular signaling domain, and BamHI restriction site were added to the 5' end in sequence by extension PCR, and the CAR T120BBZ was amplified by PCR. The clones sequenced correctly were extracted with the NucleoBond Xtra Midi Plus EF kit without endotoxin, and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag / Pol or RSV-REV) into 293X cells. After culturing at 37°C and 5% CO2 for 48 hours, the supernatant was collected, filtered at 0.45 μM, and centrifuged at 25,000 RPM for 2 hours using a Beckman ultracentrifuge and SW28 rotor to concentrate the virus, which was the pCDH-MSCVEF-T120BBZ virus (abbreviated as T120BBZ virus) for subsequent CAR-T cell production.

[0138] At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced by the same method as that for preparing T120BBZ virus. The obtained virus was used to infect 293 cells, and the virus titer was measured by flow cytometry using anti-mouse Fab antibody (Jackson ImmunoResearch #115-605-006). Figure 2 shown. Figure 2 Shown in the figure are the flow cytometry detection results when 1 μL, 3 μL, and 9 μL of the T120BBZ virus were added, with no virus added as a blank control.

[0139] The results showed that as the added virus dose increased, the expression level of CART120BBZ also increased.

[0140] 1.2 Preparation of T120BBZ CAR-T cells and 20BBZ CAR-T cells

[0141] After human PBMCs were purified by a Stemcell T cell isolation kit (purchased from stem cell Catlog #19671), they were inoculated into 96-well culture plates coated with anti-hCD3 (purchased from Bioxcell #BE0001-2) and anti-hCD28 (purchased from Bioxcell #BE0248). After 2 days, they were infected with the T120BBZ virus and 20BBZ virus prepared in step 1.1 of this example at an MOI of 10-20. After 1 day, the medium was changed to continue cell culture. The culture medium was RPMI complete medium containing 10% FBS, IL2 (50 IU / ml), IL21 (4 ng / ml). Artificial antigen presenting cells (Raji cells irradiated with 100 Gray X-rays) or anti-hCD3 (0.1 μg / ml) or anti-hCD28 (0.25 μg / ml) were used for stimulation every 6 days. After 2 rounds of stimulation, the obtained cells were T120BBZ CAR-T cells and 20BBZ CAR-T cells were stained by flow cytometry and T120BBZ CAR-T cells were stained with Alexa Fluor 647 AffiniPure F(ab′)2Fragment Goat Anti-Human IgG, Fab fragmentspecific secondary antibody staining, the results are as follows Figure 3 shown.

[0142] The results showed that all the obtained cells were CAR positive.

[0143] Example 2 Preparation of anti-Vβ8 CAR-T (F231mBBZ) cells

[0144] 2.1. Production of anti-Vβ8 CAR-T (F231mBBZ) mRNA

[0145] Similar to Example 1, a CAR targeting mouse Vβ8 was prepared, and the following sequences were artificially synthesized: scFv F231 (SEQ ID NO: 19), mBBZ (SEQ ID NO: 56), wherein mBBZ is a mouse 4-1BB costimulatory factor and a CD3ζ intracellular signaling domain connected end to end.

[0146] The scFv F231 (SEQ ID 19) and mBBZ (SEQ ID NO: 56) that can specifically bind to TCR Vβ8 were cloned into the pCDH-MSCVEF vector by overlap PCR, and EcoRI and BamHI restriction sites were added at both ends. PCR amplification was performed, and the EcoRI restriction site (including protective bases), hinge region, transmembrane region, 4-1BB co-stimulatory factor, CD3ζ intracellular signaling domain, and BamHI restriction site were added to the 5' end in sequence by extension PCR. The CAR F231mBBZ (SEQ ID NO: 51) was obtained by PCR amplification, and the T7 RNA polymerase promoter sequence was introduced at the 5' end by primers. The PCR product was recovered and purified, and mMESSAGE was used. F231mBBZ mRNA was produced using the T7 Ultra Kit and control 20BBZ mRNA was produced using the same method.

[0147] 2.2 Preparation of F231mBBZ CAR-T cells and 20BBZ CAR-T cells

[0148] Mouse spleen cells were purified using the Mouse CD3T Cell Isolation Kit (purchased from biolegend #480031), inoculated into 96-well culture plates, activated with ConA (purchased from sigma #C2010) for 2 days, and then electroporated with F231mBBZ mRNA and 20BBZ mRNA using ECM830. The resulting cells were F231mBBZ CAR-T cells and 20BBZ CAR-T cells.

[0149] Example 3 Preparation of anti-1168 CAR-T cells (1168BBZ CAR-T cells)

[0150] 3.1. Construction of lentiviral vector pCDH-MSCVEF-1168BBZ and virus production

[0151] According to the method of Example 1, a CAR targeting human Vβ8 was first prepared, and the following sequences were artificially synthesized: scFv 1168 (SEQ ID NO: 62), hinge region (SEQ ID NO: 38), transmembrane region (SEQ ID NO: 40), 4-1BB co-stimulatory factor (SEQ ID NO: 42), CD3ζ intracellular signaling domain (SEQ ID NO: 44). Among them, BBZ can be obtained by connecting the 4-1BB co-stimulatory factor and the CD3ζ intracellular signaling domain end to end, and its sequence is shown in SEQ ID NO: 54.

[0152] Meanwhile, scFv 20 was constructed as a control, and its sequence is shown in SEQ ID NO:37.

[0153] The scFv 1168 (SEQ ID NO: 62) and BBZ (SEQ ID NO: 54) that can specifically bind to human Vβ8 were cloned into the pCDH-MSCVEF vector by overlap PCR, and EcoRI and BamHI restriction sites were added at both ends. PCR amplification was performed, and the EcoRI restriction site (including protective bases), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3ζ intracellular signaling domain, and BamHI restriction site were added to the 5' end in sequence by extension PCR, and the CAR1168BBZ was obtained by PCR amplification. The clones with correct sequencing were extracted with NucleoBond Xtra Midi Plus EF kit without endotoxin, and co-transfected with lentiviral packaging plasmids (VSV-g, pMDGag / Pol or RSV-REV) into 293X cells. After culturing at 37°C and 5% CO2 for 48 hours, the supernatant was collected, filtered at 0.45 μM, and centrifuged at 25,000 RPM for 2 hours using a Beckman ultracentrifuge and SW28 rotor to concentrate the virus, which was pCDH-MSCVEF-1168BBZ virus (referred to as 1168BBZ virus) for subsequent CAR-T cell production.

[0154] At the same time, the control pCDH-MSCVEF-20BBZ virus (referred to as 20BBZ virus) was produced by the same method as that for preparing 1168BBZ virus. The obtained virus was used to infect 293 cells, and the virus titer was measured by flow cytometry using anti-mouse Fab antibody (Jackson ImmunoResearch #115-605-006). Fig. 9 shown. Fig. 9 Shown in the figure are the flow cytometry detection results when 1 μL, 3 μL, and 9 μL of the 1168BBZ virus were added, with no virus added as a blank control.

[0155] The results showed that as the added virus dose increased, the expression level of the CAR 1168BBZ also increased.

[0156] 3.2. Preparation of 1168BBZ CAR-T cells and 20BBZ CAR-T cells

[0157] After human PBMCs were purified by a Stemcell T cell isolation kit (purchased from stem cell Catlog #19671), they were inoculated into 96-well culture plates coated with anti-hCD3 (purchased from Bioxcell #BE0001-2) and anti-hCD28 (purchased from Bioxcell #BE0248). After 2 days, they were infected with the 1168BBZ virus and 20BBZ virus prepared in step 1.1 of this example at an MOI of 10-20. After 1 day, the medium was changed to continue cell culture. The culture medium was RPMI complete medium containing 10% FBS, IL2 (50 IU / ml), IL21 (4 ng / ml). Artificial antigen presenting cells (Raji cells irradiated with 100 Gray X-rays) or anti-hCD3 (0.1 μg / ml) or anti-hCD28 (0.25 μg / ml) were used for stimulation every 6 days. After 2 rounds of stimulation, the obtained cells were 1168BBZ CAR-T cells and 20BBZ CAR-T cells were stained by flow cytometry using anti-mouse Fab antibody (Jackson ImmunoResearch #115-605-006) as a secondary antibody. Fig.10 shown.

[0158] The results showed that all the obtained cells were CAR positive.

[0159] Example 4 In vitro killing of tumor cells by T120BBZ CAR-T and release of effector molecules

[0160] The T120BBZ CAR-T cells prepared in Example 1 were used, and 20BBZ CAR-T cells were used as a control, and NALM6-Vα24Jα18 + The cells were co-cultured at 37°C with 5% CO2, and NALM6-Vα24Jα18 was detected by flow cytometry after 24 hours. + T120BBZ CAR-T can selectively kill Vα24Jα18 + The tumor cells release more TNF. Figure 4 As shown, the results of TNF release were Figure 5 shown.

[0161] The results showed that compared with control CAR-T cells, T120BBZ CAR-T cells had a significant effect on Vα24Jα18 + The tumor killing effect of Vα24Jα18 - Compared with control CAR-T cells, after administration of T120BBZ CAR-T cells, Vα24Jα18 - Tumor cells decreased by 17.4%, Vα24Jα18 + The tumor cells were reduced by 72.2%, proving that T120BBZ CAR has specificity for Vα24Jα18. Moreover, after administration of T120BBZ CAR-T cells, Vα24Jα18 - The amount of TNF released by tumor cells increased slightly (by 23.7%), Vα24Jα18 + The amount of TNF released by tumor cells was significantly increased (increased by 188.6%), which also proved that T120BBZ CAR was specific for Vα24Jα18.

[0162] Example 5 In vitro killing of tumor cells by F231mBBZ CAR-T cells

[0163] The F231mBBZ CAR-T cells and 20BBZ CAR-T cells prepared in step 2.2 of Example 2 were inoculated into 96-well plates, and T cells not infected by the virus were used as negative controls, and C1498Vβ8 - CD20 - or C1498Vβ8 + CD20 - Tumor cells were co-cultured at 37°C and 5% CO2. After 24 hours, flow cytometry was used to stain TCRVb8.1 / 8.2 (Biolegend#118406) to compare the survival rate of tumor cells and the release of IFNγ. The results are shown in Figure 2. Figure 6A-6B shown.

[0164] The results showed that F231mBBZ CAR-T cells can selectively kill Vβ8 + of tumor cells and release more IFNγ.

[0165] Example 6 In vitro killing of tumor cells by 1168BBZ CAR-T and release of effector molecules

[0166] The 1168BBZ CAR-T cells prepared in Example 3 were used, and the 20BBZ CAR-T cells were used as a control, and Vβ8 +Jurkat cells were co-cultured at 37°C and 5% CO2. After 24 hours, the survival of tumor cells was detected by flow cytometry. 1168BBZCAR-T can selectively kill Vβ8 + Tumor cells. The killing effect on tumors is tested as follows Fig.11 shown.

[0167] The results showed that 1168BBZ CAR-T cells had a significantly higher expression of Vβ8 compared with control CAR-T cells. + The tumor killing effect of 20BBZ on Vβ8 + Compared with control CAR-T cells, the number of surviving tumor cells decreased after administration of 1168BBZ CAR-T cells, proving that 1168BBZ CAR has specificity for Vβ8.

[0168] The results showed that 1168BBZ CAR-T cells can selectively kill human Vβ8 + of tumor cells.

[0169] Example 7 Analysis of the killing ability of T120BBZ CAR-T cells on tumor cells in vivo

[0170] 10 6 NALM6 Vα24Jα18 + Tumor cells were inoculated into B-NDG mice (Biocytogen) by intravenous injection, and 10 7 T120BBZ CAR-T cells were used for treatment, and T cells not infected with the virus were used as negative control. On the 7th day, 5 drops of peripheral blood (100 μL) were taken for flow cytometry detection of the content of tumor cells and CAR-T cells in the peripheral blood. The results were as follows: Figures 7A-7C shown.

[0171] The results showed that compared with the control group, T120BBZ CAR-T cells reduced the tumor size in mice by 82.9% (3.798 mm 3 To 0.646mm 3 ). T120BBZ CAR-T cells significantly prolonged the tumor burden in mice and were able to expand and prolong the survival of mice in vivo.

[0172] Example 8 Analysis of TCR Composition of T120BBZ CAR-T Cells

[0173] The T120BBZ CAR-T cells prepared in Example 1 were used as controls, and 20BBZ CAR-T cells were used as controls. Anti-TCRαβ (Biolegend#306708), anti-CD3 (Biolegend#317306) and anti-Vα24Jα18 + (Biolegend#342912) staining, flow cytometry analysis of its specific targeting TCR (Vα24Jα18 + ) composition changes, the results are as follows Figures 8A-8C The results showed that compared with the control CAR-T cells, Vα24Jα18 in T120BBZ CAR-T + The number of tumor cells was significantly reduced, proving that T120BBZ CAR-T can selectively and specifically kill Vα24Jα18 + T cells, TCRαβ cells, CD3 + There was no significant difference in the cells (NS, not siginificant), proving that the T cells of other TCRαβ subtypes and the CD3 component in the TCR complex were retained, indicating that it was retained for the vast majority of T cell subtypes and had little effect on the function of T cell immunity.

[0174] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments currently listed herein will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents. Sequence Listing <110> Shanghai Jiao Tong University <120> Chimeric antigen receptor and its application <130> 0104-PA-002CN <160> 73 <170> PatentIn version 3.5 <210> 1 <211> 266 <212> PRT <213> Artificial Sequence <220> <223> scFv T120 <400> 1 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln 100 105 110 His Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 165 170 175 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 180 185 190 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 210 215 220 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Thr Arg Asn Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 <210> 2 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> scFv T120 (nucleotide) <400> 2 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 accggtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120 gtcactatca cctgcaaagc cagtcaggat gtgagcactg ctgtagcctg gtatcaacag 180 aaacccggac aggccccgcg tctgctgatt tactgggcat ccacccgtca taccggagtc 240 ccttctcgct tctctggttc cggctctggg acggatttca ctctgaccat cagctccctg 300 cagccggaag acttcgcact gtattactgt cagcaacact atagcactcc gtggacgttc 360 ggacagggta ccaagctgga gatcaaaggc ggaggcggat caggcggcgg tggatctggg 420 ggtggcggat ctgaagtaca gctggtggag tctggaggag gcttggtgca acctggagga 480 tccctgcgtc tctcctgtgt tgcctctgga ttcactttca gtaactactg gatgaactgg 540 gtccgccagg cgccaggtaa ggggcttgag tgggttgctg aaattagatt gaaatctaat 600 aattatgcaa cacattatgc ggagtctgtg aaagggaggt tcaccatctc aagagatgat 660 tccaaaaata ccgtctacct gcaaatgaac agcttaagag ctgaagacac tgcggtttat 720 tactgtaccc ggaacggtaa ttacgtggat tatgctatgg actactgggg tcaaggaacc 780 ctggtcaccg tctcctca 798 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> T120 LCDR1 <400> 3 Gln Asp Val Ser Thr Ala 1 5 <210> 4 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> T120 LCDR2 <400> 4 Trp Ala Ser 1 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T120 LCDR3 <400> 5 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> T120 LCDR1 (nucleotide) <400> 6 caggatgtga gcactgct 18 <210> 7 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> T120 LCDR2 (nucleotide) <400> 7 tgggcatcc 9 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> T120 LCDR3 (Nucleotide) <400> 8 cagcaacact atagcactcc gtggacg 27 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> T120 VL <400> 9 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 Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 321 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> T120 VL(Version Vehicle) <400> 10 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcact atcacctgca aagccagtca ggatgtgagc actgctgtag cctggtatca acagaaaccc ggacaggccc cgcgtctgct gatttactgg gcatccaccc gtcataccgg agtcccttct 180 cgcttctctg gttccggctc tgggacggat ttcactctga ccatcagctc cctgcagccg 240 gaagacttcg cactgtatta ctgtcagcaa cactatagca ctccgtggac gttcggacag ggtaccaagc tggagatcaa a <210> 11 <211> 8 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> T120 HCDR1 <400> 11 Gly Phe Thr Phe Ser Asn Tyr Trp 1 5 <210> 12 <211> 10 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> T120 HCDR2 <400> 12 Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr 1 5 10 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> T120 HCDR3 <400> 13 Thr Arg Asn Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 14 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> T120 HCDR1 (nucleotides) <400> 14 ggattcactt tcagtaacta ctgg 24 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> T120 HCDR2 (nucleotides) <400> 15 attagattga aatctaataa ttatgcaaca 30 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> T120 HCDR3 (nucleotides) <400> 16 acccggaacg gtaattacgt ggattatgct atggactac 39 <210> 17 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> T120 VH <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Asn Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> T120 VH (nucleotide) <400> 18 gaagtacagc tggtggagtc tggaggaggc ttggtgcaac ctggaggatc cctgcgtctc 60 tcctgtgttg cctctggatt cactttcagt aactactgga tgaactgggt ccgccaggcg 120 ccaggtaagg ggcttgagtg ggttgctgaa attagattga aatctaataa ttatgcaaca 180 cattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaaatacc 240 gtctacctgc aaatgaacag cttaagagct gaagacactg cggtttatta ctgtacccgg 300 aacggtaatt acgtggatta tgctatggac tactggggtc aaggaaccct ggtcaccgtc 360 tcctca 366 <210> 19 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> scFv F231 <400> 19 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe 20 25 30 Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser 35 40 45 Gln Ile Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ser Pro Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val 65 70 75 80 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln 100 105 110 His Trp Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 145 150 155 160 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 165 170 175 Trp Met Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 180 185 190 Gly Tyr Ile Asn Pro Thr Thr Gly Tyr Thr Glu Tyr Thr Gln Lys Phe 195 200 205 Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Ser Ala Tyr 210 215 220 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Gly Ser Pro Tyr Tyr Asp Ser Thr Tyr Ser Tyr Trp Tyr Phe 245 250 255 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 <210> 20 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> scFv F231 (nucleotide) <400> 20 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 accggtgata tcgtgatgac ccagagccag aagttcatga gcacatccgt gggcgacaga 120 gtgagcatca cctgtaaggc cagccagatc gtgaggaccg ccgtggcctg gtaccagcag 180 aaacccggac aaagccccaa ggccctgatc tacctggcca gcaacagaca taccggcgtg 240 cccgatagat tcaccggcag cggcagcggc accgacttta ccctcaccat ctccaacgtg 300 cagtccgagg atctggccga ctacttctgc ctccagcact ggaactaccc cttaccttc 360 ggcagcggca ccaagctgga gattaaaggc ggcggcggca gcggcggagg cggaagcgga 420 ggaggcggaa gccaagtgca gctccagcag agcggagccg aactggctaa gcccggcgct 480 agcgtgaaga tgagctgcaa ggccagcggc tacaccttca cctcctactg gatgtactgg 540 ctgaagcaga gacctggcca gggcctggag tggatcggct acatcaaccc caccaccggc 600 tacaccgagt acacccagaa gttcaaggat aaggccaccc tgaccgctga caccagcagc 660 aacagcgctt atatgcagct gtccagcctg accagcgagg actccgccgt gtactactgc 720 gctaggggca gccttacta cgactccacc tactcctact ggtacttcga cgtgtggggc 780 gccggcacca cagtgacagt gagctcc 807 <210> 21 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> F231 LCDR1 <400> twenty one Gln Ile Val Arg Thr Ala 1 5 <210> twenty two <211> 3 <212> PRT <213> Artificial Sequence <220> <223> F231 LCDR2 <400> twenty two Leu Ala Ser 1 <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <220> <223> F231 LCDR3 <400> twenty three Leu Gln His Trp Asn Tyr Pro Phe Thr 1 5 <210> twenty four <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F231 LCDR1 (nucleotide) <400> twenty four cagatcgtga ggaccgcc 18 <210> 25 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> F231 LCDR2 (nucleotide) <400> 25 ctggccagc 9 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F231 LCDR3 (nucleotide) <400> 26 ctccagcact ggaactaccc ctttacc 27 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> F231 VL <400> 27 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Ile Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 28 <211> 321 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> F231 VL(Vault Line) <400> 28 gatatcgtga tgacccagag ccagaagttc atgagcacat ccgtgggcga cagagtgagc atcacctgta aggccagcca gatcgtgagg accgccgtgg cctggtacca gcagaaaccc 120 180. ggacaaagcc ccaaggccct gatctacctg gccagcaaca gacataccgg cgtgcccgat agttcaccg gcagcggcag cggcaccgac tttaccctca ccatctccaa cgtgcagtcc 240 gaggatctgg ccgactactt ctgcctccag cactggaact acccctttac cttcggcagc ggcaccaagc tggagata a <210> 29 <211> 8 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> F231 HCDR1 <400> 29 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> F231 HCDR2 <400> 30 Ile Asn Pro Thr Thr Gly Tyr Thr 1 5 <210> 31 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> F231 HCDR3 <400> 31 Ala Arg Gly Ser Pro Tyr Tyr Asp Ser Thr Tyr Ser Tyr Trp Tyr Phe 1 5 10 15 Asp Val <210> 32 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> F231 HCDR1 (nucleotides) <400> 32 ggctacacct tcacctccta ctgg 24 <210> 33 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> F231 HCDR2 (nucleotides) <400> 33 atcaacccca ccaccggcta cacc 24 <210> 34 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> F231 HCDR3 (Nucleotide) <400> 34 gctaggggca gcccttacta cgactccacc tactcctact ggtacttcga cgtg 54 <210> 35 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> F231 VH <400> 35 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Thr Thr Gly Tyr Thr Glu Tyr Thr Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Ser Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Pro Tyr Tyr Asp Ser Thr Tyr Ser Tyr Trp Tyr Phe 100 105 110 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 36 <211> 375 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> F231 VH(Version Vehicle) <400> 36 60. caagtgcagc tccagcagag cggagccgaa ctggctaagc ccggcgctag cgtgaagatg agctgcaagg ccagcggcta caccttcacc tcctactgga tgtactggct gaagcagaga cctggccagg gcctggagtg gatcggctac atcaacccca ccaccggcta caccgagtac 180 acccagaagt tcaaggataa ggccaccctg accgctgaca ccagcagcaa cagcgcttat atgcagctgt ccagcctgac cagcgaggac tccgccgtgt actactgcgc taggggcagc 300 ccttactacg actccaccta ctcctactgg tacttcgacg tgtggggcgc cggcaccaca 360 gtgacagtga gctcc 375 <210> 37 <211> 264 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> scFv 20 <400> 37 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Thr Ser Asn Pro Pro 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 Gln 130 135 140 Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 145 150 155 160 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn 165 170 175 Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ala 260 <210> 38 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Hinge Region <400> 38 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 39 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Hinge region (nucleotides) <400> 39 actacaactc cagctccacg accaccaaca cctgctccaa ccatcgcttc gcagcctctg 60 tccctgcgcc cagaggcatg ccggccagct gcaggaggtg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 40 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Transmembrane region <400> 40 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 41 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Transmembrane region (nucleotides) <400> 41 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 42 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB co-stimulatory domain <400> 42 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 43 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> 4-1BB co-stimulatory domain (nucleotide) <400> 43 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 44 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3ζ intracellular signaling domain <400> 44 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 45 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> CD3ζ intracellular signaling domain (nucleotide) <400> 45 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgg 336 <210> 46 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> CAR T120BBZ <400> 46 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45 Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln 100 105 110 His Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 165 170 175 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 180 185 190 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 210 215 220 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Thr Arg Asn Gly Asn Tyr Val Asp Tyr Ala Met Asp Tyr Trp 245 250 255 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala 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 Gln 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 <210> 47 <211> 1479 <212> DNA <213> Artificial Sequence <220> <223> CAR T120BBZ (Nucleotide) <400> 47 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 accggtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120 gtcactatca cctgcaaagc cagtcaggat gtgagcactg ctgtagcctg gtatcaacag 180 aaacccggac aggccccgcg tctgctgatt tactgggcat ccacccgtca taccggagtc 240 ccttctcgct tctctggttc cggctctggg acggatttca ctctgaccat cagctccctg 300 cagccggaag acttcgcact gtattactgt cagcaacact atagcactcc gtggacgttc 360 ggacagggta ccaagctgga gatcaaaggc ggaggcggat caggcggcgg tggatctggg 420 ggtggcggat ctgaagtaca gctggtggag tctggaggag gcttggtgca acctggagga 480 tccctgcgtc tctcctgtgt tgcctctgga ttcactttca gtaactactg gatgaactgg 540 gtccgccagg cgccaggtaa ggggcttgag tgggttgctg aaattagatt gaaatctaat 600 aattatgcaa cacattatgc ggagtctgtg aaagggaggt tcaccatctc aagagatgat 660 tccaaaaata ccgtctacct gcaaatgaac agcttaagag ctgaagacac tgcggtttat 720 tactgtaccc ggaacggtaa ttacgtggat tatgctatg actactgggg tcaaggaacc 780 ctggtcaccg tctctcagc ggccgcaact acaactccag ctccacgacc accaacacct 840 gctccaacca tcgcttcgca gcctctgtcc ctgcgcccag aggcatgccg gccagctgca 900 ggaggtgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 960 ttggccggga cttgtggggt cttctcctg tcactggtta tcacccttta ctgcaaacgg 1020 ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1080 caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1140 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440 tacgacgccc ttcacatgca ggccctgccc cctcggtaa 1479 <210> 48 <211> 490 <212> PRT <213> Artificial Sequence <220> <223> CAR 20BBZ <400> 48 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Thr Ser Asn Pro Pro 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 Gln 130 135 140 Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 145 150 155 160 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn 165 170 175 Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly 180 185 190 Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 195 200 205 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220 Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 225 230 235 240 Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala 245 250 255 Gly Thr Thr Val Thr Val Ser Ala Ala Ala Ala Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 370 375 380 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 385 390 395 400 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425 430 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440 445 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 450 455 460 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 465 470 475 480 Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 49 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> CAR 20BBZ (Nucleotide) <400> 49 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 accggtcaaa ttgttctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120 gtcacaatga cttgcagggc cagctcaagt gtaagttaca tccactggtt ccagcagaag 180 ccaggctctt cccccaaacc ctggatttat gccacatcca acctggcttc tggagtccct 240 gttcgcttca gtggcagtgg gtctgggact tcttactctc tcacaatcag cagagtggag 300 gctgaagatg ctgccactta ttactgccag cagtggacta gtaacccacc cacgttcgga 360 ggggggacca agctggaaat caaaggcgga ggcggatcag gcggcggtgg atctgggggt 420 ggcggatctc aggtacaact gcagcagcct ggggctgagc tggtgaagcc tggggcctca 480 gtgaagatgt cctgcaaggc ttctggctac acatttacca gttacaatat gcactgggta 540 aaacagacac ctggtcgggg cctggaatgg attggagcta tttatcccgg aaatggtgat 600 acttcctaca atcagaagtt caaaggcaag gccacattga ctgcagacaa atcctccagc 660 acagcctaca tgcagctcag cagcctgaca tctgaggact ctgcggtcta ttactgtgca 720 agatcgactt actacggcgg tgactggtac ttcaatgtct ggggcgcagg gaccacggtc 780 accgtttctg cagcggccgc aactacaact ccagctccac gaccaccaac acctgctcca 840 accatcgctt cgcagcctct gtccctgcgc ccagaggcat gccggccagc tgcaggaggt 900 gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960 gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020 aagaaactcc tgtatatattt caaacaacca tttatgagac cagtacaaac tactcaagag 1080 gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140 aagttcagca ggagcgcaga cgcccccgg taccagcagg gccagaacca gctctataac 1200 gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260 cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320 cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380 ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440 gcccttcaca tgcaggccct gccccctcgg taa 1473 <210> 50 <211> 497 <212> PRT <213> Artificial Sequence <220> <223> CAR F231mBBZ <400> 50 With Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe 20 25 30 Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser 35 40 45 Gln Ile Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Ser Pro Lys Ala Leu Ile Tyr Leu Ala Ser Asn Arg His Thr Gly Val 65 70 75 80 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln 100 105 110 His Trp Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 115 120 125 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 145 150 155 160 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 165 170 175 Trp Met Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 180 185 190 Gly Tyr Ile Asn Pro Thr Thr Gly Tyr Thr Glu Tyr Thr Gln Lys Phe 195 200 205 Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Ser Ala Tyr 210 215 220 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Gly Ser Pro Tyr Tyr Asp Ser Thr Tyr Ser Tyr Trp Tyr Phe 245 250 255 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ala Ala 260 265 270 Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr 275 280 285 Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser 290 295 300 Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr 325 330 335 Leu Ile Cys Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln 340 345 350 Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser 355 360 365 Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 370 375 380 Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp 385 390 395 400 Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro 485 490 495 Arg <210> 51 <211> 1494 <212> DNA <213> Artificial Sequence <220> <223> CAR F231mBBZ (Nucleotide) <400> 51 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 accggtgata tcgtgatgac ccagagccag aagttcatga gcacatccgt gggcgacaga 120 gtgagcatca cctgtaaggc cagccagatc gtgaggaccg ccgtggcctg gtaccagcag 180 aaacccggac aaagccccaa ggccctgatc tacctggcca gcaacagaca taccggcgtg 240 cccgatagat tcaccggcag cggcagcggc accgacttta ccctcaccat ctccaacgtg 300 cagtccgagg atctggccga ctacttctgc ctccagcact ggaactaccc ctttaccttc 360 ggcagcggca ccaagctgga gattaaaggc ggcggcggca gcggcggagg cggaagcgga 420 ggaggcggaa gccaagtgca gctccagcag agcggagccg aactggctaa gcccggcgct 480 agcgtgaaga tgagctgcaa ggccagcggc tacaccttca cctcctactg gatgtactgg 540 ctgaagcaga gacctggcca gggcctggag tggatcggct acatcaaccc caccaccggc 600 tacaccgagt acacccagaa gttcaaggat aaggccaccc tgaccgctga caccagcagc 660 aacagcgctt atatgcagct gtccagcctg accagcgagg actccgccgt gtactactgc 720 gctaggggca gccttacta cgactccacc tactcctact ggtacttcga cgtgtggggc 780 gccggcacca cagtgacagt gagctccgcg gccgcaacta ctaccaagcc agtgctgcga 840 actccctcac ctgtgcaccc taccgggaca tctcagcccc agagaccaga agattgtcgg 900 ccccgtggct cagtgaaggg gaccggattg gacttcgcct gtgatattta catctgggca 960 cccttggccg gaatctgcgt ggcccttctg ctgtccttga tcatcactct catctgcaaa 1020 tggatcagga aaaaattccc ccacatattc aagcaaccat ttaagaagac cactggagca 1080 gctcaagagg aagatgcttg tagctgccga tgtccacagg aagaagaagg aggaggagga 1140 ggctatgagc tgagagcaaa attcagcagg agtgcagaga ctgctgccaa cctgcaggac 1200 cccaaccagc tctacaatga gctcaatcta gggcgaagag aggaatatga cgtcttggag 1260 aagaagcggg ctcgcgaccc agagatggga ggcaaacagc agaggaggag gaacccccag 1320 gaaggcgtat acaatgcact gcagaaagac aagatggcag aagcctacag tgagatcggc 1380 acaaaaggcg agaggcggag aggcaagggg cacgatggcc tttaccaggg tctcagcact 1440 gccaccaagg acacctatga tgccctgcat atgcagaccc tggcccctcg ctaa 1494 <210> 52 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> VL-VH connecting peptide <400> 52 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 53 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Connector peptide (VL-VH) (nucleotide) <400> 53 ggcggaggcg gatcaggcgg cggtggatct gggggtggcg gatct 45 <210> 54 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> BBZ <400> 54 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 65 70 75 80 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 85 90 95 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 100 105 110 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 115 120 125 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 130 135 140 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 145 150 155 160 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 165 170 175 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 180 185 190 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 195 200 205 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 210 215 220 <210> 55 <211> 672 <212> DNA <213> Artificial Sequence <220> <223> BBZ (Nucleotide) <400> 55 actacaactc cagctccacg accaccaaca cctgctccaa ccatcgcttc gcagcctctg 60 tccctgcgcc cagaggcatg ccggccagct gcaggaggtg cagtgcacac gagggggctg 120 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 240 aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 300 tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 360 gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 420 gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 480 agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 540 gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 600 taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 660 ccccctcggt aa 672 <210> 56 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> mBBZ <400> 56 Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr 1 5 10 15 Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser 20 25 30 Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 35 40 45 Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr 50 55 60 Leu Ile Cys Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln 65 70 75 80 Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser 85 90 95 Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 100 105 110 Arg Ala Lys Phe Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp 115 120 125 Pro Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 130 135 140 Asp Val Leu Glu Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys 145 150 155 160 Gln Gln Arg Arg Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln 165 170 175 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu 180 185 190 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 195 200 205 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro 210 215 220 Arg 225 <210> 57 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> mBBZ (nucleotide) <400> 57 actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60 ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120 gcctgtgata tttacatctg ggcacccttg gccggaatct gcgtggccct tctgctgtcc 180 ttgatcatca ctctcatctg caaatggatc aggaaaaaat tcccccacat attcaagcaa 240 ccatttaaga agaccactgg agcagctcaa gaggaagatg cttgtagctg ccgatgtcca 300 caggaagaag aaggaggagg aggaggctat gagctgagag caaaattcag caggagtgca 360 gagactgctg ccaacctgca ggaccccaac cagctctaca atgagctcaa tctagggcga 420 agagaggaat atgacgtctt ggagaagaag cgggctcgcg acccagagat gggaggcaaa 480 cagcagagga ggaggaaccc ccaggaaggc gtatacaatg cactgcagaa agacaagatg 540 gcagaagcct acagtgagat cggcacaaaa ggcgagaggc ggagaggcaa ggggcacgat 600 ggcctttacc agggtctcag cactgccacc aaggacacct atgatgccct gcatatgcag 660 accctggccc ctcgctaa 678 <210> 58 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Jα18 (Human) <400> 58 Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe Gly Arg Gly Thr Gln Leu 1 5 10 15 Thr Val Trp Pro Phe 20 <210> 59 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> Vα24Jα18 (Human) <400> 59 Met Lys Lys His Leu Thr Thr Phe Leu Val Ile Leu Trp Leu Tyr Phe 1 5 10 15 Tyr Arg Gly Asn Gly Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu 20 25 30 Ile Ile Leu Glu Gly Lys Asn Cys Thr Leu Gln Cys Asn Tyr Thr Val 35 40 45 Ser Pro Phe Ser Asn Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly 50 55 60 Pro Val Ser Leu Thr Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn 65 70 75 80 Gly Arg Tyr Thr Ala Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu 85 90 95 His Ile Thr Ala Ser Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val 100 105 110 Val Ser Asp Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe Gly Arg Gly 115 120 125 Thr Gln Leu Thr Val Trp Pro 130 135 <210> 60 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Vβ8 (mouse) <400> 60 Glu Ala Ala Val Thr Gln Ser Pro Arg Asn Lys Val Ala Val Thr Gly 1 5 10 15 Gly Lys Val Thr Leu Ser Cys Asn Gln Thr Asn Asn His Asn Asn Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Thr Gly His Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Tyr Gly Ala Gly Ser Thr Glu Lys Gly Asp Ile Pro Asp Gly Tyr 50 55 60 Lys Ala Ser Arg Pro Ser Gln Glu Asn Phe Ser Leu Ile Leu Glu Leu 65 70 75 80 Ala Thr Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Gly Asp Trp 85 90 95 Phe Ser Gln Asn Thr Leu Tyr Phe Gly Ala Gly Thr Arg Leu Ser Val 100 105 110 Leu <210> 61 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> Vβ8 (Human) <400> 61 Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly 1 5 10 15 Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asn Ser Leu 20 25 30 Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr 35 40 45 Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg 50 55 60 Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln 65 70 75 80 Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu 85 90 95 <210> 62 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> 1168scFv amino acid sequence <400> 62 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 130 135 140 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 145 150 155 160 Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly 165 170 175 Met His Trp Val Arg Gln Val Pro Glu Lys Gly Leu Glu Trp Val Ala 180 185 190 Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 195 200 205 Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu 210 215 220 Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 225 230 235 240 Arg Arg Pro Met Gly Gly Ala Gly Gly Ala Met Asp Tyr Trp Gly Gln 245 250 255 Gly Thr Ser Val Thr Val Ser Ser 260 <210> 63 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> 1168scFv nucleic acid sequence <400> 63 atggagaccg acaccctctt attatgggtg ctgttattat gggtccccgg aagcaccgga 60 accggtcaga ttgtgctgac ccagagcccc gctatcatga gcgcctcccc cggcgagaag 120 gtgaccatca gctgctccgc cagcagcagc gtgagctaca tctactggta ccagcagaag 180 cctggaagca gccccaagcc ttggatctat agaacatcca atctggccag cggcgtgccc 240 gccagattta gcggctccgg cagcggcaca tcctactctc tgacaatcag ctccatggag 300 gctgaagatg ccgccgccta ctattgccag cagtaccaca cctaccctct gacatttggc 360 gccggcacca agctggagct caaaggcggc ggaggaagcg gaggaggagg cagcggagga 420 ggcggaagcg atgtgcagct ggtggaatcc ggaggcggac tggtgcaacc cggcggctct 480 aggaagctga gctgcgctgc cagcggcttc accttctcct ccttcggcat gcactgggtg 540 agacaagtgc ccgagaaagg actggagtgg gtggcctaca tctcctccgg cagctccacc 600 atctactacg ccgacaccgt caagggaaga ttcaccatct ctagggacaa ccccaagaac 660 acactgtttc tgcagatgac atctctgaga agcgaggaca cagccatgta ctactgcgcc 720 agaagaccta tgggcggagc tggcggcgcc atggattact ggggacaagg cacctccgtg 780 accgtgtcca gc 792 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 LCDR1 <400> 64 Dear Here Dear Dear Dear Val Dear Tyr Ile Tyr 1 5 10 <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 LCDR2 <400> 65 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SCFV1168 LCDR3 <400> 66 Gln Gln Tyr His Thr Tyr Pro Leu Thr 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 HCDR1 <400> 67 Gly Phe Thr Phe Ser Ser Phe 1 5 <210> 68 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 HCDR2 <400> 68 Ser Ser Gly Ser Ser Thr 1 5 <210> 69 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 HCDR3 <400> 69 Arg Pro Met Gly Gly Ala Gly Gly Ala Met Asp Tyr 1 5 10 <210> 70 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 light chain variable region <400> 70 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Ala Tyr Tyr Cys Gln Gln Tyr His Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 71 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> scFv1168 heavy chain variable region <400> 71 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Val Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Pro Met Gly Gly Ala Gly Gly Ala Met Asp Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 72 <211> 490 <212> PRT <213> Artificial Sequence <220> <223> Full-length amino acid sequence of CAR 1168BBZ <400> 72 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Thr Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Ser Cys Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys Gln Gln Tyr 100 105 110 His Thr Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 130 135 140 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 145 150 155 160 Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly 165 170 175 Met His Trp Val Arg Gln Val Pro Glu Lys Gly Leu Glu Trp Val Ala 180 185 190 Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 195 200 205 Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe Leu 210 215 220 Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala 225 230 235 240 Arg Arg Pro Met Gly Gly Ala Gly Gly Ala Met Asp Tyr Trp Gly Gln 245 250 255 Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 370 375 380 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 385 390 395 400 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425 430 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440 445 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 450 455 460 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 465 470 475 480 Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 73 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> Full-length nucleotide sequence of CAR 1168BBZ <400> 73 atggagaccg acaccctctt attatgggtg ctgttattat gggtccccgg aagcaccgga 60 accggtcaga ttgtgctgac ccagagcccc gctatcatga gcgcctcccc cggcgagaag 120 gtgaccatca gctgctccgc cagcagcagc gtgagctaca tctactggta ccagcagaag 180 cctggaagca gccccaagcc ttggatctat agaacatcca atctggccag cggcgtgccc 240 gccagattta gcggctccgg cagcggcaca tcctactctc tgacaatcag ctccatggag 300 gctgaagatg ccgccgccta ctattgccag cagtaccaca cctaccctct gacatttggc 360 gccggcacca agctggagct caaaggcggc ggaggaagcg gaggaggagg cagcggagga 420 ggcggaagcg atgtgcagct ggtggaatcc ggaggcggac tggtgcaacc cggcggctct 480 aggaagctga gctgcgctgc cagcggcttc accttctcct ccttcggcat gcactgggtg 540 agacaagtgc ccgagaaagg actggagtgg gtggcctaca tctcctccgg cagctccacc 600 atctactacg ccgacaccgt caagggaaga ttcaccatct ctagggacaa ccccaagaac 660 acacctgtttc tgcagatgac atctctgaga agcgaggaca cagccatgta ctactgcgcc 720 agagaccta tgggcggagc tggcggcgcc atgattact ggggacagg cacctccgtg 780 accgtgtcca gcgcggccgc aactacaact ccagctccac gaccaccac acctgctcca 840 accatcgctt cgcagccctct gtccctgcgc ccagaggcat gccggccagc tgcaggaggt 900 gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960 gggacttgtg gggtccttct cctgtcactg gttatcacc tttactgca acggggcaga 1020 aagaaactcc tgtatattat caacaacca tttatgagac cagtacaac tactcagag 1080 gaagatggct gtagctgccg atttccagaa gagagaag gaggatgtga actgagagtg 1140 aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 1200 gagctcaatc taggacgaag agaggagtac gatgttttgg acagagacg tggccgggac 1260 cctgagatgg ggggaagcc gagaggag aaccctcagg aaggctgta caatgaactg 1320 cagaaagata agatggcgga ggcctacagt gagattggga tgaaggcga gcgccggagg 1380 ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440 gcccttcaca tgcaggccct gccccctcgg taa 1473

Claims

1. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises a targeting portion, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the targeting portion specifically binds to the variable region Vβ of a T cell receptor or a fragment thereof, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

72.

2. An isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

3. The isolated nucleic acid molecule according to claim 2, wherein the nucleic acid sequence is shown in SEQ ID NO:

73.

4. A vector comprising the nucleic acid molecule according to any one of claims 2 to 3.

5. The vector according to claim 4, comprising an mRNA vector, a plasmid vector, a retroviral vector and / or a lentiviral vector.

6. An immune effector cell comprising and / or capable of expressing the chimeric antigen receptor of claim 1, the nucleic acid molecule of any one of claims 2-3, or the vector of any one of claims 4-5.

7. The cell according to claim 6, wherein the immune effector cell is selected from T lymphocytes and natural killer cells.

8. The cell according to claim 7, wherein the T lymphocytes and / or natural killer cells are of natural origin or induced by stem cells.

9. A method for preparing immune effector cells, comprising introducing the vector according to any one of claims 4 to 5 into the immune effector cells.

10. A composition comprising the immune effector cell of any one of claims 6-8.

11. Use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of any one of claims 2-3, the vector of any one of claims 4-5, or the immune effector cell of any one of claims 6-8 for preparing a medicament, wherein the medicament is used to treat T-cell lymphoma or leukemia, and the T-cell lymphoma or leukemia is selected from one or more of the following groups: peripheral T-cell lymphoma, angioimmunoblastic lymphoma, extranodal NKT-cell lymphoma, anaplastic T-cell lymphoma, T-lymphoblastic lymphoma, cytotoxic T-cell lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma, T-prolymphocytic leukemia, T-large granular lymphocytic leukemia, hepatosplenic T-cell lymphoma, Sezary syndrome, subcutaneous tonsillar T-cell lymphoma, and unclassified T-cell lymphoma.

12. The use according to claim 11, wherein the peripheral T-cell lymphoma is selected from unspecified peripheral T-cell lymphoma.

13. The use according to claim 11, wherein the anaplastic T-cell lymphoma is selected from ALK-positive anaplastic cell lymphoma and ALK-negative anaplastic cell lymphoma.

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