Bispecific chimeric antigen receptor and application thereof

Abstract

The invention relates to a bispecific chimeric antigen receptor (CAR) and application thereof, the CAR comprises a binding domain, and the binding domain comprises a CD30 binding domain and a CD20 binding domain. The invention further relates to an immune cell for expressing the chimeric antigen receptor and application of the immune cell.

Description

Bispecific chimeric antigen receptors and their applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a bispecific chimeric antigen receptor and its application. Background Art

[0002] CD30, also known as Ki-1 or TNFRSF8, is a member of the tumor necrosis factor (TNF) receptor superfamily. In healthy individuals, CD30 expression is restricted to a small subset of activated T and B lymphocytes. However, CD30 is widely expressed in classical Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL), and is also expressed to varying degrees in some other lymphoproliferative disorders. CD20 (also known as LEU-16, MS4A1) is a transmembrane 4A family protein expressed on the surface of B cells from the pro-B stage to the mature B cell stage and plays a role in B cell development and differentiation. The CD20 antigen is also expressed on a variety of hematological tumors.

[0003] Chimeric antigen receptors (CARs) specifically recognize antigens expressed on the surface of tumor cells to guide CAR-expressing immune cells to eliminate tumors. For example, when cells positive for the antigen targeted by the CAR are present, cells expressing the CAR can recognize and kill these antigen-positive cells. Currently, various CD30- and CD20-targeted therapies have been proposed, but there have been no reports of simultaneously integrating CD20- and CD30-targeting binding domains into the CAR structure to construct a bispecific chimeric antigen receptor targeting both CD20 and CD30.

[0004] Summary of the Invention

[0005] The present application provides a chimeric antigen receptor, which has bispecificity for targeting CD20 and CD30. Bispecificity means that the chimeric antigen receptor can specifically bind to and immune-recognize two different antigens, CD20 and CD30, and the chimeric antigen receptor can generate an immune response by binding to either CD20 or CD30, or can simultaneously bind to CD20 and CD30 to generate an immune response.

[0006] Compared with chimeric antigen receptors targeting a single antigen, the chimeric antigen receptor provided in the present application can target CD20 and CD30. The cell activity of immune cells expressing the chimeric antigen receptor is enhanced, and it has a stronger killing effect. It can effectively kill cells expressing CD20 and / or CD30. The CD20 and CD30 targets have additive or synergistic effects and have a wider therapeutic range. Simultaneously targeting CD20 and CD30 on the cell surface can reduce the possibility of antigen escape caused by downregulation or deletion of a single surface antigen.

[0007] The bispecific chimeric antigen receptor targeting CD20 and CD30 provided in the present application can have one or more effects selected from the following: (1) excellent expression efficiency; (2) cells expressing the chimeric antigen receptor have significant target cell killing effect; (3) cells expressing the chimeric antigen receptor have sustained target cell killing effect; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

[0008] In one aspect, the present application provides a bispecific chimeric antigen receptor (CAR), comprising a binding domain comprising a CD30 binding domain and a CD20 binding domain, wherein:

[0009] 1) the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 5 (amino acid sequence SAS), and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 6;

[0010] 2) the CD20 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 7, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 8, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 9, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 11 (amino acid sequence is ATS), and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 12; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 45, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 46, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 47, and the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: The amino acid sequence of the LCDR2 is shown in SEQ ID NO: 48, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 49 (amino acid sequence is DAS), and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 50.

[0011] In certain embodiments, the CD30 binding domain comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 13 or a functional variant thereof.

[0012] In certain embodiments, the CD30 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

[0013] In certain embodiments, the VH and VL in the CD30 binding domain are directly or indirectly linked.

[0014] In certain embodiments, the CD30 binding domain is a single-chain antibody.

[0015] In certain embodiments, the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises, from N-terminus to C-terminus: a VL domain-linker-VH domain, a VH domain-linker-VL domain, a VL domain-VH domain, or a VH domain-VL domain.

[0016] In certain embodiments, the CD30 binding domain comprises the amino acid sequence shown in SEQ ID NO: 15 or a functional variant thereof.

[0017] In certain embodiments, the CD20 binding domain comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 16 or 32 or a functional variant thereof.

[0018] In certain embodiments, the CD20 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 17 or 33 or a functional variant thereof.

[0019] In certain embodiments, VH and VL in the CD20 binding domain are directly or indirectly linked.

[0020] In certain embodiments, the CD20 binding domain is a single-chain antibody.

[0021] In certain embodiments, the CD20 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises, from N-terminus to C-terminus: a VL domain-linker-VH domain, a VH domain-linker-VL domain, a VL domain-VH domain, or a VH domain-VL domain.

[0022] In certain embodiments, the CD20 binding domain comprises the amino acid sequence shown in SEQ ID NO: 18, 34 or 52, or a functional variant thereof.

[0023] In certain embodiments, the CD30 binding domain is directly or indirectly linked to the CD20 binding domain.

[0024] In certain embodiments, the C-terminus of the CD30 binding domain is directly or indirectly linked to the N-terminus of the CD20 binding domain.

[0025] In certain embodiments, the N-terminus of the CD30 binding domain is directly or indirectly linked to the C-terminus of the CD20 binding domain.

[0026] In certain embodiments, the indirect connection comprises connection through a linker.

[0027] In certain embodiments, the linker comprises the sequence shown in SEQ ID NO: 20.

[0028] In certain embodiments, the linker comprises a cleavage peptide.

[0029] In certain embodiments, the cleavage peptide comprises the sequence shown in SEQ ID NO: 29.

[0030] In certain embodiments, the CAR further comprises a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0031] In certain embodiments, wherein the CAR comprises one or more hinge regions, wherein the hinge region comprises a hinge region derived from CD28, CD8, IgG1 and / or IgG4.

[0032] In certain embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 23 and / or SEQ ID NO: 24 or a functional variant thereof.

[0033] In certain embodiments, wherein the CAR comprises one or more transmembrane domains, the transmembrane domains comprising a transmembrane domain derived from one or more proteins in the following group: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and variants thereof.

[0034] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD28 and / or CD8.

[0035] In certain embodiments, the transmembrane domain of the CAR comprises the amino acid sequence shown in SEQ ID NO: 21 and / or SEQ ID NO: 22 or a functional variant thereof.

[0036] In certain embodiments, the CAR further comprises one or more intracellular costimulatory domains, which are derived from costimulatory domains selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM, and ZAP70.

[0037] In certain embodiments, the intracellular costimulatory domain of the CAR is derived from CD28, OX40 and / or 4-1BB.

[0038] In certain embodiments, the intracellular costimulatory domain of the CAR comprises an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 26 and / or SEQ ID NO: 27 or a functional variant thereof.

[0039] In certain embodiments, the CAR comprises one or more intracellular signaling domains, which are derived from the following intracellular signaling domains: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and a domain containing at least one ITAM.

[0040] In certain embodiments, the intracellular signaling domain of the CAR is derived from CD3ζ.

[0041] In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof.

[0042] In certain embodiments, the CAR further comprises a signal peptide, wherein the signal peptide comprises a signal peptide selected from CD8 and / or CD45.

[0043] In certain embodiments, the CAR further comprises a signal peptide, and the signal peptide comprises a CD8α signal peptide.

[0044] In certain embodiments, the CAR further comprises a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 19 or a functional variant thereof.

[0045] In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a signal peptide, a CD30 binding domain, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain.

[0046] In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a signal peptide, a CD20 binding domain, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain.

[0047] In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a signal peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain.

[0048] In certain embodiments, from N-terminus to C-terminus, the CAR comprises, in sequence, a signal peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain.

[0049] In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NOs: 37-42, SEQ ID NO: 54, and SEQ ID NO: 56.

[0050] In certain embodiments, the CAR comprises a tandem CAR targeting CD20 and a tandem CAR targeting CD30.

[0051] In certain embodiments, the tandem CAR comprises a CD20-targeting CAR and a CD30-targeting CAR that are directly or indirectly linked.

[0052] In certain embodiments, the indirect linkage comprises linkage via a cleavage peptide.

[0053] In certain embodiments, the cleavage peptide comprises the sequence shown in SEQ ID NO: 29.

[0054] On the other hand, the present application provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR described in the present application.

[0055] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0056] In certain embodiments, it is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

[0057] On the other hand, the present application provides an immune effector cell comprising the CAR described in the present application, the nucleic acid molecule described in the present application, or the vector described in the present application.

[0058] In certain embodiments, the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, NKT cells, and macrophages.

[0059] In another aspect, the present application provides a method for preparing immune effector cells, comprising introducing the vector described in the present application into the immune effector cells.

[0060] In certain embodiments, the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, NKT cells, DC cells, and macrophages.

[0061] In another aspect, the present application provides a composition comprising the immune effector cells described herein.

[0062] On the other hand, the present application provides the use of the CAR described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein in the preparation of a medicament for treating CD20 and / or CD30-related disorders.

[0063] On the other hand, the present application provides a method for preventing and / or treating CD20 and / or CD30-related disorders, which comprises administering an effective amount of the CAR described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein.

[0064] On the other hand, the present application provides the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application, which is used to prevent and / or treat CD20 and / or CD30-related disorders.

[0065] In certain embodiments, the CD20 and / or CD30-related disorders include disorders in which CD20-positive and / or CD30-positive cells participate in or are caused by the disorder.

[0066] In certain embodiments, the CD20 and / or CD30-associated disorders include tumors, lymphoproliferative diseases, and infectious mononucleosis.

[0067] In certain embodiments, the CD30-associated disorder comprises a CD30-expressing tumor.

[0068] In certain embodiments, the CD20-associated disorder comprises a tumor expressing CD20.

[0069] In certain embodiments, the CD20 and / or CD30-associated disorder comprises a CD30-expressing and CD20-expressing tumor.

[0070] In certain embodiments, the CD20 and / or CD30-associated disorder comprises lymphoma.

[0071] In certain embodiments, the CD20 and / or CD30-associated disorder comprises a CD20-expressing and CD30-expressing lymphoma.

[0072] In certain embodiments, the CD20 and / or CD30-associated disorder comprises Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0073] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0075] FIG1 shows a schematic diagram of the chimeric antigen receptor molecule described in the present application.

[0076] Figure 2 shows the expression of CD30 and CD20 on the target cells described in this application. Figure 2A: CD30 expression on L-540.LUC and Raji-CD30.LUC cells; Figure 2B: CD20 expression on Raji.LUC and L-540-CD20.LUC cells.

[0077] Figure 3 shows the expression efficiency of the viral vector of the chimeric antigen receptor described in this application after transduction of T cells. Figure 3A shows the CAR expression of T cells transduced by P420, P422, and P037 CAR viral vectors on the sixth day; Figure 3B shows the CAR expression of T cells transduced by P411, P423, P421, P422, and P037 CAR viral vectors on the fifth day; Figure 3C shows the CAR expression of T cells transduced by P421, P424, P425, and P037 CAR viral vectors on the fifth day; Figure 3D shows the CAR expression of T cells transduced by P432, P433, P434, and P435 CAR viral vectors on the sixth day.

[0078] Figure 4 shows the in vitro killing of L540 cells and cytokine secretion by the CD20 / CD30 CAR-T cells described in this application. Figures 4A, 4D, and 4H show the in vitro killing of L540 tumor cells by the CAR-T cells described in this application; Figures 4B, 4E, and 4I show the release of IL-2 detected by enzyme-linked immunosorbent assay after incubation of the CAR-T cells described in this application with L540 cells; Figures 4C, 4F, and 4J show the release of IFN-γ detected by enzyme-linked immunosorbent assay after incubation of the CAR-T cells described in this application with L540 cells.

[0079] Figure 5 shows the in vitro killing and cytokine secretion of L540-CD20 tumor cells by the CD20 / CD30 CAR-T cells described in this application. Figures 5A and 5D show the in vitro killing of L540-CD20 tumor cells by the CAR-T cells described in this application; Figures 5B and 5E show the release of IL-2 detected by enzyme-linked immunosorbent assay after incubation of L540-CD20 cells with the CAR-T cells described in this application; and Figures 5C and 5F show the release of IFN-γ detected by enzyme-linked immunosorbent assay after incubation of L540-CD20 cells with the CAR-T cells described in this application.

[0080] Figure 6 shows the in vitro killing and cytokine secretion of Raji tumor cells by CD20 / CD30 CAR-T cells described in this application. Figures 6A, 6B, 6E, and 6H show the in vitro killing of Raji tumor cells by CAR-T cells described in this application; Figures 6C, 6F, and 1 show the release of IL-2 detected by enzyme-linked immunosorbent assay after incubation of Raji cells with CAR-T cells described in this application; Figures 6D, 6G, and 6J show the release of IFN-γ detected by enzyme-linked immunosorbent assay after incubation of Raji tumor cells with CAR-T cells described in this application.

[0081] Figure 7 shows the in vitro killing and cytokine secretion of Raji-CD30 tumor cells by the CD20 / CD30 CAR-T cells described in this application. Figures 7A and 7D show the in vitro killing of Raji-CD30 tumor cells by the CAR-T cells described in this application; Figures 7B and 7E show the release of IL-2 detected by enzyme-linked immunosorbent assay after incubation of the CAR-T cells described in this application with Raji-CD30 cells; Figures 7C and 7F show the release of IFN-γ detected by enzyme-linked immunosorbent assay after incubation of the CAR-T cells described in this application with Raji-CD30 cells.

[0082] FIG8 shows the CAR-T cell phenotype of the CD20 / CD30 CAR-T cells described in the present application 7 days after viral vector transduction.

[0083] Figure 9 shows the in vivo killing effect of CD20 / CD30 CAR-T cells described herein on immunodeficient tumor-bearing mice subcutaneously inoculated with human L540 tumor cells. Figure 9A: Changes in L540 tumor cell volume; Figure 9B: Changes in mouse body weight; Figure 9C: Mouse survival curve; Figure 9D: CAR-T cell expansion in mice.

[0084] Figure 10 shows the killing effect of CD20 / CD30 CAR-T cells described herein in immunodeficient tumor-bearing mice subcutaneously inoculated with human Raji tumor cells. Figure 10A: Changes in Raji tumor cell volume; Figure 10B: Changes in mouse body weight; Figure 10C: Mouse survival curve; Figure 10D: Changes in tumor size and fluorescence after CAR-T administration.

[0085] Figure 11 shows the cytotoxic effect of the CD20 / CD30 CAR-T cells described herein in immunodeficient tumor-bearing mice subcutaneously inoculated with human Raji tumor cells. Figures 11A and 11E show changes in Raji tumor cell volume; Figures 11B and 11F show changes in mouse body weight; Figures 11C and 11G show mouse survival curves; and Figure 11D shows the expansion of CAR-T cells in mice.

[0086] Figure 12 shows the killing effect of the CD20 / CD30 CAR-T cells described herein on immunodeficient tumor-bearing mice subcutaneously inoculated with human L540-CD20 tumor cells. Figure 12A: Changes in L540-CD20 tumor cell volume; Figure 12B: Changes in mouse body weight; Figure 12C: Mouse survival curve; Figure 12D: In vivo expansion of CAR-T cells in mice; Figure 12E: Changes in tumor size and fluorescence after CAR-T administration.

[0087] Figure 13 shows the in vivo killing effect of CD20 / CD30 CAR-T cells described herein on immunodeficient tumor-bearing mice subcutaneously inoculated with human Raji-CD30 tumor cells. Figure 13A: Changes in Raji-CD30 tumor cell volume; Figure 13B: Changes in mouse body weight; Figure 13C: Mouse survival curve; Figure 13D: CAR-T cell expansion in mice. DETAILED DESCRIPTION

[0088] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0089] Definition of terms

[0090] Throughout this application, the term "CD30" generally refers to the CD30 protein or the gene encoding it. CD30, also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family. As used herein, CD30 encompasses any native CD30 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats), unless otherwise indicated. For example, the complete amino acid sequence of human CD30 has a Genbank accession number of NP_001234, and the complete cDNA sequence encoding human CD30 has a Genbank accession number of NM_001243.

[0091] In this application, the term "CD20" generally refers to the CD20 protein or the gene encoding it. CD20 is also known as transmembrane 4-domain subfamily A member 1 (MS4A1), B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; CD20 in this application includes any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. For example, the amino acid sequence of human CD20 is shown in Uniprot Accession No. P11836. For example, the amino acid sequence of human CD20 can be found under Genbank Accession Nos. NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found under Genbank Accession Nos. NM_152866.2 and NM_021950.3, respectively.

[0092] In this application, the term "chimeric antigen receptor (CAR)" generally refers to an artificial cell receptor that is engineered to express and specifically bind to an antigen on an immune effector cell. CAR can be used as a therapy for adoptive cell transfer. CAR can include an extracellular domain, a transmembrane domain, and an intracellular domain.

[0093] In this application, the term "bispecific" generally refers to a chimeric antigen receptor that can bind to at least two targets and has specificity for two different targets. For example, a bispecific CAR can contain two antigen binding domains, each of which is specific for a different antigenic determinant. For example, the two targets can be located on the same cell surface or on different cell surfaces.

[0094] In the present application, the term "tandem CAR" generally refers to a conjugated form of two or more chimeric antigen receptor molecules. In the present application, tandem CAR may include two or more chimeric antigen receptors with independent antigen binding domains, transmembrane domains, and intracellular domains that are conjugated in a direct or indirect manner. For example, two chimeric antigen receptors may have different antigen binding domains, different transmembrane domains, and different intracellular domains, wherein the N-terminus of one chimeric antigen receptor is conjugated to the C-terminus of another chimeric antigen receptor in a direct or indirect manner. For example, indirect conjugation may include a connection through a linker. For example, the linker may include a polypeptide. In the present application, tandem CAR may include the conjugation of a domain in two or more chimeric antigen receptors. For example, tandem CAR may include the conjugation of two or more antigen binding domains, wherein the antigen binding domains are conjugated directly or indirectly to form a tandem CAR, and the tandem CAR may contain only one transmembrane domain and one intracellular domain.

[0095] As used herein, the term "cleavage peptide" generally refers to a class of polypeptides capable of cleaving a protein. For example, the cleavage peptide can cleave a protein via ribosome skipping rather than proteolysis. For example, the cleavage peptide can be a cleavage 2A peptide, which can include T2A, F2A, P2A, and / or E2A. For example, the cleavage peptide can comprise the amino acid sequence set forth in SEQ ID NO: 29.

[0096] In this application, the term "signal peptide" generally refers to a propeptide present as an N-terminal peptide on a protein precursor. The function of the signal peptide is to promote the translocation of the expressed polypeptide connected to the endoplasmic reticulum and to express the target protein on the cell membrane. The signal peptide can usually be removed during this process. For example, the chimeric antigen receptor of the present application may include a signal peptide. For example, the antigen chimeric receptor of the present application may not have a signal peptide and its biological activity may not be affected. The signal peptide can be heterologous or homologous to the organism used to produce the polypeptide.

[0097] In this application, the term "antigen-binding domain" generally refers to a domain that is capable of binding to a target antigen. An antigen-binding domain may comprise a chimeric antigen receptor or fragment thereof, an antibody or antigen-binding fragment thereof that specifically binds to an antigen. An antigen-binding domain may be of natural, synthetic, semi-synthetic, or recombinant origin. For example, the antigen-binding domain may comprise a single-chain antibody.

[0098] As used herein, the term "CD30 binding domain" generally refers to a domain that specifically binds to CD30. The CD30 binding domain may comprise an anti-CD30 antibody or antigen-binding fragment thereof that specifically binds to CD30. For example, the CD30 binding domain may comprise an anti-CD30 single-chain antibody.

[0099] In this application, the term "CD20 binding domain" generally refers to a domain that can specifically bind to CD30. The CD20 binding domain can comprise an anti-CD20 antibody or antigen-binding fragment thereof that can specifically bind to CD20. For example, the CD20 binding domain can comprise an anti-CD20 single-chain antibody.

[0100] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies. The term "antibody" may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function.

[0101] In this application, the term "CDR" generally refers to the complementary determining region, which is primarily responsible for binding to the antigen epitope. The CDRs of the heavy chain are generally referred to as HCDR1, HCDR2, and HCDR3, and are numbered sequentially starting from the N-terminus. In this application, CDRs can be defined or identified by conventional methods. For example, according to the IMGT numbering method of Lefranc et al. (Lefranc MP, C Pommié, Ruiz M, et al. Developmental & Comparative Immunology, 2003, 27 (1): 55-77.). For example, according to the method of Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132 (2): 211-50, (1970); Borden, P. and Kabat EA, PNAS, 84: 2440-2443 (1987), or by Chothia et al. (Chothia, C. and Lesk, AM, J Mol.Biol., 196(4):901-917(1987) method. For example, in this application, CDRs can be defined by the IMGT numbering method. The boundaries of the CDRs of the variable regions of the same antibody obtained based on different coding systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different coding systems are different. Therefore, when it comes to defining antibodies using the specific CDR sequences defined in the present invention, the scope of the antibodies also covers such antibodies, whose variable region sequences contain the specific CDR sequences, but due to the application of different schemes (such as different coding systems), the CDR boundaries claimed are different from the specific CDR boundaries defined in the present invention. The CDRs of the antibodies of the present invention can be manually evaluated to determine the boundaries according to any scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above ways.

[0102] In the present application, the term "single-chain antibody (scFv)" may be an antibody composed of the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody or comprising an antibody connected by a linker.

[0103] In this application, the term "hinge region" generally refers to the binding domain in a chimeric antigen receptor that plays a role in positioning the binding domain away from the effector cell surface to enable appropriate cell / cell contact, antigen binding, and activation. In this application, the hinge region can be located between the binding domain and the transmembrane domain. The hinge region can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0104] In this application, the term "CD28" generally refers to the T-cell-specific surface glycoprotein CD28. For example, CD28 can be found under the UniProt number P10747. CD28 can include its variants, homologs, and functionally active fragments.

[0105] In this application, the term "CD28 hinge region" generally refers to any polypeptide comprising an amino acid sequence that has sequence identity or similarity to a naturally occurring CD28 hinge region sequence. For example, the CD28 hinge region may comprise the amino acid sequence from positions 114 to 152 of the UniProt number P10747. For example, the CD28 hinge region may comprise a functional variant of the CD28 hinge region sequence.

[0106] In this application, the term "transmembrane domain" can be used interchangeably with "transmembrane region (abbreviated as TM)", which refers to a part of CAR that fuses the extracellular binding portion and the intracellular signal transduction domain and anchors CAR to the plasma membrane of the immune effector cell. The transmembrane region can be obtained from natural proteins or can be synthesized, semi-synthesized or recombinantly obtained. The TM domain may include at least the transmembrane domain of the following proteins: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

[0107] In this application, the term "intracellular domain" is used interchangeably with "intracellular domain" and generally refers to the inner intracellular part of a molecule.

[0108] In the present application, the term "costimulatory domain" generally refers to the intracellular signal transduction domain of a costimulatory molecule. For example, a costimulatory molecule can be a cell surface molecule other than an antigen receptor or an Fc receptor, which can provide a second signal required for the efficient activation and function of T lymphocytes when the antigen is bound. For example, the costimulatory domain can be selected from the following groups: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70.

[0109] In this application, the term "intracellular signaling domain" is generally a part of CAR that participates in transducing information of effective CAR binding antigen polypeptides into the interior of immune effector cells to trigger the functions of effector cells, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or other cellular responses triggered by antigens bound to extracellular CAR domains. In this application, the intracellular signaling domain may include a signaling domain of CD3ζ.

[0110] In this application, the term "CD3ζ" generally refers to CD247 or T-cell surface glycoprotein CD3 zeta chain. CD3ζ can be found under UniProt number P20963. CD3ζ can include variants, homologs, and functionally active fragments thereof.

[0111] In the present application, the term "directly or indirectly linked" refers to direct linkage via a peptide bond, or indirect linkage via a linker or via a non-peptide linkage.

[0112] In this application, the term "linker" generally refers to a portion or group of portions that join or connect two or more discrete, separate monomer domains. When connected together in a multimer, the linker allows the discrete, separate monomer domains to remain separate. The linker portion is generally a substantially linear portion. Suitable linkers include polypeptides, polynucleic acids, peptide nucleic acids, and the like. For example, the linker may include a peptide linker of natural and / or synthetic origin, which may be composed of a linear amino acid chain, wherein the 20 naturally occurring amino acids are monomer building blocks. The amino acid chain may have a length of 1-50 amino acids, for example, between 1 and 28 amino acids, particularly preferably between 3 and 25 amino acids. For example, the linker connecting VH and VL may be a linear amino acid chain. For example, the linker connecting VH and VL may comprise the amino acid sequence shown in SEQ ID NO: 30 or 51. For example, the linker connecting the various components of CAR may be a linear amino acid chain. For example, the linker connecting the various components of CAR may comprise the amino acid sequence shown in SEQ ID NO: 20 or 29.

[0113] As used herein, the term "functional variant" generally refers to an amino acid sequence that has substantially the same function (e.g., can possess the properties of a chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto. In certain embodiments, the amino acid sequence variant is an amino acid sequence that has substantially the same function (e.g., can possess the properties of a chimeric antigen receptor) and comprises one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid substitutions, deletions, or additions thereto.

[0114] In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cell they are introduced into (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides that cannot replicate autonomously, naked DNA polynucleotides, polynucleotides composed of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, some vectors can guide the expression of genes effectively connected to them. This type of vector is referred to as a "recombinant expression vector" (or simply "expression vector") in this application. Generally speaking, the expression vector used in recombinant DNA technology is typically in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably, as plasmid is the most commonly used form of vector.

[0115] In this application, the term "viral vector" generally refers to a non-wild-type recombinant viral particle that acts as a gene delivery vehicle and comprises a recombinant viral genome packaged in a viral capsid. Animal virus species used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAVs), herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (e.g., SV40).

[0116] In this application, the term "immune cell" generally refers to cells that participate in an immune response, such as cells that promote immune effector responses. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0117] In this application, the terms "T lymphocyte" and "T cell" can be used interchangeably and generally refer to thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be helper T (Th) cells, such as helper T1 (Th1) cells or helper T2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells) CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells or CD4-CD8- T cells. Alternatively, the T lymphocytes can include naive T cells and memory T cells.

[0118] In this application, the term "natural killer (NK) cell" generally refers to a subtype of white blood cells that are part of the innate immune system. NK cells play a major role in host rejection of tumors and virally infected cells. NK cells have cytotoxicity and induce apoptosis. NK cells can be used to inhibit viral infections and to clear infections by producing antigen-specific cytotoxic T cells through adaptive immune responses.

[0119] In this application, the term "killing ability" generally refers to killing of cells by contacting the cells with an effective amount of an antibody, immunoconjugate, bispecific / multispecific molecule or composition. The method can include killing cells that express an antigen, optionally in the presence of effector cells, such as by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0120] In this application, the term "cytokine" generally refers to a protein released by one cell population that acts as an intercellular regulator on another cell. For example, cytokines can include interleukins (IL), interferons (IFN), chemokines, tumor necrosis factors (TNF), and transforming growth factors (TGF).

[0121] In this application, the term "CD20-related disorder" generally refers to a disorder associated with CD20 expression. For example, CD20-related disorders include disorders in which cells expressing CD20 are involved or caused. For example, a CD20-related disorder may include a tumor associated with CD20 expression. For example, CD20 expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, wherein any immunohistochemical staining above background levels in a tumor sample indicates that the tumor is a CD20-expressing tumor. Methods for detecting CD20 expression in tumors are known in the art and include immunohistochemical assays. CD20-related disorders may include lymphomas and lymphocytic leukemias. For example, the lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma; b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma and non-Burkitt lymphoma); c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B cell lymphoma and splenic marginal zone lymphoma; d) mantle cell lymphoma (MCL); e) large cell lymphoma (including diffuse large cell B cell lymphoma); f) Hairy cell leukemia; g) Lymphocytic lymphoma, Waldenstrom's macroglobulinemia; h) Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia; i) Plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytoma; j) Hodgkin's disease.

[0122] In this application, the term "CD30-related disorder" generally refers to a disorder associated with CD30 expression. For example, CD30-related disorders include disorders in which cells expressing CD30 are involved or caused. For example, a CD30-related disorder may include a tumor associated with CD30 expression. For example, CD30 expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, wherein any immunohistochemical staining above background levels in a tumor sample indicates that the tumor is a CD30-expressing tumor. Methods for detecting CD30 expression in tumors are known in the art and include immunohistochemical assays. CD30-related disorders may include Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL), angioimmunoblastic T-cell lymphoma (AITL), lymphomatoid papulosis, degenerative atypical histiocytosis, germ cell tumors, plasma cell lymphoma, myeloma, pancreatic and salivary gland tumors.

[0123] As used herein, the term "CD20- and CD30-related disorders" generally refers to disorders associated with both CD20 and CD30 expression. For example, CD20- and CD30-related disorders may include disorders in which cells expressing CD20 and CD30 are involved or contribute to the disorder. For example, CD20- and CD30-related disorders may include disorders in which immune cells expressing CD20 and CD30 are abnormally overproliferated and / or multiplied during the course of the disorder. For example, CD20- and CD30-related disorders may include lymphoma, lymphoproliferative diseases, infectious mononucleosis, and the like.

[0124] As used herein, the term "tumor" generally refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and pancreatic islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, and melanomas.

[0125] In this application, the term "cancer" generally refers to a neoplasm formed when a cell in a local tissue loses normal regulation of its growth at the genetic level under the action of various carcinogenic factors, resulting in its clonal abnormal proliferation. Because this neoplasm is mostly a space-occupying, block-like protrusion, it is also called a neoplasm. In this application, the cancer may include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B cell lymphoma (including low-grade / follicular The present invention relates to a group of cancers that are classified as non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade small non-cleaved cell NHL, AIDS-related lymphoma, Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD). In the present application, the cancer may include cancers or malignancies associated with the expression of CD30.

[0126] In this application, the term "carrier" refers to a diluent, adjuvant, or excipient with which the polypeptides disclosed herein are administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers can be saline solutions, gum arabic, gelatin, starch paste, mica, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants can also be used. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, mica, sodium chloride, dry skim milk, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the compositions of the present invention can also contain a small amount of wetting agents or emulsifiers or pH buffers.

[0127] In the present application, the term "prevention and / or treatment" includes not only preventing and / or treating a disease, but also generally includes preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and generally any pharmacological action that is beneficial to the treated patient.

[0128] In this application, the term "subject" generally refers to an animal, typically a mammal, such as a human, non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects can include fetuses, newborns, infants, adolescents, and adult subjects. Subjects can include animal disease models.

[0129] In this application, the term "therapeutically effective amount" or "effective amount" generally refers to an amount of the antibody of the present application sufficient to prevent or alleviate the symptoms associated with a disease or condition (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0130] In this application, the term "drug" generally refers to a chemical compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient.

[0131] In this application, the term "composition" refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The therapeutic composition should generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersant, liposome, or other ordered structure suitable for high antibody concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount together with one of the ingredients or combinations of ingredients listed above in a suitable solvent, followed by filtration and sterilization as needed.

[0132] As used herein, the term "anti-tumor activity" refers to a decrease in the proliferation rate, viability, or metastatic activity of tumor cells. For example, anti-tumor activity can be demonstrated by a decrease in the growth rate of abnormal cells that develop during treatment, or by stabilization or reduction in tumor size, or by a prolonged survival period resulting from treatment compared to a control without treatment. This activity can be assessed using recognized in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models for studying anti-tumor activity known in the art.

[0133] In this application, the term "adjuvant" generally refers to any substance that assists or modulates the effects of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen.

[0134] In this application, the term "comprises" or "comprising" generally means that the features specifically stated are included, but other elements are not excluded.

[0135] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 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 the specified value.

[0136] Detailed Description of the Invention

[0137] In one aspect, the present application provides a chimeric antigen receptor (CAR), comprising a binding domain comprising a CD30 binding domain and a CD20 binding domain.

[0138] In certain embodiments, the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO:2, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO:4, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO:5 (the amino acid sequence of SEQ ID NO:5 is: SAS), and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:6;

[0139] In certain embodiments, the CD20 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of the HCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO:8, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO:9, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO:10, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO:11 (the amino acid sequence of SEQ ID NO:11 is: ATS), and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO:12; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO:45, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO:46, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: NO: 47, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 48, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 49 (the amino acid sequence of SEQ ID NO: 49 is: DAS), and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 50.

[0140] In the present application, the CD30 binding domain may comprise an anti-CD30 antibody or antigen-binding fragment thereof. For example, the CD30 binding domain may comprise a heavy chain variable region. For example, the heavy chain variable region comprises HCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 1 to 3. For example, the heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 13 or a functional variant thereof. For example, the CD30 binding domain may comprise a light chain variable region. For example, the light chain variable region comprises LCDRs 1 to 3. For example, the light chain variable region comprises LCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 4 to 6. For example, the light chain variable region may comprise the sequence set forth in SEQ ID NO: 14 or a functional variant thereof. For example, the light chain variable region and the heavy chain variable region may comprise a linker. For example, the linker sequence between the light chain variable region and the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 30.

[0141] In the present application, the CD20 binding domain may comprise an anti-CD20 antibody or antigen-binding fragment thereof. For example, the CD30 binding domain may comprise a heavy chain variable region. For example, the heavy chain variable region comprises HCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 7 to 9. For example, the heavy chain variable region comprises HCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 45 to 47. For example, the heavy chain variable region may comprise the sequence set forth in SEQ ID NOs: 16 or 32, or a functional variant thereof. For example, the CD20 binding domain may comprise a light chain variable region. For example, the light chain variable region comprises LCDRs 1 to 3. For example, the light chain variable region comprises LCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 7 to 9. For example, the light chain variable region comprises LCDRs 1 to 3 of the sequences set forth in SEQ ID NOs: 48 to 50. For example, the light chain variable region may comprise the sequence set forth in SEQ ID NOs: 17 or 33, or a functional variant thereof. For example, a linker is included between the light chain variable region and the heavy chain variable region.

[0142] In the present application, the CD30-binding domain can be a single-chain antibody. For example, the CD30-binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises, from the N-terminus to the C-terminus, a VL domain-linker-VH domain, a VH domain-linker-VL domain, a VL domain-VH domain, or a VH domain-VL domain. For example, the CD30-binding domain comprises, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 15, or a functional variant thereof. The functional variant is selected from the group consisting of: a protein or polypeptide having one or more amino acids substituted, deleted, or added in the CD30-binding domain; and a protein or polypeptide having a sequence identity of greater than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) to the CD30-binding domain.

[0143] In the present application, the CD20 binding domain can be a single-chain antibody. For example, the CD20 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises, from the N-terminus to the C-terminus, a VL domain-linker-VH domain, a VH domain-linker-VL domain, a VL domain-VH domain, or a VH domain-VL domain. For example, the CD20 binding domain comprises, but is not limited to, an amino acid sequence as shown in SEQ ID NO: 18, 34, or 52, or a functional variant thereof. The functional variant is selected from the following group: a protein or polypeptide having one or more amino acids substituted, deleted, or added in the CD20 binding domain; and a protein or polypeptide having a sequence homology of 90% or more (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) to the CD20 binding domain.

[0144] In the present application, the CD30 binding domain may be greater than or equal to about 10 5 M -1 (For example, greater than or equal to about 10 5 M - 1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , greater than or equal to about 10 9 M - 1 , greater than or equal to about 10 10 M -1, greater than or equal to about 10 11 M -1 , greater than or equal to about 10 12 M -1 , greater than or equal to about 10 13 M - 1 or greater) of Ka (i.e., the equilibrium association constant for the binding interaction, which is 1 / M); or, less than or equal to about 10 - 5 M (e.g., less than or equal to about 10 -5 M, less than or equal to about 10 -6 M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, less than or equal to about 10 -9 M, less than or equal to about 10 -10 M, less than or equal to about 10 -11 M, less than or equal to about 10 - 12 M, less than or equal to about 10 -13 4 M or less) binds or associates with CD30.

[0145] In the present application, the CD20 binding domain may be greater than or equal to about 10 5 M -1 (For example, greater than or equal to about 10 5 M - 1 , greater than or equal to about 10 6 M -1 , greater than or equal to about 10 7 M -1 , greater than or equal to about 10 8 M -1 , greater than or equal to about 10 9 M - 1 , greater than or equal to about 10 10 M -1 , greater than or equal to about 10 11 M -1 , greater than or equal to about 10 12 M -1 , greater than or equal to about 10 13 M - 1 or greater) of Ka (i.e., the equilibrium association constant for the binding interaction, which is 1 / M); or, less than or equal to about 10 - 5 M (e.g., less than or equal to about 10 -5 M, less than or equal to about 10 -6M, less than or equal to about 10 -7 M, less than or equal to about 10 -8 M, less than or equal to about 10 -9 M, less than or equal to about 10 -10 M, less than or equal to about 10 -11 M, less than or equal to about 10 - 12 M, less than or equal to about 10 -13 The present invention binds or associates with CD20 with an equilibrium dissociation constant, Kd, ​​of 4 M or less.

[0146] In the present application, the affinity of the CD30 binding domain and CD30, or the affinity of the CD20 binding domain and CD20 can be determined using conventional techniques in the art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assay using a labeled ligand, or using a surface plasmon resonance device (such as Biacore T100, which is available from Biacore, Inc., Piscataway, NJ) or optical biosensor technology.

[0147] In the present application, the chimeric antigen receptor comprises the CD30 binding domain and the CD20 binding domain, without being limited to any connection method or connection order.

[0148] For example, the antigen binding domain of the chimeric antigen receptor is composed of the CD30 binding domain and the CD20 binding domain. For example, the CD30 binding domain and the CD20 binding domain in the chimeric antigen receptor can be directly or indirectly connected. For example, the C-terminus of the CD30 binding domain is directly or indirectly connected to the N-terminus of the CD20 binding domain. For example, the N-terminus of the CD30 binding domain is directly or indirectly connected to the C-terminus of the CD20 binding domain.

[0149] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus, a CD30-binding scFv, a linker, and a CD20-binding scFv. For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus, a CD20-binding scFv, a linker, and a CD30-binding scFv. For example, the CD30-binding scFv and the CD20-binding scFv may not contain a linker.

[0150] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VH that binds to CD30 scFv, a linker, a VL that binds to CD30 scFv, a linker, a VH that binds to CD20 scFv, a linker, and a VL that binds to CD20 scFv. For example, one or more linkers may be absent.

[0151] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VH that binds to CD30 scFv, a linker, a VL that binds to CD30 scFv, a linker, a VL that binds to CD20 scFv, a linker, and a VH that binds to CD20 scFv. For example, one or more linkers may be absent.

[0152] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus, a VL that binds to CD30 scFv, a linker, a VH that binds to CD30 scFv, a linker, a VH that binds to CD20 scFv, a linker, and a VL that binds to CD20 scFv. For example, one or more linkers may be absent.

[0153] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VL that binds to CD30 scFv, a linker, a VH that binds to CD30 scFv, a linker, a VL that binds to CD20 scFv, a linker, and a VH that binds to CD20 scFv. For example, one or more linkers may be absent.

[0154] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VH that binds to CD20 scFv, a linker, a VL that binds to CD20 scFv, a linker, a VH that binds to CD30 scFv, a linker, and a VL that binds to CD30 scFv. For example, one or more linkers may be absent.

[0155] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VH that binds to CD20 scFv, a linker, a VL that binds to CD20 scFv, a linker, a VL that binds to CD30 scFv, a linker, and a VH that binds to CD30 scFv. For example, one or more linkers may be absent.

[0156] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus, a VL that binds to CD20 scFv, a linker, a VH that binds to CD20 scFv, a linker, a VH that binds to CD30 scFv, a linker, and a VL that binds to CD30 scFv. For example, one or more linkers may be absent.

[0157] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VL that binds to CD20 scFv, a linker, a VH that binds to CD20 scFv, a linker, a VL that binds to CD30 scFv, a linker, and a VH that binds to CD30 scFv. For example, one or more linkers may be absent.

[0158] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VH that binds to CD30 scFv, a linker, a VL that binds to CD20 scFv, a linker, a VH that binds to CD20 scFv, a linker, and a VL that binds to CD30 scFv. For example, one or more linkers may be absent.

[0159] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: VH that binds to CD20 scFv, a linker, VL that binds to CD30 scFv, a linker, VH that binds to CD30 scFv, a linker, and VL that binds to CD20 scFv.

[0160] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VL that binds to CD30 scFv, a linker, a VH that binds to CD20 scFv, a linker, a VL that binds to CD20 scFv, a linker, and a VH that binds to CD30 scFv. For example, one or more linkers may be absent.

[0161] For example, the antigen-binding domain of the chimeric antigen receptor may comprise, from N-terminus to C-terminus: a VL that binds to CD20 scFv, a linker, a VH that binds to CD30 scFv, a linker, a VL that binds to CD30 scFv, a linker, and a VH that binds to CD20 scFv. For example, one or more linkers may be absent.

[0162] For example, the linker may include a polypeptide. For example, the linker may include a sequence as shown in SEQ ID NO: 20. For example, the linker may include a cleavage peptide. For example, the cleavage peptide may include a sequence as shown in SEQ ID NO: 29.

[0163] In the present application, the CAR may further comprise a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0164] In the present application, the binding domain of the CAR can be connected to the transmembrane domain through a hinge region. For example, the CAR may include one or more hinge regions between the binding domain and the transmembrane domain. For example, the hinge region may include a hinge region derived from CD28, CD8 and / or IgG1. For example, the hinge region may include an amino acid sequence shown in SEQ ID NO: 23 and / or SEQ ID NO: 24 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that has substituted, deleted or added one or more amino acids in the hinge region; and a protein or polypeptide with more than 90% (e.g., at least 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 hinge region.

[0165] In the present application, the CAR may include one or more transmembrane domains. For example, the transmembrane domain may be connected to the C-terminus of the hinge region. In the present application, the transmembrane domain may include a polypeptide selected from the following proteins: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM. For example, the transmembrane domain of the CAR may include a transmembrane domain derived from CD28 and / or CD8. For example, the transmembrane domain of the CAR includes an amino acid sequence as shown in SEQ ID NO: 21 and / or SEQ ID NO: 22 or a functional variant thereof. Wherein the functional variant is selected from the following group: a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the transmembrane domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the transmembrane domain.

[0166] In the present application, the CAR may include one or more intracellular costimulatory domains. For example, the costimulatory domain may be connected to the C-terminal end of the transmembrane domain. In the present application, the costimulatory domain may include a costimulatory domain selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM, and ZAP70. For example, the CAR may include a costimulatory domain. In the present application, the intracellular costimulatory domain may comprise a costimulatory domain derived from CD28, OX40 and / or 4-1BB. For example, the costimulatory domain may comprise an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 26 and / or SEQ ID NO: 27 or a functional variant thereof. Wherein the functional variant is selected from the following group: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the intracellular costimulatory domain; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with the intracellular costimulatory domain.

[0167] In the present application, the CAR may comprise one or more intracellular signaling domains. For example, the intracellular signaling domain may be connected to the C-terminus of the costimulatory domain. In the present application, the signal transduction domain may comprise an intracellular signaling domain selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the CAR comprises an intracellular signaling domain. In the present application, the intracellular signaling domain may comprise a CD3ζ intracellular signaling domain. For example, the intracellular signaling domain may comprise an amino acid sequence as shown in SEQ ID NO: 28 or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the intracellular signaling domain; and a protein or polypeptide having more than 90% (e.g., at least 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 intracellular signaling domain.

[0168] In the present application, the CAR may include a signal peptide. For example, the signal peptide may include a signal peptide selected from CD8 and CD45. For example, the signal may include a CD8α signal peptide. For example, the signal peptide may include an amino acid sequence as shown in SEQ ID NO: 19 or a functional variant thereof. Wherein the functional variant is selected from the following groups: a protein or polypeptide that is substituted, deleted or added with one or more amino acids in the signal peptide; and a protein or polypeptide having more than 90% (e.g., at least 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 signal peptide.

[0169] In the present application, the CAR may include a linker between the domains. For example, a linker is added for proper spacing and conformation between the domains. In the present application, the linker may include the following amino acid sequences: GGG (SEQ ID NO: 57), (GGGGS)n (SEQ ID NO: 58), and GGRRGGGS (SEQ ID NO: 59).

[0170] In the present application, the CAR may comprise a signal peptide, a CD30 binding domain, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain from N-terminus to C-terminus.

[0171] In the present application, the CAR may comprise a signal peptide, a CD20 binding domain, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain from N-terminus to C-terminus.

[0172] In the present application, the CAR comprises a tandem CAR targeting CD20 and a tandem CAR targeting CD30. For example, the tandem CAR may comprise a CAR targeting CD20 and a CAR targeting CD30 that are directly or indirectly connected. For example, the tandem CAR may comprise a CAR targeting CD20 and a CAR targeting CD30 that are connected by a cleavage peptide.

[0173] In the present application, the tandem CAR may comprise a signal peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a signal peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain from N-terminus to C-terminus.

[0174] In the present application, the tandem CAR may comprise a signal peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a signal peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain from N-terminus to C-terminus.

[0175] For example, the hinge region, transmembrane domain, intracellular co-stimulatory domain and / or intracellular signaling domain in the tandem CAR may be the same or different.

[0176] In the present application, the CAR may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 37-42, SEQ ID NO: 54 and SEQ ID NO: 56, or a functional variant thereof. The functional variant is selected from the following groups: a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the CAR; and a protein or polypeptide having a sequence homology of more than 90% (e.g., at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) with CAR.

[0177] In another aspect, the present application provides an isolated nucleic acid molecule that can encode the CAR.

[0178] On the other hand, the present application provides a vector, which may contain the nucleic acid molecule.

[0179] In the present application, the vector may comprise one or more of a replication origin, a selection cassette, a promoter, an enhancer, a translation initiation signal (Shine Dalgarno sequence or Kozak sequence), an intron, a polyadenylation sequence, and 5' and 3' untranslated regions.

[0180] In the present application, the vector can be selected from plasmids, phages, artificial chromosomes (e.g., yeast artificial chromosomes YACs) and animal viruses. For example, the vector can be selected from DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors and retroviral vectors.

[0181] On the other hand, the present application provides a cell comprising the CAR, the nucleic acid molecule and / or the vector.

[0182] In the present application, the cells may comprise immune cells. For example, the immune cells may comprise cells selected from T cells, natural killer (NK) cells, and NKT cells. For example, the immune cells are T cells.

[0183] On the other hand, the present application provides a method for preparing cells, which comprises introducing the CAR, the nucleic acid molecule and / or the vector into the cells.

[0184] In the present application, the cells may comprise immune cells. For example, the immune cells may comprise T cells, natural killer (NK) cells, NKT cells, DC cells and / or macrophages. For example, the immune cells are T cells. In the present application, the method may include a step of obtaining the immune effector cells from a subject. For example, the T lymphocytes may be obtained from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue and a tumor. In addition, the method may further include a step of selecting a specific cell subset from the immune effector cells. For example, a specific T cell subset may be selected by the specific expression of CD3, CD28, CD4, CD8, CD45RA and CD45RO.

[0185] On the other hand, the present application provides a composition, which may comprise the CAR, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0186] In the present application, the pharmaceutically acceptable carrier can be selected from the following groups: excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants and emulsifiers.

[0187] In the present application, the composition may further comprise other active ingredients, such as one or more of cytokines, growth factors, hormones, small molecule chemical active ingredients, prodrugs and antibodies.

[0188] In the present application, the amount of cells in the composition can be an effective amount. The effective amount can be the minimum dose required to achieve a beneficial or desired preventive or therapeutic effect. For example, the effective amount can be affected by factors such as the subject's disease severity, age, weight, and gender.

[0189] On the other hand, the present application provides a chimeric antigen receptor (CAR), a nucleic acid molecule encoding the CAR, a vector comprising a nucleic acid molecule encoding the CAR, and / or a cell expressing the CAR for use in the preparation of a drug, wherein the CAR comprises a binding domain comprising a CD30 binding domain and a CD20 binding domain. In certain embodiments, the drug can be used to treat CD20 and / or CD30 related disorders.

[0190] In certain embodiments, the bispecific chimeric antigen receptor may comprise a CAR described herein.

[0191] On the other hand, the present application provides a method for preventing and / or treating CD20 and / or CD30 related disorders, which may include the use of a bispecific chimeric antigen receptor (CAR), a nucleic acid molecule encoding the CAR, a vector comprising a nucleic acid molecule encoding the CAR, and / or a cell expressing the bispecific CAR, wherein the CAR comprises a binding domain comprising a CD30 binding domain and a CD20 binding domain. In certain embodiments, the method may include administering an effective amount of the CAR described herein.

[0192] On the other hand, the present application provides a bispecific chimeric antigen receptor (CAR), a nucleic acid molecule encoding the CAR, a vector comprising a nucleic acid molecule encoding the CAR, and / or a cell expressing the CAR for preventing and / or treating CD20 and / or CD30-related disorders, wherein the CAR comprises a binding domain, and the binding domain comprises a CD30 binding domain and a CD20 binding domain.

[0193] In certain embodiments, the bispecific chimeric antigen receptor may comprise a CAR described herein.

[0194] In the present application, the CD20 and / or CD30 related conditions may include conditions in which CD20-positive and / or CD30-positive cells are involved or caused. For example, the CD20 and / or CD30 related conditions may include tumors, lymphoproliferative diseases, and infectious mononucleosis. For example, the CD30 related conditions include tumors that express CD30. For example, the CD20 related conditions include tumors that express CD20. For example, the CD20 and / or CD30 related conditions include tumors that express CD30 and / or CD20. For example, wherein the CD20 and / or CD30 related conditions include lymphomas. For example, the CD20 and / or CD30 related conditions include lymphomas that express CD20 and CD30. For example, the CD20 and / or CD30 related conditions include Hodgkin lymphoma or non-Hodgkin lymphoma. For example, such CD20 and / or CD30-associated disorders include Hodgkin lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma, adult T-cell lymphoma (ATL), angioimmunoblastic T-cell lymphoma (AITL), lymphomatoid papulosis, degenerative atypical histiocytosis, germ cell tumors, plasma cell lymphoma, myeloma, pancreatic and salivary gland tumors.

[0195] In the present application, the mode of administration of the drug may include aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. For example, the injection may include intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection, or may be directly injected into a tumor or lymph node.

[0196] The bispecific chimeric antigen receptor targeting CD20 and CD30 provided in the present application can have one or more effects selected from the following: (1) excellent expression efficiency; (2) cells expressing the chimeric antigen receptor have significant target cell killing effect; (3) cells expressing the chimeric antigen receptor have sustained target cell killing effect; (4) cells expressing the chimeric antigen receptor have significant cell activation ability; and (5) cells expressing the chimeric antigen receptor have significant cytokine secretion ability.

[0197] For example, the effect can be relative to a chimeric antigen receptor having only a CD30 binding domain or only a CD20 binding domain and an immune cell expressing the same. For example, the effect can be relative to an immune cell expressing a CAR having a CD30 binding domain and a CAR having a CD20 binding domain in the same cell.

[0198] For example, the expression efficiency may refer to the CAR positive rate of cells expressing the CAR. For example, the expression efficiency may be detected by technical means known in the art. For example, the expression efficiency may be detected by flow cytometry. For example, compared to the expression efficiency of a chimeric antigen receptor having only a CD30 binding domain or only a CD20 binding domain, the expression efficiency of the CAR described in the present application is comparable or higher. For example, compared to the expression efficiency of immune cells expressing a CAR having a CD30 binding domain and a CAR having a CD20 binding domain, respectively, the expression efficiency of the CAR described in the present application is comparable or higher. For example, the expression efficiency of the CAR may be higher than at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

[0199] For example, cells expressing the CAR may have a significant target cell killing effect. For example, the target cell killing effect may include a reduction in target cell proliferation rate, viability, or metastatic activity. For example, immune cells expressing the CAR described herein may have a killing effect on cells expressing CD20, cells expressing CD30, and / or cells expressing CD20 and CD30 simultaneously. For example, the target cell killing effect may be detected by technical means known in the art. For example, the target cell killing effect may be detected by in vitro experiments. For example, the target cell killing effect may be detected by in vivo experiments. For example, the target cell killing effect may refer to cells that do not express CAR. For example, the target cell killing effect may refer to cells before expressing the CAR. For example, compared to immune cells expressing a chimeric antigen receptor having only a CD30 binding domain or only a CD20 binding domain, immune cells expressing the CAR described herein have a stronger target cell killing effect. For example, compared to immune cells expressing CARs with CD30 binding domains and CARs with CD20 binding domains, respectively, immune cells expressing the CARs described herein have a stronger target cell killing effect. For example, immune cells expressing the CARs described herein may have a target cell killing effect greater than at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

[0200] For example, the cells expressing the CAR may have a sustained target cell killing effect. For example, the sustained target cell killing effect may be manifested as the cells expressing the CAR can still be detected after a period of time. For example, the sustained target cell killing effect can be detected by technical means known in the art. For example, the cells expressing the CAR may be detected by technical means known in the art. For example, compared with immune cells expressing a chimeric antigen receptor having only a CD30 binding domain or only a CD20 binding domain, the target cell killing effect of the immune cells expressing the CAR described in the present application lasts longer. For example, compared with immune cells expressing a CAR having a CD30 binding domain and a CAR having a CD20 binding domain, the target cell killing effect of the immune cells expressing the CAR described in the present application lasts longer. For example, the target cell killing effect of the immune cells expressing the CAR may last for at least 1 day, 7 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months, 18 months, 24 months, 30 months, 36 months or longer.

[0201] For example, the cell expressing the CAR may have a significant cytokine secretion capacity. For example, the cytokine secretion capacity may be expressed as a cytokine secretion amount. For example, the cytokine secretion capacity may be detected by technical means known in the art. For example, the measurable activity of activated cells may be detected by technical means known in the art. For example, the cytokine secretion capacity may refer to a cell relative to a cell that does not express CAR. For example, the cytokine secretion capacity may refer to a cell relative to that before expressing the CAR. For example, compared with immune cells expressing a chimeric antigen receptor having only a CD30 binding domain or only a CD20 binding domain, immune cells expressing the CAR described in the present application have a stronger cytokine secretion capacity. For example, compared with immune cells expressing a CAR having a CD30 binding domain and a CAR having a CD20 binding domain, respectively, immune cells expressing the CAR described in the present application have a stronger cytokine secretion capacity. For example, the cytokine secretion capacity of the cells expressing the CAR can be higher than at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300% or more.

[0202] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0203] Example

[0204] Example 1 Target cell construction and target cell CD30 and CD20 expression

[0205] 1) Construction of target cell lines L-540.LUC, L540-CD20.LUC, Raji.LUC, and Raji-CD30.LUC

[0206] HEK293T cells in good growth condition were digested and centrifuged to change the medium. 2×10 7 Each cell was seeded in a 15 cm dish and cultured for 24 hours. A luciferase-expressing plasmid (purchased from NovoPro, Catalog No. V006878) or a luciferase-expressing plasmid with an inserted CD20 or CD30 gene fragment was mixed with a lentiviral packaging kit (Aikangde Biotechnology, Catalog No. LVP2MIX) and transfected into HEK293T cells. The cells were incubated at 37°C, 5% CO2, and the supernatant was collected after 48 hours. Well-grown L-540 (purchased from DSMZ, Catalog No. ACC 72) and Raji (purchased from the Chinese Academy of Sciences Cell Bank, Catalog No. TCHu 44) cells were transduced with the virus and cultured for 3-5 days. Luciferase-expressing single clones were isolated by flow cytometry and cultured for subsequent experiments.

[0207] 2) Target cell CD30 and CD20 expression

[0208] L-540.LUC and Raji-CD30.LUC were centrifuged and the medium was replaced. 1E+05 cells were centrifuged and 100 μl of anti-CD30 APC (BD Biosciences) diluted 1:100 in DPBS was added; incubated at 4°C for 45 min. The cells were washed three times and resuspended in DPBS, and CD30 expression was detected on a flow cytometer. Raji.LUC and L540-CD20.LUC were centrifuged and the medium was replaced. 1E+05 cells were centrifuged and 100 μl of Pacific Blue diluted 1:100 in DPBS was added. TM Anti-human CD20 (Biolegend) was incubated at 4°C for 45 min. Cells were washed three times and resuspended in DPBS. CD20 expression was measured by flow cytometry.

[0209] The results are shown in Figure 2. The present application successfully constructed the L540-CD20.LUC cell line expressing CD20, and the L540-CD20.LUC cell line simultaneously expressed CD20 and CD30; and successfully constructed the Raji-CD30.LUC cell line expressing CD30, and the Raji-CD30.LUC cell line simultaneously expressed CD20 and CD30.

[0210] Example 2 CAR virus preparation and CAR-T cell preparation

[0211] 1) CAR retroviral packaging

[0212] HEK293T cells in good growth condition were digested and centrifuged to change the medium. 2×10 7 The cells were seeded in a 15 cm culture dish and cultured for 24 h. The CAR plasmid (inserted into the vector after full gene synthesis) and the auxiliary plasmid gag-pol plasmid (purchased from NovoPro, product number V006593) and GALV plasmid (purchased from NovoPro, product number V000819) were pre-mixed with polyetherimide (PEI) at a ratio of 4:3:1, and then 0.5 ml of the transfection mixture was added dropwise to HEK293T cells (approximately 4×10 7 Incubate at 37°C, 5% CO2, and collect the supernatant after 24 hours.

[0213] 2) CAR-T cell preparation

[0214] Fresh blood from healthy individuals was collected and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. The separated cells were resuspended in T cell culture medium (X-VIVO 15 culture medium containing 300 IU / mL IL-2) and T Cell TransAct TM (Miltenyi) reagent and placed in a cell culture incubator for activation for 48 h.

[0215] Novonectin protein (Nearshore) was diluted to 20 ng / mL with DPBS and added to an untreated 12-well plate at 1 mL / well for overnight coating. The next day, the coating solution was removed and 2 mL / well of viral supernatant was added. The cells were centrifuged at 2000 g for 120 min at 32°C. T cells activated for 2 days were centrifuged, resuspended in T cell culture medium, and counted, and diluted to 2.5 × 10 5 cells / mL. Add 1mL / well to a 12-well plate and centrifuge at 400g for 10 minutes at 32°C. Place in an incubator and culture overnight. Transfer to a new 12-well plate the next day and change the culture medium every 3 days. On the 6th day, centrifuge 1.2E+05 cells and add 100ul of FITC-Protein L (genscript) diluted 1:100 with DPBS or FITC-Labeled Human CD30 / TNFRSF8 Protein (ACROBiosystems) diluted 1:50 with DPBS. Incubate at 4°C for 45 minutes. Wash the cells 3 times and resuspend them in DPBS. Detect CAR expression on a flow cytometer.

[0216] The results are shown in FIG3 . The present application successfully constructed a series of chimeric antigen receptors comprising CD20 and CD30 binding domains and immune cells expressing the chimeric antigen receptors.

[0217] Example 3 CD20 / CD30 CAR-T to CD20 + and / or CD30 + In vitro cell killing and cytokine secretion

[0218] 1) CD20 / CD30 CAR-T versus CD20 + and / or CD30 + In vitro cell killing assay

[0219] In this example, the luciferase assay was used to analyze the effect of CD20 / CD30 CAR effector cells on CD20 + and / or CD30 + The function of cells, this example illustrates through in vitro experiments that T cells expressing the CAR described in this application have significant anti-tumor activity.

[0220] L540 cells or L540-CD20 cells expressing luciferase were used as target cells. The target cells were diluted to 2×10 5 cells / mL were seeded into 96-well plates at 50 μL / well. CAR-T and blank control cells were counted according to effector-target ratios (E:T) of 2:1, 1:2, and 1:4, and added to the corresponding 96-well plates. After incubation for 16 hours, 50 μL of luciferase substrate (Promega) was added, and the remaining live target cells in each well were quantified by assessing the residual luciferase activity in each well.

[0221] Raj or Raji-CD30 cells expressing luciferase were used as target cells. The target cells were diluted to 2×10 5 cells / mL were inoculated into 96-well plates at 50 μL / well. CAR-T and blank control cells were counted according to effector-target ratios (E:T) of 12:1, 6:1, 2:1, and 1:2, and added to the corresponding 96-well plates. After incubation for 16 hours, 50 μL of luciferase substrate (Promega) was added, and the remaining live target cells in each well were quantified by assessing the residual luciferase activity in each well.

[0222] 2) CD20 / CD30 CAR-T against CD20 + and / or CD30 + Cytokine secretion by cells

[0223] ELISA was used to analyze the expression of CD20 / CD30 CAR-T cells in CD20 + and / or CD30 +The release of IL-2 and IFN-γ under cell stimulation indicates that the CAR-T cells described in this application have significant cytokine secretion capacity.

[0224] CAR-T cells were co-incubated with L540 cells or L540-CD20 cells. The cells were incubated for 16 hours at effector-to-target ratios (E:T) of 2:1, 1:2, and 1:4. The supernatant was then collected by centrifugation and cytokine levels were quantified using the Human IL-2 ELISA Kit II (BD Biosciences) and the Human IFN-γ ELISA Set (BD Biosciences). The absorbance of each well of the test sample at 450 nm was read using a multimode microplate reader (Thermoscientific #Varioskan LUX). The cytokine concentration corresponding to the absorbance of the test sample was calculated based on a standard curve of standard absorbance. For specific procedures, refer to the instructions for the above kits.

[0225] CAR-T cells were co-incubated with Raji cells or Raji-CD30. The cells were incubated for 16 hours at effector-to-target ratios (E:T) of 12:1, 6:1, 2:1, and 1:2. The supernatant was then centrifuged and cytokine levels were quantified using the Human IL-2 ELISA Kit II (BD Biosciences) and the Human IFN-γ ELISA Set (BD Biosciences). The absorbance of each well of the test sample was read at 450 nm using a multimode microplate reader (Thermoscientific #Varioskan LUX). The cytokine concentration corresponding to the absorbance of the test sample was calculated using a standard curve based on the absorbance of the standard. For detailed procedures, refer to the kit instructions.

[0226] The results are shown in FIG4 . + Cells, such as L540 cells in vitro killing effect; in CD30 + Under the stimulation of cells such as L540 cells, T cells expressing the chimeric antigen receptor described in the present application secrete and release cytokines such as IL-2 and IFN-γ.

[0227] The results are shown in FIG6 . T cells expressing the chimeric antigen receptor described in this application showed a strong affinity for CD20. + In vitro killing of cells, such as Raji cells; + Under the stimulation of cells such as Raji cells, T cells expressing the chimeric antigen receptor described in the present application secrete and release cytokines such as IL-2 and IFN-γ.

[0228] The results are shown in Figures 5 and 7. T cells expressing the chimeric antigen receptor described in this application showed CD30 + CD20 + In vitro killing of cells, such as L540-CD20 cells or Raji-CD30 cells; + CD20 + Under the stimulation of cells such as L540-CD20 or Raji-CD30 cells, T cells expressing the chimeric antigen receptor described in the present application secrete and release cytokines such as IL-2 and IFN-γ.

[0229] Example 4 Phenotype of CAR-T cells after 7 days of viral transduction

[0230] This example detects the CD20 / CD30 CAR-T cell phenotype and distinguishes CAR-T cells T T cm, T em, T emra ratio.

[0231] The T cells were gently blown evenly, 1.2E+05 cells were centrifuged, and 100ul of PE anti-human CD45RA Antibody, Pacific Blue were diluted 1:100 with DPBS. TM Anti-human CD197 (CCR7) was incubated with FITC-labeled Human CD30 / TNFRSF8 Protein or FITC-Protein L at 4°C for 45 min. Cells were washed three times with DPBS and resuspended in DPBS. The cells were analyzed by flow cytometry. The results are shown in Figure 8.

[0232] Example 5 In vivo efficacy evaluation of CD20 / CD30 CAR-T cells in L540 tumor xenograft mice

[0233] This example tests the killing / clearing ability of CD20 / CD30 CAR-T cells against mouse xenografted Hodgkin lymphoma in NOG mice. This example demonstrates through in vivo experiments that cells expressing the CAR described in this application have significant anti-tumor activity.

[0234] NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that met the requirements of adaptive observation were selected and injected subcutaneously with 4×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected into the tail vein with 8×10 6CAR-T cells (prepared according to the method of Example 2, the solvent is X-VIVO 15 medium) and non-virus-infected control T cells (the solvent is X-VIVO 15 medium) were cultured. The tumor diameter was measured twice a week with a vernier caliper. The tumor volume was calculated as follows: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0235] Example 6 In vivo efficacy evaluation of CD20 / CD30 CAR-T cells in Raji tumor xenograft mice

[0236] This example tests the killing / clearing ability of CD20 / CD30 CAR-T cells against xenografted human Burkitt's lymphoma in NOG mice. This example demonstrates through in vivo experiments that cells expressing the CAR described in this application have significant anti-tumor activity.

[0237] NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that met the requirements of adaptive observation were selected and injected subcutaneously with 1×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the mice were randomly divided into groups and injected with 5×10 6 CAR-T cells (prepared according to the method of Example 2, the solvent is X-VIVO 15 medium) and non-virus-infected control T cells (the solvent is X-VIVO 15 medium) were cultured. The tumor diameter was measured twice a week with a vernier caliper. The tumor volume was calculated as follows: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0238] Example 7 In vivo efficacy evaluation of CD20 / CD30 CAR-T cells in L540-CD20 tumor xenograft mice

[0239] This example tests the killing / clearing ability of CD20 / CD30 CAR-T cells against mouse xenografted L540-CD20 in NOG mice. This example demonstrates through in vivo experiments that cells expressing the CAR described in this application have significant anti-tumor activity.

[0240] NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that met the requirements of adaptive observation were selected and injected subcutaneously with 5×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected with 5×10 6CAR-T cells (prepared according to the method of Example 1, the solvent is X-VIVO 15 medium) and non-virus-infected control T cells (the solvent is X-VIVO 15 medium) were cultured. The tumor diameter was measured twice a week with a vernier caliper. The tumor volume was calculated as follows: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

[0241] Example 8 In vivo efficacy evaluation of CD20 / CD30 CAR-T cells in Raji-CD30 tumor xenograft mice

[0242] This example tests the killing / clearing ability of CD20 / CD30 CAR-T cells against mouse xenografted Raji-CD30 in NOG mice. This example demonstrates through in vivo experiments that cells expressing the CAR described in this application have significant anti-tumor activity.

[0243] NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) that met the requirements of adaptive observation were selected and injected subcutaneously with 5×10 6 After 5 days of feeding, when the tumor volume was about 50 cubic millimeters, the cells were randomly divided into groups and injected into the tail vein with 5×10 6 CAR-T cells (prepared according to the method of Example 1, the solvent is X-VIVO15 medium) and non-virus-infected control T cells (the solvent is X-VIVO15 medium), the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated as follows: V = 0.5 × a × b 2 , where a and b are the length and width of the tumor, respectively.

Claims

1. A bispecific chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain, the antigen binding domain comprising a CD30 binding domain and a CD20 binding domain, wherein: 1) the CD30 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), the amino acid sequence of the HCDR1 being as shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 being as shown in SEQ ID NO: 2 and the amino acid sequence of the HCDR3 being as shown in SEQ ID NO: 3, the amino acid sequence of the LCDR1 being as shown in SEQ ID NO: 4, the amino acid sequence of the LCDR2 being as shown in SEQ ID NO: 5 (the amino acid sequence is SAS), and the amino acid sequence of the LCDR3 being as shown in SEQ ID NO: 6; 2) the CD20 binding domain comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), a heavy chain complementary determining region 3 (HCDR3), a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), the amino acid sequence of the HCDR1 being as shown in SEQ ID NO:7, the amino acid sequence of the HCDR2 being as shown in SEQ ID NO:8 and the amino acid sequence of the HCDR3 being as shown in SEQ ID NO:9, the amino acid sequence of the LCDR1 being as shown in SEQ ID NO:10, the amino acid sequence of the LCDR2 being as shown in SEQ ID NO:11 (the amino acid sequence is ATS), and the amino acid sequence of the LCDR3 being as shown in SEQ ID NO:12; or the amino acid sequence of the HCDR1 being as shown in SEQ ID NO:45, the amino acid sequence of the HCDR2 being as shown in SEQ ID NO:46 and the amino acid sequence of the HCDR3 being as shown in SEQ ID NO:47, the amino acid sequence of the LCDR1 being as shown in SEQ ID NO: The amino acid sequence of LCDR2 is shown in SEQ ID NO: 48, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 49 (the amino acid sequence is DAS), and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

50.

2. The CAR according to claim 1, wherein the CD30 binding domain comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 13 or a functional variant thereof.

3. The CAR according to any one of claims 1-2, wherein the CD30 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 14 or a functional variant thereof.

4. The CAR according to any one of claims 1-3, wherein VH and VL are directly or indirectly connected in the CD30 binding domain.

5. The CAR of any one of claims 1-4, wherein the CD30 binding domain is a single-chain antibody.

6. The CAR according to any one of claims 1-5, wherein the CD30 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises from N-terminus to C-terminus: VL domain-linker-VH domain, VH domain-linker-VL domain, VL domain-VH domain or VH domain-VL domain.

7. The CAR according to any one of claims 1 to 6, wherein the CD30 binding domain comprises the amino acid sequence shown in SEQ ID NO: 15 or a functional variant thereof.

8. The CAR according to any one of claims 1 to 7, wherein the CD20 binding domain comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 16 or 32 or a functional variant thereof.

9. The CAR according to any one of claims 1 to 8, wherein the CD20 binding domain comprises a light chain variable region VL, and the light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 17 or 33 or a functional variant thereof.

10. The CAR of any one of claims 1-9, wherein VH and VL are directly or indirectly connected in the CD20 binding domain.

11. The CAR of any one of claims 1-10, wherein the CD20 binding domain is a single-chain antibody.

12. The CAR according to any one of claims 1-11, wherein the CD20 binding domain is a single-chain scFv antibody, wherein the single-chain scFv comprises from N-terminus to C-terminus: VL domain-linker-VH domain, VH domain-linker-VL domain, VL domain-VH domain or VH domain-VL domain.

13. The CAR according to any one of claims 1 to 12, wherein the CD20 binding domain comprises the amino acid sequence shown in SEQ ID NO: 18, 34 or 52 or a functional variant thereof.

14. The CAR of any one of claims 1-13, wherein the CD30 binding domain is directly or indirectly connected to the CD20 binding domain.

15. The CAR of any one of claims 1-14, wherein the C-terminus of the CD30 binding domain is directly or indirectly connected to the N-terminus of the CD20 binding domain.

16. The CAR of any one of claims 1-15, wherein the N-terminus of the CD30 binding domain is directly or indirectly connected to the C-terminus of the CD20 binding domain.

17. The CAR of any one of claims 1-16, wherein the indirect connection comprises connection through a linker.

18. The CAR according to claim 17, wherein the linker comprises the sequence shown in SEQ ID NO:

20.

19. The CAR of claim 17, wherein the linker comprises a cleavage peptide.

20. The CAR according to claim 19, wherein the cleavage peptide comprises the sequence shown in SEQ ID NO:

29.

21. The CAR of any one of claims 1-20, wherein the CAR further comprises a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and / or an intracellular signaling domain.

22. The CAR according to claim 21, wherein the CAR comprises one or more hinge regions, wherein the hinge region comprises a hinge region derived from CD28, CD8, IgG1 and / or IgG4.

23. The CAR according to any one of claims 21-22, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO: 23 and / or SEQ ID NO: 24 or a functional variant thereof.

24. A CAR according to any one of claims 21-23, wherein the CAR comprises one or more transmembrane domains, and the transmembrane domain comprises a transmembrane domain derived from one or more proteins in the following group: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

25. The CAR of any one of claims 21-24, wherein the transmembrane domain of the CAR comprises a transmembrane domain derived from CD28 and / or CD8.

26. The CAR according to any one of claims 21-25, wherein the transmembrane domain of the CAR comprises the amino acid sequence shown in SEQ ID NO: 21 and / or SEQ ID NO: 22 or a functional variant thereof.

27. The CAR of any one of claims 21-26, wherein the CAR further comprises one or more intracellular co-stimulatory domains, wherein the co-stimulatory domains are derived from co-stimulatory domains selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKG2C, SLP76, TRIM, and ZAP70.

28. The CAR of any one of claims 21-27, wherein the intracellular co-stimulatory domain of the CAR is derived from CD28, OX40 and / or 4-1BB.

29. The CAR according to any one of claims 21-28, wherein the intracellular co-stimulatory domain of the CAR comprises an amino acid sequence as shown in SEQ ID NO: 25, SEQ ID NO: 26 and / or SEQ ID NO: 27 or a functional variant thereof.

30. The CAR of any one of claims 21-29, wherein the CAR comprises one or more intracellular signaling domains, which are derived from the following intracellular signaling domains: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and a domain comprising at least one ITAM.

31. The CAR of any one of claims 21-30, wherein the intracellular signaling domain of the CAR is derived from CD3ζ.

32. The CAR according to any one of claims 21-31, wherein the intracellular signaling domain of the CAR comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof.

33. The CAR of any one of claims 21-32, wherein the CAR further comprises one or more signal peptides, the signal peptides comprising a signal peptide selected from CD8 and / or CD45.

34. The CAR of any one of claims 21-33, wherein the CAR further comprises one or more signal peptides, wherein the signal peptides comprise a CD8α signal peptide.

35. The CAR according to any one of claims 21-34, wherein the CAR further comprises one or more signal peptides, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 19 or a functional variant thereof.

36. The CAR according to any one of claims 1-35, from N-terminus to C-terminus, the CAR comprises a signal peptide, a CD30 binding domain, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain in sequence.

37. The CAR according to any one of claims 1-36, from N-terminus to C-terminus, the CAR comprises a signal peptide, a CD20 binding domain, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain in sequence.

38. The CAR according to any one of claims 1-37, from N-terminus to C-terminus, the CAR comprises a signal peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain.

39. The CAR according to any one of claims 1-38, from N-terminus to C-terminus, the CAR comprises a signal peptide, a CD20 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, a signaling domain, a cleavage peptide, a CD30 binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a signaling domain.

40. The CAR of any one of claims 1-39, wherein the CAR comprises the amino acid sequence shown in any one of SEQ ID NOs: 37-42.

41. The CAR of any one of claims 1-40, wherein the CAR comprises a tandem CAR targeting CD20 and a tandem CAR targeting CD30.

42. The CAR of claim 41, wherein the tandem CAR comprises a CAR targeting CD20 and a CAR targeting CD30 that are directly or indirectly linked.

43. The CAR of claim 42, wherein the indirect connection comprises connection via a cleavage peptide.

44. The CAR of claim 43, wherein the cleavage peptide comprises the sequence shown in SEQ ID NO:

29.

45. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the CAR of any one of claims 1-44.

46. ​​A vector comprising the nucleic acid molecule of claim 45.

47. The vector of claim 46, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.

48. An immune effector cell comprising the CAR of any one of claims 1-44, the nucleic acid molecule of claim 45, or the vector of any one of claims 46-47.

49. The immune effector cell of claim 48, wherein the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a NKT cell, a DC cell, and a macrophage.

50. A method for preparing immune effector cells, comprising introducing the vector of any one of claims 46-47 into the immune effector cells.

51. The method of claim 50, wherein the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, NKT cells, DC cells, and macrophages.

52. A composition comprising the immune effector cell of any one of claims 48-49.

53. Use of the CAR according to any one of claims 1-44, the nucleic acid molecule according to claim 45, the vector according to any one of claims 46-47 and / or the immune effector cell according to any one of claims 48-49 in the preparation of a medicament for treating CD20 and / or CD30-related diseases.

54. A method for preventing and / or treating CD20 and / or CD30-related disorders, comprising administering an effective amount of the CAR of any one of claims 1-44, the nucleic acid molecule of claim 45, the vector of any one of claims 46-47, and / or the immune effector cell of any one of claims 48-49.

55. The CAR of any one of claims 1-44, the nucleic acid molecule of claim 45, the vector of any one of claims 46-47 and / or the cell of any one of claims 48-49, for preventing and / or treating CD20 and / or CD30 related disorders.

56. The use as claimed in claim 53 or 55 or the method as claimed in claim 54, wherein the CD20 and / or CD30-related disorder comprises a disorder in which CD20-positive and / or CD30-positive cells are involved or caused.

57. The use according to claim 53 or 55 or the method according to claim 54, wherein the CD20 and / or CD30 related disorders comprise tumors, lymphoproliferative diseases, infectious mononucleosis.

58. The use of claim 53 or 55 or the method of claim 54, wherein the CD30-related disorder comprises a tumor expressing CD30.

59. The use of claim 53 or 55 or the method of claim 54, wherein the CD20-related disorder comprises a tumor expressing CD20.

60. The use of claim 53 or 55 or the method of claim 54, wherein the CD20 and / or CD30 related disorder comprises a CD30 expressing and CD20 expressing tumor.

61. The use of claim 53 or 55 or the method of claim 54, wherein the CD20 and / or CD30 related disorder comprises lymphoma.

62. The use of claim 53 or 55 or the method of claim 54, wherein the CD20 and / or CD30 related disorder comprises a CD20-expressing and CD30-expressing lymphoma.

63. The use according to claim 53 or 55 or the method according to claim 54, wherein the CD20 and / or CD30 related disorder comprises Hodgkin's lymphoma or non-Hodgkin's lymphoma.

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