A CD19 CD20 car / truc-t cell and related products and uses thereof

Abstract

The application discloses a CD19CD20CAR / TRuC-T cell and related products and application thereof, and the CD19CD20CAR / TRuC-T cell is a CD19CAR&CD20TRuC-T which simultaneously targets CD19 and CD20, the CD19CAR&CD20TRuC-T has stronger recognition ability to target cells, is favorable to prevent the emergence of antigen escape phenomenon, and has lower self-activation, better anti-depletion capacity, more comprehensive T cell activation signal, higher target point sensitivity and better tumor infiltration capacity, and has wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a CD19 CD20 CAR / TRuC-T cell and a related product and application thereof, and more particularly relates to a CD19 CAR&CD20 TRuC-T cell and a related product and application thereof. BACKGROUND

[0002] Although CD19 CAR-T cell therapy significantly improves the prognosis of patients with relapsed / refractory B-cell non-Hodgkin lymphoma (R / R B-NHL), about 50% of patients relapse after treatment. Studies show that about 30% of relapsed patients are caused by low expression or deletion of tumor cell CD19 antigen (i.e. antigen escape), which leads to treatment failure. This antigen escape mechanism mainly includes CD19 gene mutation, epigenetic modification (such as promoter hypermethylation), and biological processes such as lineage switching.

[0003] In recent years, in response to the antigen escape problem, many clinical studies have focused on introducing a multi-target strategy to enhance the treatment coverage by simultaneously targeting B-cell specific antigens such as CD20, CD22, and BCMA. Among them, CD20 is the most potential combination target point selection due to its wide expression in more than 90% of B-NHL and the synergistic effect with rituximab verified in clinical practice. Clinical trial data shows that CD19 / CD20 dual-target CAR-T therapy exhibits the best objective response rate (ORR) and progression-free survival (PFS) in patients with R / R B-NHL.

[0004] However, the traditional dual-target structure still faces major technical challenges: 1) spatial hindrance of tandem structure: when the expression abundance of the target antigen is insufficient, the spatial distance of the two targets may hinder the recognition of the other target after the binding of a single target, resulting in incomplete T cell activation; 2) signal competition of parallel structure: the double CAR structure designed by the traditional bicistronic vector has the problems of low virus packaging efficiency and competitive consumption of T cell activation signals by intracellular signal domains, which weakens the overall anti-tumor effect. SUMMARY

[0005] Therefore, in order to overcome the above technical problems in the prior art, the purpose of the present application is to provide a CD19 CD20 CAR / TRuC-T cell and a related product and application thereof.

[0006] The TCR fusion construct (TRuC) utilized by the present application is a structure connecting a single-chain antibody with a CD3 complex (CD3 gamma, delta, epsilon), recognizing a specific target through the single-chain antibody, and killing the target cell by utilizing the activation signal of the natural T cell evoked by the TCR-CD3 complex. On this basis, the present application designs a class of CD19 CD20 CAR / TRuC-T cells, and the modified CAR-T (CD19 CAR & CD20 TRuC-T) can improve the double-target T cell therapy in the following aspects, including: lower self-activation, better anti-exhaustion ability, more comprehensive T cell activation signal, higher target sensitivity and better tumor infiltration ability.

[0007] The present application realizes the above-mentioned application purposes by adopting the following technical solutions:

[0008] The first aspect of the present application provides a fusion protein targeting CD19 and CD20.

[0009] Further, the fusion protein comprises a chimeric antigen receptor targeting CD19, a TCR fusion construct targeting CD20;

[0010] The chimeric antigen receptor targeting CD19 comprises an antibody targeting CD19;

[0011] The TCR fusion construct targeting CD20 comprises a single-domain antibody targeting CD20;

[0012] The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are respectively shown as SEQ ID NO:1-3;

[0013] The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody targeting CD19 are respectively shown as SEQ ID NO:5, HTS and SEQ ID NO:6.

[0014] Further, the chimeric antigen receptor targeting CD19 is in front, and the TCR fusion construct targeting CD20 is behind;

[0015] Alternatively, the fusion protein is obtained in series by the chimeric antigen receptor targeting CD19, T2A and the TCR fusion construct targeting CD20;

[0016] Alternatively, the chimeric antigen receptor targeting CD19 further comprises a hinge region, a transmembrane region, a costimulatory signal domain and an intracellular signaling domain;

[0017] Optionally, the hinge region is selected from the hinge region of CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3 epsilon, IL-2 receptor, IL-7 receptor, or IL-11 receptor;

[0018] Optionally, the transmembrane region is selected from the transmembrane region of CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3 epsilon, IL-2 receptor, IL-7 receptor, or IL-11 receptor;

[0019] Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domain of 41BB, CD27, CD19, CD4, CD28, CD278, CD8 alpha, CD8 beta, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, or B7H3;

[0020] Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, TCR zeta, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fc epsilon RI, DAP10, DAP12, or CD66d;

[0021] Optionally, the hinge region is a CD8 hinge region;

[0022] Optionally, the transmembrane region is a CD8 transmembrane region;

[0023] Optionally, the co-stimulatory signaling domain is a 41BB co-stimulatory signaling domain;

[0024] Optionally, the intracellular signaling domain is a CD3 zeta intracellular signaling domain;

[0025] Optionally, the CD19-targeting chimeric antigen receptor is obtained by concatenating, in sequence, an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signaling domain, and a CD3 zeta intracellular signaling domain;

[0026] Optionally, the amino acid sequences of the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, and the CD3 zeta intracellular signaling domain are shown in SEQ ID NOs: 15-18, respectively;

[0027] Optionally, the CD20-targeting TCR fusion construct further comprises a Linker, a TCR complex subunit fusion moiety;

[0028] Optionally, the Linker is selected from G4S, (G4S)2, (G4S)3 or EAAAK.

[0029] Optionally, the TCR complex subunit fusion moiety is selected from CD3ε, CD3γ or CD3δ.

[0030] Optionally, the Linker is G4S.

[0031] Optionally, the TCR complex subunit fusion moiety is CD3ε.

[0032] Optionally, the TCR fusion construct targeting CD20 is obtained by connecting in series a single-domain antibody targeting CD20, G4S, CD3ε in order.

[0033] Optionally, the amino acid sequence of the CD3ε is shown in SEQ ID NO: 19.

[0034] Optionally, the fusion protein is obtained by connecting in series an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signal domain, a CD3ζ intracellular signaling domain, T2A, a single-domain antibody targeting CD20, G4S, CD3ε in order.

[0035] Optionally, the N-terminus of the fusion protein further comprises a membrane exit signal peptide.

[0036] Optionally, the membrane exit signal peptide is selected from the membrane exit signal peptide CD8a-SP or the membrane exit signal peptide GMCSF-SP.

[0037] Optionally, the amino acid sequences of the membrane exit signal peptide CD8a-SP and the membrane exit signal peptide GMCSF-SP are shown in SEQ ID NOs: 22-23, respectively.

[0038] In the present application, the CD3z is CD3ζ and the CD3e is CD3ε.

[0039] In some embodiments, the Linker is a linker commonly used in the art, which is not limited to G4S used in the embodiments of the present application, but can be any one of (GGGGS)n, (GGGS)n, (SSSSG)n, (GSGSA)n, (GGSGG)n or other linkers, wherein n can be any integer between 1 and 10.

[0040] In a specific embodiment of the present application, the present application experimentally verifies for the first time that in the fusion protein, the connection order of the chimeric antigen receptor targeting CD19 in front and the TCR fusion construct targeting CD20 behind has a greater impact on the effect of the fusion protein modified T cells, and the treatment effect of the fusion protein modified T cells (CD19 CAR & CD20 TRuC-T) prepared based on the connection order of the chimeric antigen receptor targeting CD19 in front and the TCR fusion construct targeting CD20 behind is significantly better, that is, the fusion protein of the first aspect of the present application achieves an unexpected technical effect.

[0041] The second aspect of the present application provides a nucleic acid molecule.

[0042] Further, the nucleic acid molecule encodes the fusion protein of the first aspect of the present application;

[0043] Optionally, the nucleotide sequence of the antibody targeting CD19 in the fusion protein is as shown in SEQ ID NO: 13;

[0044] Optionally, the nucleotide sequence of the single-domain antibody targeting CD20 in the fusion protein is as shown in SEQ ID NO: 14;

[0045] Optionally, the nucleotide sequences of the CD8 hinge region, CD8 transmembrane region, 41BB costimulatory signal domain, CD3 zeta intracellular signaling domain and CD3 epsilon in the fusion protein are as shown in SEQ ID NO: 24-28, respectively.

[0046] In some embodiments, the nucleic acid molecule can comprise natural, unnatural or altered nucleotides; and it can comprise natural, unnatural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of the phosphodiester present between the nucleotides of unmodified oligonucleotides.

[0047] In some embodiments, the nucleic acid does not comprise any insertion, deletion, inversion and / or substitution. However, in some cases, it can be appropriate for the nucleic acid to comprise one or more insertions, deletions, inversions and / or substitutions, and therefore, the nucleic acid formed by the insertions, deletions, inversions and / or substitutions based on the nucleic acid molecule provided by the present application is also included in the protection scope of the present application.

[0048] The third aspect of the present application provides an expression vector.

[0049] Further, the expression vector comprises the nucleic acid molecule of the second aspect of the present application;

[0050] Optionally, the expression vector is a DNA vector or an RNA vector;

[0051] Optionally, the DNA vector is a plasmid.

[0052] Optionally, the RNA vector is a viral-derived vector.

[0053] Optionally, the viral-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxviral vector, or a herpesviral vector.

[0054] In some embodiments, examples of vectors that can be used in the present application include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, viral-derived vectors. Various vectors known in the art can be used, for example, commercially available vectors can be used, and then a nucleotide sequence encoding the fusion protein of the first aspect of the present application is operably linked to an expression control sequence to form an expression vector.

[0055] In some embodiments, the viral-derived vector includes, but is not limited to, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector, a baculoviral vector, a papillomaviral vector, a papovaviral vector.

[0056] The fourth aspect of the present application provides a genetically engineered host cell.

[0057] Further, the genetically engineered host cell comprises the nucleic acid molecule of the second aspect of the present application or the expression vector of the third aspect of the present application.

[0058] Optionally, the host cell is a mammalian cell.

[0059] Optionally, the host cell is an immune cell.

[0060] Optionally, the immune cell is a T cell, a B cell, an NK cell, an iNKT cell, a gd T cell, an NK92 cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, a neutrophil, or any combination thereof.

[0061] Optionally, the immune cell is a T cell.

[0062] Optionally, the genetically engineered host cell is a T cell modified by the fusion protein of the first aspect of the present application.

[0063] In some embodiments, the genetically engineered host cell is an autologous cell or an allogeneic cell.

[0064] In some embodiments, the genetically engineered immune cell is obtained from a subject having a CD19 and / or CD20 related disease. In some embodiments, the genetically engineered immune cell is obtained from a healthy donor.

[0065] A fifth aspect of the present application provides a derivative.

[0066] Further, the derivative is selected from the group consisting of:

[0067] (1) the fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application or the genetically engineered host cell of the third aspect of the present application, which contains a detectable label;

[0068] (2) the fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application or the genetically engineered host cell of the third aspect of the present application, which confers antibiotic resistance; or

[0069] (3) the fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application or the genetically engineered host cell of the third aspect of the present application, which is conjugated or coupled to a therapeutic agent;

[0070] Optionally, the detectable label is selected from the group consisting of a fluorescent dye, colloidal gold, a chemiluminescent label or a chemiluminescent catalyst.

[0071] Optionally, the antibiotic resistance gene is selected from the group consisting of a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene or a kanamycin resistance gene.

[0072] Optionally, the therapeutic agent is selected from the group consisting of a radionuclide, a cytokine, a gold nanoparticle, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme or a chemotherapeutic agent.

[0073] A sixth aspect of the present application provides a pharmaceutical composition or a biological agent.

[0074] Further, the pharmaceutical composition comprises the fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the genetically engineered host cell of the fourth aspect of the present application and / or the derivative of the fifth aspect of the present application.

[0075] Optionally, the biological agent comprises the pharmaceutical composition.

[0076] Optionally, the dosage form of the biological agent is selected from the group consisting of a lyophilized powder injection, a liquid suspension, a liposome preparation, a microsphere preparation or a gel preparation.

[0077] In some embodiments, the pharmaceutical composition or biologic can further comprise a pharmaceutically acceptable carrier and / or adjuvant, which are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995), which are used as necessary to aid stability of the formulation or to enhance bioavailability of the active or active substance. In some embodiments, the use of the pharmaceutical composition or biologic means that a safe and effective amount of the pharmaceutical composition or biologic of the present application as described above is administered to a human.

[0078] In some embodiments, suitable administration forms of the pharmaceutical composition or biologic include forms suitable for parenteral administration, e.g. by injection or infusion, e.g. by rapid injection or continuous infusion, intravenously, inhalable or subcutaneously. In the case of products for injection or infusion, they can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and they can contain formulation agents such as suspending, preserving, stabilizing and / or dispersing agents.

[0079] In some embodiments, the pharmaceutical composition or biologic can be prepared in various dosage forms as necessary and can be administered by a physician in a dose that is beneficial to the patient according to the patient's species, age, weight and general disease condition, administration method, etc. A skilled physician can usually easily determine the prescription and the administration dose and administration method of the prescription that are effective for the desired treatment and / or prevention.

[0080] A seventh aspect of the present application provides a kit.

[0081] Further, the kit comprises the fusion protein according to the first aspect of the present application, the nucleic acid molecule according to the second aspect of the present application, the expression vector according to the third aspect of the present application, the genetically engineered host cell according to the fourth aspect of the present application and / or the derivative according to the fifth aspect of the present application.

[0082] An eighth aspect of the present application provides any one of the following methods:

[0083] (1) A method of preparing the genetically engineered host cell according to the fourth aspect of the present application, the method comprising the step of introducing the nucleic acid molecule according to the second aspect of the present application or the expression vector according to the third aspect of the present application into a host cell;

[0084] Optionally, the introduction is by a method selected from the group consisting of transfection, microinjection, electroporation, a DNA vector, a retroviral vector, a lentiviral vector, a poxviral vector, a herpes simplex viral vector, an adenoviral vector or an adeno-associated viral vector;

[0085] (2) A method for inhibiting the activity of CD19 and / or CD20 protein for non-therapeutic purposes in vitro, the method comprising the step of contacting the genetically engineered host cell of the fourth aspect of the present application, the derivative of the fifth aspect of the present application, the pharmaceutical composition or biological preparation of the sixth aspect of the present application with cells of an organism.

[0086] In addition, the present application also provides a method for treating and / or preventing CD19 and / or CD20 related diseases, the method comprising administering to a subject in need thereof an effective amount of the genetically engineered host cell of the fourth aspect of the present application, the derivative of the fifth aspect of the present application and / or the pharmaceutical composition or biological preparation of the sixth aspect of the present application.

[0087] In specific embodiments of the present application, the administration mode of the fusion protein modified T cells is infusion. It should be noted that the scope of protection of the present application is not limited by the specific administration mode and administration dose, as long as the fusion protein modified T cells, the pharmaceutical composition or the biological preparation of the present application produce the expected therapeutic and / or preventive effect in the subject, which all fall within the scope of protection of the present application.

[0088] The ninth aspect of the present application provides the use of any one of the following:

[0089] (1) The fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the genetically engineered host cell of the fourth aspect of the present application, the derivative of the fifth aspect of the present application and / or the kit of the seventh aspect of the present application for the preparation of a medicament for treating and / or preventing CD19 and / or CD20 related diseases;

[0090] (2) The fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the genetically engineered host cell of the fourth aspect of the present application, the derivative of the fifth aspect of the present application and / or the kit of the seventh aspect of the present application for the preparation of a biological preparation for treating and / or preventing CD19 and / or CD20 related diseases;

[0091] (3) The fusion protein of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the genetically engineered host cell of the fourth aspect of the present application and / or the derivative of the fifth aspect of the present application for the preparation of a kit for preparing fusion protein modified immune cells for treating and / or preventing CD19 and / or CD20 related diseases;

[0092] (4) The use of the kit of the seventh aspect of the present application in the preparation of fusion protein modified immune cells for treating and / or preventing CD19 and / or CD20 related diseases;

[0093] (5) The use of the fusion protein of the first aspect of the present application in the preparation of fusion protein modified immune cells for treating and / or preventing CD19 and / or CD20 related diseases;

[0094] Optionally, the CD19 and / or CD20 related disease is non-Hodgkin's lymphoma, chronic myelogenous leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, megakaryocytic leukemia, Burkitt's lymphoma, anaplastic large cell lymphoma, mucosa-associated lymphoid tissue lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma or follicular lymphoma.

[0095] In the present application, the CD19 and / or CD20 related disease is not limited to the specific diseases listed in the present application, and any disease related to CD19 and / or CD20 expression is within the scope of protection of the present application.

[0096] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0097] (1) The present application provides a brand new CD19 CAR&CD20 TRuC-T that simultaneously targets CD19 and CD20 in the art, and the CD19 CAR&CD20 TRuC-T has been confirmed to have a significant killing effect on tumors expressing CD19 and / or CD20 through in vivo and in vitro experiments.

[0098] (2) Compared with ordinary single-target CAR, the dual-target CD19 CAR&CD20 TRuC-T provided by the present application has stronger recognition ability for target cells, which is beneficial to prevent the occurrence of antigen escape phenomenon, and has lower self-activation, better anti-exhaustion ability, more comprehensive T cell activation signal, higher target sensitivity and better tumor infiltration ability, and has broad clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 The CD19 CAR / CD20 TRuC-T provided by the present application corresponds to the structural design schematic diagram;

[0100] Figure 2: The structure diagram corresponding to CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T and T cells;

[0101] Figure 3 : The comparison result diagram of the killing situation of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T and T cells to Raji and Daudi cell lines which express CD19 and CD20;

[0102] Figure 4 : The comparison result diagram of the killing situation of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T and T cells to K562 cell lines which only overexpress CD19 or CD20, Raji cell lines with CD19 or CD20 knockout;

[0103] Figure 5 : The comparison result diagram of the response rate of each group to CD20 or CD19 by activation detection of CD19 CAR-T, CD20 CAR-T, CD19 CAR & CD20 CAR-T, CD19-CD20 CAR-T, CD19 CAR & CD20 TRuC-T, CD20 CAR & CD19 TRuC-T and T cells;

[0104] Figure 6 : The result diagram corresponding to the evaluation of the inhibitory effect of CAR-T cells using CD19 CAR-T (CAR19), CD19 CAR & CD20 CAR-T (DUAL), CD19-CD20 CAR-T (Tandem CAR 20 & 19) and CD19 CAR & CD20 TRuC-T (CAR19 & TRuC20) structures on mouse lymphoma cells. DETAILED DESCRIPTION

[0105] The present application is further described in conjunction with the specific embodiments thereof, which are intended to be illustrative only and not limiting of the present application. Those skilled in the art will understand that modifications, variations, substitutions and changes in the embodiments can be made without departing from the spirit and scope of the present application, which is defined solely by the claims and their equivalents.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In order to facilitate the understanding of this application, terms related to the present application are explained here:

[0107] As used herein, the terms "comprise" or "comprising" means including, but not limited to, any one or more of the stated elements or integers.

[0108] As used herein, the term "Chimeric Antigen Receptor (CAR)" is an artificially synthesized receptor that combines a single-chain antibody fragment that recognizes a specific antigen with a signal domain that activates the function of immune cells, enabling immune cells to specifically recognize and kill target cells expressing the corresponding antigen. The structural composition includes an extracellular antigen binding region, a transmembrane region, and an intracellular signal transduction region.

[0109] CAR-modified immune cells specifically recognize antigens on the surface of target cells through the extracellular antigen binding region, and this recognition is independent of the major histocompatibility complex (MHC), so it can bypass MHC restriction and directly recognize target cells. When CAR binds to the target antigen, the intracellular signal transduction region is activated, initiating the activation signal transduction pathway of immune cells, allowing immune cells to proliferate, secrete cytokines, and exert cytotoxic effects, killing target cells.

[0110] As used herein, the term "TCR fusion construct (TRuC)" is a new type of antigen receptor design aimed at reprogramming T cells to specifically recognize and kill tumor cells. TRuC is composed of a specific ligand antibody fused to a T cell receptor (TCR) subunit. It usually contains a single-domain antibody or other antibody fragment that can specifically recognize tumor surface antigens, and is fused to the CD3 epsilon subunit of TCR through a flexible linker peptide.

[0111] The TRuC construct is cloned into a lentivirus or the like vector, and after transduction into T cells, the expressed fusion protein is integrated into the endogenous TCR complex, replacing the native CD3 epsilon subunit. When the TRuC-T cells recognize the specific antigen on the surface of the tumor cells, they can activate the T cells in a human leukocyte antigen (HLA)-independent manner, starting the killing function of the T cells, and specifically killing the tumor cells. Due to the use of the entire TCR signaling ability, the TRuC-T cells can trigger more effective signal transduction in the process of tumor cell recognition and killing, and promote the transportation, long-term persistence function and anti-tumor activity of T cells.

[0112] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. Nucleic acid molecules are "operably linked" when they are functionally connected. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0113] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) into which a recombinant polynucleotide is incorporated.

[0114] In some embodiments, the expression vector according to the present application is capable of directing the replication and expression of the nucleic acid molecule of the present application in a host, and thus ensures the expression of the fusion protein of the present application as provided previously in a selected host. The expression vector may, for example, be a cloning vector, a binary vector, or an integrating vector. Expression includes transcription of the nucleic acid molecule, for example into translatable mRNA.

[0115] In some embodiments, non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen), or pCR TOPO (Invitrogen), lambda gtll, pJOE, pBBRl-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACTl, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-l, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORTl (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for use in Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the versatile expression vector pCS2+.

[0116] In some embodiments, the vector can contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes. Additionally, the coding sequences contained in the vector can be linked to transcriptional regulatory elements and / or to other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure initiation of transcription, internal ribosome entry sites (IRES), and, optionally, regulatory elements that ensure termination of transcription and stability of the transcript. Non-limiting examples of such regulatory elements that ensure initiation of transcription include promoters, translation initiation codons, enhancers, insulators, and / or regulatory elements that ensure termination of transcription, which are included downstream of the nucleic acid molecules of the application. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding a secretion signal, or signal sequences depending on the expression system used, which are capable of directing the expressed protein to a cellular compartment or to the culture medium. The vector can also contain additional expressible polynucleotides encoding one or more chaperones to facilitate proper protein folding.

[0117] As used herein, the term "host cell" refers to a cell that can be used for the introduction of an expression vector, including but not limited to: a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, an animal cell such as fibroblast cell, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK293 cell, or human cell.

[0118] In a specific embodiment of the application, the host cell is preferably an immune cell.

[0119] In some embodiments, the immune cell includes but is not limited to: a T cell, a B cell, an NK cell, an iNKT cell, a CTL cell, a dendritic cell, a myeloid cell, a monocyte, a macrophage, or any combination thereof, preferably a T cell. Furthermore, the "host cell" of the application can include a single cell or a population of cells, i.e., the "genetically modified host cell" of the application described above includes a single genetically modified host cell and a population of genetically modified host cells.

[0120] As used herein, the term "treatment" refers to complete or partial amelioration or lessening of a disease or condition or disorder, or a symptom, adverse effect or consequence, or a phenotype associated therewith. Desired therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, ameliorating or palliating the disease state, and relieving or ameliorating prognosis. The term does not imply complete cure or complete elimination of any symptoms or effects on all symptoms or consequences.

[0121] As used herein, the term "prevention" includes providing a prophylactic effect in a subject with respect to occurrence or recurrence of a disease, which individual can be susceptible to the disease but has not yet been diagnosed with the disease.

[0122] As used herein, the term "effective amount" includes "therapeutically effective amount", "prophylactically effective amount". Wherein, "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest a disease and its complications in an individual already suffering from the disease. The therapeutically effective amount can vary according to factors such as the severity of the disease to be treated, the overall state of the patient's immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other treatments used concurrently, etc. "Prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the onset of a disease.

[0123] In some embodiments, the dose and frequency (single or multiple doses) of the pharmaceutical composition or biologic administered to a subject can vary according to a variety of factors, such as, for example, whether the mammal is suffering from another disease and its route of administration; the age, sex, health, weight, body mass index, and diet of the subject; the nature and extent of the symptoms of the disease being treated (e.g., cancer symptoms and severity of such symptoms), the kind of concurrent treatment, complications or other health-related problems arising from the disease being treated, etc. Other therapeutic regimens or agents can be used in conjunction with the pharmaceutical composition or biologic and methods of treatment described herein. Adjustments and manipulations of established dosages (e.g., frequency and duration) are well within the capabilities of those skilled in the art.

[0124] In some embodiments, the pharmaceutical composition or biologic can have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. In addition, it can be administered once or multiple times. At this time, the pharmaceutical composition or biologic is administered in the form of a liquid formulation, a powder, an aerosol, a capsule, a vaginal tablet, a capsule, or a suppository. The administration route can include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, etc. When administered orally, it can be formulated to protect the active ingredients in the pharmaceutical composition or biologic from degradation in the stomach. In addition, the active ingredients can be administered by any device capable of transferring to the target cells. In specific embodiments, the pharmaceutical composition or biologic provided by the present application can be prepared into various dosage forms as needed, and the dosage that is beneficial to the patient can be administered by a clinician according to the species, age, weight, and general disease condition of the subject patient, administration method, etc. The administration method can be, for example, injection or any suitable administration method known to those skilled in the art.

[0125] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. In certain embodiments, the "subject" is preferably a human.

[0126] The reagents and materials used in the present application are readily available to one of ordinary skill in the art, and, unless otherwise stated, are available from commercial sources. The experimental methods of the present application, for which specific conditions are not specified, are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are merely illustrative of the present application and should not be construed as limiting the scope of the application as described in the claims. It should be noted that the experimental conditions and results described in the following examples are merely illustrative of the present application and should not and will not limit the present application as described in detail in the claims.

[0127] Example 1 Construction of a new type of CD19 CD20 CAR / TRuC-T cell and verification of its killing effect

[0128] 1. Experimental methods

[0129] According to the structural design diagram in Figure 1 , the corresponding different types of CAR-T structure were designed and synthesized into pCDH-EF1a lentiviral expression plasmid (see Figure 2The schematic diagram of plasmid structure in the application is shown in Figure 1. The plasmid design is as follows: (1) CD19 CAR-T; (2) CD20 CAR-T; (3) CD19 CAR & CD20 CAR-T; (4) CD19-CD20 CAR-T; (5) CD19 CAR & CD20 TRuC-T; (6) CD20 CAR & CD19 TRuC-T.

[0130] wherein (1) the CD19 CAR-T is obtained by CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z in turn; (2) the CD20 CAR-T is obtained by CD20 vhh, CD8 hinge, CD8 Tm, 41BB, CD3z in turn; (3) the CD19 CAR & CD20 CAR-T is obtained by CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD20 vhh, CD8 hinge, CD8 Tm, 41BB, CD3z in turn; (4) the CD19-CD20 CAR-T is obtained by CD20 vhh, G4S, CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z in turn; (5) the CD19 CAR & CD20 TRuC-T is obtained by CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD20 vhh, G4S, CD3e in turn; (6) the CD20 CAR & CD19 TRuC-T is obtained by CD20 vhh, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD19 scfv, G4S, CD3e in turn.

[0131] The protein can be selected from a membrane-out signal peptide CD8a-SP or a membrane-out signal peptide GMCSF-SP. The amino acid sequence information of the CD19 scfv and the CD20 vhh is shown in Table 1, the nucleotide sequence of the CD19 scfv and the CD20 vhh is shown in SEQ ID NO: 13-14, the amino acid sequence of the CD8 Hinge, the CD8 Tm, the 41BB, the CD3z, the CD3e, the G4S linker, the T2A, the membrane-out signal peptide CD8a-SP, and the membrane-out signal peptide GMCSF-SP is shown in SEQ ID NO: 15-23, and the nucleotide sequence of the CD8 Hinge, the CD8 Tm, the 41BB, the CD3z, the CD3e, the G4S linker, the T2A, the membrane-out signal peptide CD8a-SP, and the membrane-out signal peptide GMCSF-SP is shown in SEQ ID NO: 24-32.

[0132] Table 1 Amino acid sequences of CD19 scfv and CD20 vhh

[0133]

[0134]

[0135] The lentivirus system plasmid (pCDH-EF1a lentivirus expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed in a mass ratio of 3:2:1) was transfected into logarithmic growth phase adherent 293T cells, and the cell culture supernatant of the cells transfected for 48-72 hours was harvested. After concentration filtration, the CAR lentivirus was obtained and stored at -80°C.

[0136] Peripheral blood was collected by leukapheresis of patients or healthy volunteers, and then PBMCs were obtained by ficoll density gradient centrifugation. EasySep TM T cells were isolated using Human T Cell Isolation Kit (STEMCELL, #17951). The X-vivo (Lonza) culture medium containing 10 ng / mL IL-7 (Coastal Protein, GMP-C086), 5 ng / mL IL-15 (Coastal Protein, GMP-C016), ImmunoCult TM After normal activation of T cells by Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibodies for 2-3 days, lentivirus transduction was performed. The medium without activation antibodies was replaced for expansion to 9-11 days, and the positive rate of CAR-T cells was detected. By mixing T cells of the same batch, the positive rate of CAR-T cells in each group was adjusted to be consistent.

[0137] After co-incubation of the above CAR-T cells with GFP-expressing Raji and Daudi cells at an effector-to-target ratio of 1:1 for 24 hours, the cell viability was detected, and the cells in the control group (T cells) were used as a reference.

[0138] 2. Experimental results

[0139] The results are shown in Table 2. Figure 3 As shown in Table 2, the results show that the No. 5 CD19 CAR & CD20 TRuC-T structure exhibits significant killing of cell lines Raji and Daudi expressing both CD19 and CD20, and the cell viability is significantly decreased, and is significantly better than that of other groups.

[0140] Example 2 Verification of sensitivity of CD19 CD20 CAR / TRuC-T cells constructed in Example 1 to CD19 or CD20

[0141] 1. Experimental method

[0142] The plasmid overexpressing human CD19 and CD20 was constructed by pLV3-CMV-target- 3xFLAG-CopGFP-Puro, and K562 cells were infected after packaging lentivirus, and sorted by puromycin, to construct K562 cell lines overexpressing CD19 or CD20 alone. Using the CRISPR-Cas9 system, select the sgRNA corresponding to the database CD19 / CD20, complete the construction of CD19 or CD20 knockout cell lines in Raji. Incubate the above four cells with the different structures of CAR-T cells constructed in Example 1 at a ratio of 1:1 effector target ratio overnight, repeat the above killing verification.

[0143] 2. Experimental results

[0144] The results are shown in Figure 4 , and the results show that No. 5 CD19 CAR&CD20 TRuC-T structure is most sensitive to CD20, and can effectively recognize cells expressing CD19 or CD20 alone.

[0145] Example 3 Verification of reaction rate of CD19 CD20 CAR / TRuC-T cells constructed in Example 1 to CD19 or CD20

[0146] 1. Experimental method

[0147] Biotinylated Human CD19(20-291) Protein, Fc, Avitag TM (Arco, CD9-H82F6), Biotinylated Human CD20 / MS4A1 Full Length Protein, His, Avitag TM (Arco, CD0-H82E5), adjust the activation medium to different antigen concentrations by PBS, and detect the T cell activation marker CD69 for the several groups of CAR-T constructed in the above Example 1.

[0148] 2. Experimental results

[0149] The results are shown in Figure 5 , and the results show that No. 5 CD19 CAR&CD20 TRuC-T structure has the highest reaction rate to CD20, and the reaction rate to CD19 is similar to that of conventional CD19 CAR.

[0150] Example 4: Verification of the tumor-killing effect of a class of CD19CD20 CAR / TRuC-T cells constructed in Example 1 on tumors in vivo. 1. Experimental methods

[0151] The inhibitory effects of CAR-T cells constructed in Example 1 on mouse lymphoma cells were evaluated using the CD19 CAR-T (CAR19), CD19 CAR&CD20 CAR-T (DUAL), CD19-CD20 CAR-T (Tandem CAR 20&19), and CD19 CAR&CD20 TRuC-T (CAR19&TRuC20) structures. Female NCG mice, 6-8 weeks old, were intravenously infused with 5E5 luciferase-Raji per mouse. Five days later, the corresponding CAR-T cells (1E6 per mouse) were infused. In vivo imaging was performed every seven days to observe tumor growth.

[0152] 2. Experimental Results

[0153] The results are as follows Figure 6 As shown, the results indicate that the CD19 CAR & CD20 TRuC-T structure 5 has the most significant inhibitory effect on lymphoma in mice.

Claims

1. A fusion protein targeting CD19 and CD20, characterized in that, The fusion protein comprises a chimeric antigen receptor targeting CD19 and a TCR fusion construct targeting CD20; The chimeric antigen receptor targeting CD19 contains an antibody targeting CD19; The CD20-targeting TCR fusion construct contains a single-domain antibody targeting CD20; The amino acid sequences of CDR1, CDR2, and CDR3 of the single-domain antibody targeting CD20 are shown in SEQ ID NO:9-11, respectively. The chimeric antigen receptor targeting CD19 is formed by sequentially connecting an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signaling domain, and a CD3ζ intracellular signal transduction domain. The CD20-targeting TCR fusion construct is obtained by sequentially connecting a CD20-targeting single-domain antibody, a linker, and CD3ε.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

3. The fusion protein according to claim 1, characterized in that, The chimeric antigen receptor targeting CD19 is placed first, followed by the TCR fusion construct targeting CD20.

4. The fusion protein according to claim 1, characterized in that, The fusion protein is obtained by sequentially linking a chimeric antigen receptor targeting CD19, T2A, and a TCR fusion construct targeting CD20.

5. The fusion protein according to claim 1, characterized in that, The amino acid sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signaling domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:15-18, respectively.

6. The fusion protein according to claim 1, characterized in that, The Linker is selected from G4S, (G4S)2, (G4S)3 or EAAAK.

7. The fusion protein according to claim 1, characterized in that, The amino acid sequence of CD3ε is shown in SEQ ID NO:

19.

8. The fusion protein according to claim 1, characterized in that, The fusion protein is formed by sequentially connecting an antibody targeting CD19, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, the CD3ζ intracellular signal transduction domain, T2A, a single-domain antibody targeting CD20, G4S, and CD3ε.

9. The fusion protein according to claim 8, characterized in that, The N-terminus of the fusion protein also contains an exophase signal peptide.

10. The fusion protein according to claim 9, characterized in that, The exophase signal peptide is selected from exophase signal peptide CD8a-SP or exophase signal peptide GMCSF-SP.

11. The fusion protein according to claim 10, characterized in that, The amino acid sequences of the exosome signal peptides CD8a-SP and GMCSF-SP are shown in SEQ ID NO:22-23, respectively.

12. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to any one of claims 1-11.

13. The nucleic acid molecule according to claim 12, characterized in that, The nucleotide sequence of the antibody targeting CD19 in the fusion protein is shown in SEQ ID NO:

13.

14. The nucleic acid molecule according to claim 12, characterized in that, The nucleotide sequence of the single-domain antibody targeting CD20 in the fusion protein is shown in SEQ ID NO:

14.

15. The nucleic acid molecule according to claim 12, characterized in that, The nucleotide sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signaling domain, CD3ζ intracellular signal transduction domain, and CD3ε in the fusion protein are shown in SEQ ID NO:24-28, respectively.

16. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule according to any one of claims 12-15.

17. The expression vector according to claim 16, characterized in that, The expression vector is a DNA vector or an RNA vector.

18. The expression vector according to claim 17, characterized in that, The DNA vector is a plasmid.

19. The expression vector according to claim 17, characterized in that, The RNA vector is a viral vector.

20. The expression vector according to claim 19, characterized in that, The vector from which the virus originates is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, poxvirus vector, or herpesvirus vector.

21. A genetically modified host cell, characterized in that, The genetically modified host cell comprises the nucleic acid molecule of any one of claims 12-15 or the expression vector of any one of claims 16-20.

22. The genetically modified host cell according to claim 21, characterized in that, The host cell is a mammalian cell.

23. The genetically modified host cell according to claim 21, characterized in that, The host cells are immune cells.

24. The genetically modified host cell according to claim 23, characterized in that, The immune cells are T cells, B cells, NK cells, iNKT cells, γδT cells, NK92 cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, neutrophils, or any combination thereof.

25. The genetically modified host cell according to claim 21, characterized in that, The genetically modified host cell is a T cell modified with the fusion protein as described in any one of claims 1-11.

26. A derivative, characterized in that, The derivative is a fusion protein containing a detectable label as described in any one of claims 1-11, a nucleic acid molecule as described in any one of claims 12-15, or a genetically modified host cell as described in any one of claims 21-25; The detectable marker is selected from fluorescent dyes, colloidal gold, chemiluminescent markers, or chemiluminescent catalysts.

27. A derivative, characterized in that, The derivative is the genetically modified host cell of claim 25 that is combined with or conjugated with the therapeutic agent; The therapeutic agent is selected from radionuclides, cytokines, gold nanoparticles, viral particles, liposomes, magnetic nanoparticles, prodrug-activated enzymes, or chemotherapeutic agents.

28. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the genetically modified host cell of claim 25 and / or the derivative of claim 27.

29. A biological agent, characterized in that, The biological agent comprises the pharmaceutical composition of claim 28.

30. The biological agent according to claim 29, characterized in that, The dosage form of the biological agent is selected from lyophilized powder for injection, liquid suspension, liposome formulation, microsphere formulation, or gel formulation.

31. A reagent kit, characterized in that, The kit comprises the fusion protein of any one of claims 1-11, the nucleic acid molecule of any one of claims 12-15, and / or the expression vector of any one of claims 16-20.

32. A method for preparing genetically modified host cells according to any one of claims 21-25, characterized in that, The method includes the following steps: introducing the nucleic acid molecule of any one of claims 12-15 or the expression vector of any one of claims 16-20 into a host cell.

33. The method according to claim 32, characterized in that, The method of introduction is selected from transfection, microinjection, electroporation, DNA vector, retroviral vector, lentiviral vector, poxvirus vector, herpes simplex virus vector, adenovirus vector, or adeno-associated virus vector.

34. The use of the genetically modified host cell of claim 25 and / or the derivative of claim 27 in the preparation of a medicament for the treatment and / or prevention of CD19 and / or CD20-related diseases; The CD19 and / or CD20-related diseases are non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, or follicular lymphoma. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO: 1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

35. The use of the genetically modified host cell of claim 25 and / or the derivative of claim 27 in the preparation of a biological agent for the treatment and / or prevention of CD19 and / or CD20-related diseases; The CD19 and / or CD20-related diseases are non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, or follicular lymphoma. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO: 1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

36. The use of the fusion protein of any one of claims 1-11, the nucleic acid molecule of any one of claims 12-15, and / or the expression vector of any one of claims 16-20 in a kit for preparing fusion protein-modified T cells for the treatment and / or prevention of CD19 and / or CD20-related diseases; The CD19 and / or CD20-related diseases are non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, or follicular lymphoma. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO: 1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

37. The use of the kit of claim 31 in the preparation of T cells modified with fusion proteins for the treatment and / or prevention of CD19 and / or CD20-related diseases; The CD19 and / or CD20-related diseases are non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, or follicular lymphoma. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO: 1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

38. Use of the fusion protein of any one of claims 1-11 in the preparation of fusion protein-modified T cells for the treatment and / or prevention of CD19 and / or CD20-related diseases; The CD19 and / or CD20-related diseases are non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, or follicular lymphoma. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO: 1-3, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:5, HTS, and SEQ ID NO:6, respectively.

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