CD19 CD20 CAR / TRuC-T cell as well as related product and application thereof

Abstract

The invention discloses a CD19CD20CAR / TRuC-T cell as well as a related product and an application of the CD19CD20CAR / TRuC-T cell. The CD19CD20CAR / TRuC-T cell is a CD19CARamp which is targeted to CD19 and CD20 at the same time; cD20 TRuC-T, the CD19 CARamp, the CD19 CARamp, the CD20 TRuC- The CD20TRuC-T has higher recognition capability on target cells, is beneficial to preventing the occurrence of an antigen escape phenomenon, has lower self-activation, better anti-depletion capability, more comprehensive T cell activation signals, higher target sensitivity and better tumor infiltration capability, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies. Specifically, it relates to a CD19 CD20 CAR / TRuC-T cell and its related products and applications. More specifically, it relates to a CD19 CAR&CD20 TRuC-T cell and its related products and applications. Background Art

[0002] Although the CD19 CAR-T cell therapy has significantly improved the prognosis of patients with relapsed / refractory B-cell non-Hodgkin lymphoma (R / R B-NHL), about 50% of the patients still relapse after treatment. Studies have shown that about 30% of the relapsed patients experience treatment failure due to low expression or deletion of the CD19 antigen on tumor cells (i.e., antigen escape). This antigen escape mechanism mainly includes biological processes such as CD19 gene mutation, epigenetic modification (such as promoter hypermethylation), and lineage conversion.

[0003] To address the problem of antigen escape, in recent years, a number of 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, the CD20 target has become the most promising combination target option due to its widespread expression in more than 90% of B-NHL and its proven synergistic effect with rituximab in clinical practice. Clinical trial data show that the 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 in the tandem structure: When the expression abundance of the target antigen is insufficient, the spatial distance between the two targets may cause the binding of a single target to hinder the recognition of the other target, resulting in incomplete activation of T cells; 2) Signal competition in the parallel structure: The dual CAR structure designed with a traditional bicistronic vector has problems such as low virus packaging efficiency and competitive consumption of T cell activation signals by the intracellular signal domains, weakening the overall anti-tumor effect. Summary of the Invention

[0005] In view of this, in order to overcome the above technical problems faced by the current art, the purpose of the present invention is to provide a CD19 CD20 CAR / TRuC-T cell and its related products and applications for the art.

[0006] The TCR fusion construct (TRuC) utilized in the present invention is a structure that links a single-chain antibody with the CD3 complex (CD3γ, δ, ε). It recognizes specific targets through the single-chain antibody and utilizes the property of the TCR-CD3 complex to stimulate the activation signal of natural T cells to kill target cells. On this basis, the present invention constructs and designs a class of CD19 CD20 CAR / TRuC-T cells. The modified CAR-T (CD19 CAR&CD20 TRuC-T) can enhance the dual-target T cell therapy in the following aspects, including: lower self-activation, better anti-exhaustion ability, more comprehensive T cell activation signals, higher target sensitivity, and better tumor infiltration ability.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention purpose:

[0008] In the first aspect of the present invention, a fusion protein targeting CD19 and CD20 is provided.

[0009] Furthermore, the fusion protein comprises a chimeric antigen receptor targeting CD19 and 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 as shown in 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 as shown in SEQ ID NO:5, HTS, and SEQ ID NO:6.

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

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

[0016] Optionally, the chimeric antigen receptor targeting CD19 further comprises a hinge region, a transmembrane region, a co-stimulatory signal domain, and an intracellular signal transduction domain;

[0017] Optionally, the hinge region is selected from the hinge regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor or IL-11 receptor;

[0018] Optionally, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor or IL-11 receptor;

[0019] Optionally, the co-stimulatory signal domain is selected from the co-stimulatory signal domains of the following molecules: 41BB, CD27, CD19, CD4, CD28, CD278, CD8α, CD8β, 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 domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12 or CD66d;

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

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

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

[0024] Optionally, the intracellular signaling domain is the CD3ζ intracellular signaling domain;

[0025] Optionally, the chimeric antigen receptor targeting CD19 is obtained by sequentially concatenating an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signal domain, and a CD3ζ intracellular signaling domain;

[0026] Optionally, the amino acid sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signal domain, and CD3ζ intracellular signaling domain are respectively as shown in SEQ ID NO: 15-18;

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

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

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

[0030] Optionally, the Linker is G4S;

[0031] Optionally, the TCR complex subunit fusion part is CD3ε;

[0032] Optionally, the TCR fusion construct targeting CD20 is obtained by sequentially concatenating a single-domain antibody targeting CD20, G4S, and CD3ε;

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

[0034] Optionally, the fusion protein is obtained by sequentially concatenating 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, and CD3ε;

[0035] Optionally, the N-terminus of the fusion protein further contains a transmembrane signal peptide;

[0036] Optionally, the transmembrane signal peptide is selected from transmembrane signal peptide CD8a-SP or transmembrane signal peptide GMCSF-SP;

[0037] Optionally, the amino acid sequences of the transmembrane signal peptide CD8a-SP and the transmembrane signal peptide GMCSF-SP are as shown in SEQ ID NO:22-23, respectively.

[0038] In the present invention, CD3z is the same as CD3ζ, and CD3e is the same as CD3ε.

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

[0040] In a specific embodiment of the present invention, it is experimentally verified for the first time that in the fusion protein, the connection order of the chimeric antigen receptor targeting CD19 in the front and the TCR fusion construct targeting CD20 in the back will have a greater impact on the effect of T cells modified by the fusion protein. Based on the fusion protein with the connection order of the chimeric antigen receptor targeting CD19 in the front and the TCR fusion construct targeting CD20 in the back, the therapeutic effect of the T cells modified by the fusion protein (CD19 CAR&CD20TRuC-T) is significantly better. That is, the fusion protein described in the first aspect of the present invention has achieved unexpected technical effects.

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

[0042] Furthermore, the nucleic acid molecule encodes the fusion protein described in the first aspect of the present invention;

[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 co-stimulatory signal domain, CD3ζ intracellular signaling domain, and CD3ε in the fusion protein are as shown in SEQ ID NOs:24-28 respectively.

[0046] In some embodiments, the nucleic acid molecule may comprise natural, non-natural or altered nucleotides; and it may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, in place of the phosphodiester present between the nucleotides of an unmodified oligonucleotide.

[0047] In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, in some cases, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions and / or substitutions. Therefore, nucleic acids formed by insertions, deletions, inversions and / or substitutions based on the nucleic acid molecule provided by the present invention are also included within the scope of protection of the present invention.

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

[0049] Furthermore, the expression vector contains the nucleic acid molecule described in the second aspect of the present invention;

[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 virus-derived vector;

[0053] Optionally, the virus-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 invention include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, virus-derived vectors. Various vectors known in the art can be selected, for example, commercially available vectors can be selected, and then the nucleotide sequence encoding the fusion protein described in the first aspect of the present invention is operably linked to an expression regulatory sequence to form an expression vector.

[0055] In some embodiments, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, herpesviral vectors, baculoviral vectors, papillomaviral vectors, polyomaviral vectors.

[0056] The fourth aspect of the present invention provides a genetically modified host cell.

[0057] Furthermore, the genetically modified host cell contains the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention;

[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, B cell, NK cell, iNKT cell, γδT cell, NK92 cell, CTL cell, dendritic cell, myeloid cell, monocyte, macrophage, neutrophil, or any combination thereof;

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

[0062] Optionally, the genetically modified host cell is a T cell modified with the fusion protein described in the first aspect of the present invention.

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

[0064] In some embodiments, the genetically modified immune cells are obtained from a subject suffering from a CD19- and / or CD20-related disease. In some embodiments, the genetically modified immune cells are obtained from a healthy donor.

[0065] The fifth aspect of the present invention provides a derivative.

[0066] Furthermore, the derivative is selected from:

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

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

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

[0070] Optionally, the detectable label is selected from a fluorescent dye, colloidal gold, a chemiluminescent label, or a chemiluminescent catalyst;

[0071] Optionally, the gene conferring antibiotic resistance is selected from 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 a radionuclide, a cytokine, gold nanoparticles, virus particles, liposomes, magnetic nanoparticles, a prodrug-activating enzyme, or a chemotherapeutic agent.

[0073] The sixth aspect of the present invention provides a pharmaceutical composition or a biological preparation.

[0074] Furthermore, the pharmaceutical composition contains the fusion protein according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the expression vector according to the third aspect of the present invention, the genetically modified host cell according to the fourth aspect of the present invention, and / or the derivative according to the fifth aspect of the present invention;

[0075] Optionally, the biological preparation contains the pharmaceutical composition;

[0076] Optionally, the dosage form of the biological preparation is selected from a freeze-dried powder injection, a liquid suspension, a liposome preparation, a microsphere preparation, or a gel preparation.

[0077] In some embodiments, the pharmaceutical composition or biological agent may further comprise a pharmaceutically acceptable carrier and / or excipient, which are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used as needed to help with the stability of the formulation or to enhance the bioavailability of the active or active substances. In some embodiments, when using the pharmaceutical composition or biological agent, it means administering a safe and effective amount of the pharmaceutical composition or biological agent of the present invention as described above to a human.

[0078] In some embodiments, suitable forms of administration of the pharmaceutical composition or biological agent include forms suitable for parenteral administration, such as by injection or infusion, for example by bolus injection or continuous infusion, intravenously, inhalably or subcutaneously. In the case where the product is for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulating agents such as suspending agents, preservatives, stabilizers and / or dispersing agents.

[0079] In some embodiments, the pharmaceutical composition or biological agent can be made into various dosage forms as needed, and the physician can determine the dose beneficial to the patient according to factors such as the type of patient, age, weight and general disease condition, and the mode of administration. A skilled physician can usually easily determine the prescription and the dosage and mode of administration of the prescription effective for the desired treatment and / or prevention.

[0080] The seventh aspect of the present invention provides a kit.

[0081] Furthermore, the kit comprises the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the genetically modified host cell described in the fourth aspect of the present invention and / or the derivative described in the fifth aspect of the present invention.

[0082] The eighth aspect of the present invention provides any one of the following methods:

[0083] (1) A method for preparing the genetically modified host cell described in the fourth aspect of the present invention, the method comprising the following steps: introducing the nucleic acid molecule described in the second aspect of the present invention or the expression vector described in the third aspect of the present invention into a host cell;

[0084] Optionally, the introducing method is selected from transfection, microinjection, electroporation, DNA vector, retroviral vector, lentiviral vector, poxviral vector, herpes simplex virus vector, adenoviral vector or adeno-associated viral vector;

[0085] (2) A method for non-therapeutically inhibiting the activity of CD19 and / or CD20 proteins in vitro, the method comprising the following steps: contacting the genetically modified host cell described in the fourth aspect of the present invention, the derivative described in the fifth aspect of the present invention, the pharmaceutical composition or biological agent described in the sixth aspect of the present invention with somatic cells of an organism.

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

[0087] In a specific embodiment of the present invention, the fusion protein-modified T cells are administered by infusion. It should be noted that the protection scope of the present invention is not limited by the specific administration method and dosage. As long as the fusion protein-modified T cells, pharmaceutical composition or biological agent described in the present invention produce the expected therapeutic and / or prophylactic effect in a subject, they all fall within the protection scope of the present invention.

[0088] The ninth aspect of the present invention provides the following applications in any one aspect:

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

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

[0091] (3) The application of the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the genetically modified host cell described in the fourth aspect of the present invention, and / or the derivative described in the fifth aspect of the present invention in 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) Use of the kit according to the seventh aspect of the present invention in the preparation of fusion protein-modified immune cells for the treatment and / or prevention of CD19- and / or CD20-related diseases;

[0093] (5) Use of the fusion protein according to the first aspect of the present invention in the preparation of fusion protein-modified immune cells for the treatment and / or prevention of CD19- and / or CD20-related diseases;

[0094] Optionally, the CD19- and / or CD20-related diseases are non-Hodgkin lymphoma, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, megakaryocytic leukemia, Burkitt 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 invention, the CD19- and / or CD20-related diseases are not limited to the specific diseases listed above in the present invention, and any diseases related to CD19 and / or CD20 expression are within the protection scope of the present invention.

[0096] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0097] (1) The present invention provides a novel CD19 CAR&CD20TRuC-T that simultaneously targets CD19 and CD20 in the art, and in vivo and in vitro experiments have confirmed that the CD19 CAR&CD20 TRuC-T has a significant killing effect on tumors expressing CD19 and / or CD20.

[0098] (2) Compared with ordinary single-target CARs, the dual-target CD19 CAR&CD20 TRuC-T provided by the present invention has a stronger ability to recognize target cells, is conducive to preventing the occurrence of antigen escape, and has lower self-activation, better anti-exhaustion ability, more comprehensive T cell activation signals, higher target sensitivity, and better tumor infiltration ability, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 : Schematic diagram of the corresponding structural design of the CD19 CAR / CD20 TRuC-T provided by the present invention;

[0100] Figure 2: Schematic diagrams corresponding to CD19 CAR-T, CD20 CAR-T, CD19 CAR&CD20 CAR-T, CD19-CD20 CAR-T, CD19CAR&CD20 TRuC-T, and CD20 CAR&CD19 TRuC-T;

[0101] Figure 3 : Comparative result graphs of the killing effects of CD19 CAR-T, CD20 CAR-T, CD19 CAR&CD20 CAR-T, CD19-CD20 CAR-T, CD19CAR&CD20 TRuC-T, CD20 CAR&CD19 TRuC-T, and T cells on cell lines Raji and Daudi that express both CD19 and CD20;

[0102] Figure 4 : Comparative result graphs of the killing effects of CD19 CAR-T, CD20 CAR-T, CD19 CAR&CD20 CAR-T, CD19-CD20 CAR-T, CD19CAR&CD20 TRuC-T, CD20 CAR&CD19 TRuC-T, and T cells on K562 cell lines that only overexpress CD19 or CD20 alone, and Raji cell lines with CD19 or CD20 knocked out;

[0103] Figure 5 : Comparative result graphs of the response rates of each group to CD20 or CD19 in the activation detection of CD19 CAR-T, CD20 CAR-T, CD19 CAR&CD20 CAR-T, CD19-CD20 CAR-T, CD19CAR&CD20 TRuC-T, CD20 CAR&CD19 TRuC-T, and T cells;

[0104] Figure 6 : Result graphs corresponding to the evaluation of the inhibitory effects of CAR-T cells with structures of 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) on mouse lymphoma cells. Specific implementation manners

[0105] The present invention will be further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and should not be construed as a limitation of the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined 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 invention belongs. For the purpose of facilitating the understanding of the present invention, the following terms related to the present invention are explained herein:

[0107] As used herein, the term "comprising" or "including" means including any one or more of the stated elements or components, without excluding other elements or other components.

[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 the antigen on the surface of target cells through the extracellular antigen-binding region. This recognition does not depend on the major histocompatibility complex (MHC), so it can bypass the 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, causing immune cells to proliferate, secrete cytokines, and exert cytotoxic effects to kill target cells.

[0110] As used herein, the term "TCR fusion construct (TRuC)" is a novel antigen receptor design aimed at reprogramming T cells to specifically recognize and kill tumor cells. TRuC consists 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 the tumor surface antigen, fused to the CD3ε subunit of TCR through a flexible linker peptide.

[0111] The TRuC construct is cloned into vectors such as lentiviruses. After transduction into T cells, the fusion protein it expresses will integrate into the endogenous TCR complex, replacing the native CD3ε subunit. When the TRuC-T cell recognizes a specific antigen on the surface of tumor cells, it can activate the T cell in a human leukocyte antigen (HLA)-independent manner, initiate the killing function of the T cell, and specifically kill tumor cells. Due to the utilization of the signal transduction ability of the entire TCR, during the recognition and killing of tumor cells by TRuC-T cells, more effective signal transduction can be triggered, promoting the trafficking, 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. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when placed into a functional relationship with another nucleic acid sequence. 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 containing a recombinant polynucleotide that includes expression control sequences operably linked to a nucleotide sequence to be expressed. The expression vector contains 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., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that are incorporated into the recombinant polynucleotide.

[0114] In some embodiments, the expression vector according to the present invention is capable of directing the replication and expression of the nucleic acid molecule of the present invention in a host, and thus ensuring the expression of the fusion protein of the present invention encoded thereby in a selected host. The expression vector can be, for example, a cloning vector, a binary vector, or an integrative vector. Expression includes the transcription of the nucleic acid molecule, e.g., transcription 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 pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems) pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

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

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

[0118] In specific embodiments of the present invention, the host cell is preferably an immune cell.

[0119] In some embodiments, the immune cells include but are not limited to: T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof, preferably T cells. Additionally, the "host cell" as described in the present invention can include a single cell or a cell population, that is, the "genetically modified host cell" as described above in the present invention includes a single genetically modified host cell and a population of genetically modified host cells.

[0120] As used herein, the term "treatment" refers to the complete or partial amelioration or alleviation of a disease or disorder or condition, or symptoms, adverse effects or consequences, or phenotypes associated therewith. Desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing cancer metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease condition, and alleviating or improving the prognosis. The term does not imply complete cure of the disease or complete elimination of any symptoms or effects on all symptoms or outcomes.

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

[0122] As used herein, the term "effective amount" includes "therapeutically effective amount" and "prophylactically effective amount". Among them, "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient suffering from the disease. The therapeutically effective amount can vary depending on factors such as the severity of the disease to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight and gender, the mode of administration of the drug, and other treatment methods used simultaneously, etc. "Prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease.

[0123] In some embodiments, the dose and frequency (single or multiple doses) of the pharmaceutical composition or biological agent administered to a subject can vary depending on a variety of factors, such as whether the mammal has another disease and its route of administration; the age, gender, health status, weight, body mass index and diet of the subject; the nature and degree of the symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of co-treatment, the complications caused by the disease being treated or other health-related problems, etc. Other treatment regimens or agents can be used in combination with the pharmaceutical composition or biological agent and treatment methods described herein. Adjustment and manipulation of established doses (e.g., frequency and duration) are well within the capabilities of those skilled in the art.

[0124] In some embodiments, the pharmaceutical composition or biological agent may have any one of the following formulations: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspending agents, emulsions, freeze-dried preparations, and suppositories. In addition, it can be administered one or more times. At this time, the pharmaceutical composition or biological agent is administered in the form of a liquid preparation, powder, aerosol, capsule, vaginal tablet, capsule, or suppository. The administration routes may include, but are not limited to: intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intralung, rectal, etc. When administered orally, it can be formulated with a coating that protects the active ingredient in the pharmaceutical composition or biological agent from degradation in the stomach. In addition, the active ingredient can be administered by any device capable of transferring to the target cells. In a specific embodiment, the pharmaceutical composition or biological agent provided by the present invention can be made into various dosage forms according to actual needs, and the clinician can determine the dose beneficial to the patient according to factors such as the type, age, weight, and general disease condition of the subject patient, and the administration method. The administration method can be, for example, injection or any other 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 raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained commercially. The experimental methods without specific conditions noted in the present invention are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0127] Example 1 Construction of a Novel Class of CD19 CD20 CAR / TRuC-T Cells and Verification of Their Killing Effect

[0128] 1. Experimental Method

[0129] According to Figure 1 the structural design schematic diagram in, corresponding different types of CAR-T structures were designed and synthesized into the pCDH-EF1a lentiviral expression plasmid (see Figure 2Plasmid structure schematic diagram). 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) CD19CAR&CD20 TRuC-T; (6) CD20 CAR&CD19 TRuC-T.

[0130] Among them, (1) the CD19 CAR-T is obtained by sequentially connecting CD19 scfv, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (2) the CD20 CAR-T is obtained by sequentially connecting CD20 vhh, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (3) the CD19 CAR&CD20 CAR-T is obtained by sequentially connecting CD19 scfv, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD20 vhh, CD8hinge, CD8 Tm, 41BB, and CD3z in series; (4) the CD19-CD20 CAR-T is obtained by sequentially connecting CD20 vhh, G4S, CD19scfv, CD8 hinge, CD8 Tm, 41BB, and CD3z in series; (5) the CD19 CAR&CD20 TRuC-T is obtained by sequentially connecting CD19scfv, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD20 vhh, G4S, and CD3e in series; (6) the CD20CAR&CD19 TRuC-T is obtained by sequentially connecting CD20 vhh, CD8 hinge, CD8 Tm, 41BB, CD3z, T2A, CD19 scfv, G4S, and CD3e in series.

[0131] The protein's selectable transmembrane signal peptide is transmembrane signal peptide CD8a-SP or transmembrane signal peptide GMCSF-SP. Among them, the amino acid sequence information of the CD19 scfv and CD20 vhh is shown in Table 1 below. The nucleotide sequences of the CD19 scfv and CD20 vhh are respectively as shown in SEQ ID NO:13-14. The amino acid sequences of the CD8 Hinge, CD8 Tm, 41BB, CD3z, CD3e, G4Slinker, T2A, transmembrane signal peptide CD8a-SP, and transmembrane signal peptide GMCSF-SP are respectively as shown in SEQ ID NO:15-23. The nucleotide sequences of the CD8 Hinge, CD8 Tm, 41BB, CD3z, CD3e, G4S linker, T2A, transmembrane signal peptide CD8a-SP, and transmembrane signal peptide GMCSF-SP are respectively as shown in SEQ ID NO:24-32.

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

[0133]

[0134]

[0135] The lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase. The cell culture supernatant harvested 48 - 72 hours after transfection was concentrated and filtered to obtain CAR lentivirus, which was stored at -80 °C for later use.

[0136] Peripheral blood was collected by leukapheresis from patients or healthy volunteers, and then PBMC was isolated by ficoll density gradient centrifugation. T cells were isolated using the EasySep TM Human T Cell Isolation Kit (STEMCELL, #17951). X-vivo (Lonza) medium containing 10 ng / mL IL-7 (Novoprotein, GMP-C086), 5 ng / mL IL-15 (Novoprotein, GMP-C016), and ImmunoCult TM Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody was used to normally activate T cells. After 2 - 3 days of activation, lentiviral transduction was performed. The medium without the activation antibody was replaced for amplification for 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-incubating the above CAR-T cells with Raji and Daudi cells expressing GFP at an effector-to-target ratio of 1:1 for 24 hours, the cell viability was detected, with reference to the cells in the control group (T cells).

[0138] 2. Experimental results

[0139] The results are as Figure 3 shown. The results show that the 5th CD19 CAR&CD20 TRuC-T construct showed significant killing effects on the cell lines Raji and Daudi that both express CD19 and CD20. The cell viability decreased significantly, and it was significantly better than other groups of cells.

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

[0141] 1. Experimental method

[0142] Plasmids overexpressing human CD19 and CD20 were constructed by pLV3-CMV-target-3×FLAG-CopGFP-Puro, and lentiviruses were packaged and used to infect K562 cells. After sorting with puromycin, K562 cell lines overexpressing only CD19 or CD20 were constructed. Using the CRISPR-Cas9 system, the corresponding sgRNAs of CD19 / CD20 in the database were selected to construct CD19- or CD20-knockout cell lines in Raji. The above four types of cells were incubated overnight with CAR-T cells with different structures constructed in Example 1 at an effector-to-target ratio of 1:1, and the above killing verification was repeated.

[0143] 2. Experimental results

[0144] The results are as Figure 4 shown. The results show that the 5th CD19 CAR&CD20 TRuC-T structure is the most sensitive to CD20 and can effectively recognize cells expressing only CD19 or CD20.

[0145] Verification of the response rate of a class of CD19 / CD20 CAR / TRuC-T cells constructed in Example 1 to CD19 or CD20

[0146] 1. Experimental method

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

[0148] 2. Experimental results

[0149] The results are as Figure 5 shown. The results show that the 5th CD19 CAR&CD20 TRuC-T structure has the highest response rate to CD20, and its response rate to CD19 is similar to that of conventional CD19 CAR.

[0150] Example 4 Verification of the killing effect of a class of CD19 / CD20 CAR / TRuC-T cells constructed in Example 1 on tumors in vivo 1. Experimental method

[0151] The inhibitory effects of CAR-T cells with 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 constructed in Example 1 on mouse lymphoma cells were evaluated. NCG mice, female, 6-8 weeks old, were intravenously injected with 5E5 luciferase-Raji per mouse. Five days later, the corresponding CAR-T cells (1E6 per mouse) were injected respectively, and in vivo imaging was performed every seven days to observe tumor growth.

[0152] 2. Experimental results

[0153] The results are as Figure 6 shown. The results show that the inhibitory effect of the 5th CD19 CAR&CD20 TRuC-T structure on lymphoma in mice is the most significant.

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 comprises an antibody targeting CD19; The TCR fusion construct targeting CD20 comprises a single-domain antibody targeting CD20; The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody targeting CD19 are respectively as shown in SEQ ID NO: 1-3; The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody targeting CD19 are respectively as shown in SEQ ID NO: 5, HTS, and SEQ ID NO: 6; 2. The fusion protein according to claim 1, wherein The chimeric antigen receptor targeting CD19 is in the front and the TCR fusion construct targeting CD20 is in the back; Optionally, the fusion protein is obtained by sequentially concatenating a chimeric antigen receptor targeting CD19, T2A, and a TCR fusion construct targeting CD20; Optionally, the chimeric antigen receptor targeting CD19 further comprises a hinge region, a transmembrane region, a co-stimulatory signal domain, and an intracellular signaling domain; Optionally, the hinge region is selected from the hinge regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor; Optionally, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, CD28, IgG1, IgG4, 41BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor, or IL-11 receptor; Optionally, the co-stimulatory signal domain is selected from the co-stimulatory signal domains of the following molecules: 41BB, CD27, CD19, CD4, CD28, CD278, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, or B7H3; Optionally, the intracellular signaling domain is selected from the intracellular signaling domains of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, or CD66d; Optionally, the hinge region is the CD8 hinge region; Optionally, the transmembrane region is the CD8 transmembrane region; Optionally, the co-stimulatory signal domain is the 41BB co-stimulatory signal domain; Optionally, the intracellular signaling domain is the CD3ζ intracellular signaling domain; Optionally, the chimeric antigen receptor targeting CD19 is obtained by sequentially concatenating an antibody targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 41BB co-stimulatory signal domain, and a CD3ζ intracellular signaling domain; Optionally, the amino acid sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signal domain, and CD3ζ intracellular signaling domain are shown in SEQ ID NOs: 15-18, respectively; Optionally, the CD20-targeted TCR fusion construct further comprises a Linker and a TCR complex subunit fusion portion; Optionally, the Linker is selected from G4S, (G4S)2, (G4S)3, or EAAAK; Optionally, the TCR complex subunit fusion portion is selected from CD3ε, CD3γ, or CD3δ; Optionally, the Linker is G4S; Optionally, the TCR complex subunit fusion portion is CD3ε; Optionally, the CD20-targeted TCR fusion construct is obtained by sequentially concatenating a CD20-targeted single-domain antibody, G4S, and CD3ε; Optionally, the amino acid sequence of CD3ε is shown in SEQ ID NO: 19; Optionally, the fusion protein is obtained by sequentially concatenating an anti-CD19 antibody, CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signal domain, CD3ζ intracellular signaling domain, T2A, a CD20-targeted single-domain antibody, G4S, and CD3ε; Optionally, the N-terminus of the fusion protein further comprises a transmembrane signal peptide; Optionally, the transmembrane signal peptide is selected from transmembrane signal peptide CD8a-SP or transmembrane signal peptide GMCSF-SP; Optionally, the amino acid sequences of transmembrane signal peptide CD8a-SP and transmembrane signal peptide GMCSF-SP are shown in SEQ ID NOs: 22-23, respectively.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein according to claim 1 or 2; Optionally, the nucleotide sequence of the anti-CD19 antibody in the fusion protein is shown in SEQ ID NO: 13; Optionally, the nucleotide sequence of the CD20-targeted single-domain antibody in the fusion protein is shown in SEQ ID NO: 14; Optionally, the nucleotide sequences of the CD8 hinge region, CD8 transmembrane region, 41BB co-stimulatory signal domain, CD3ζ intracellular signaling domain, and CD3ε in the fusion protein are shown in SEQ ID NOs: 24-28, respectively.

4. An expression vector, characterized in that, The expression vector comprises the nucleic acid molecule according to claim 3; Optionally, the expression vector is a DNA vector or an RNA vector; Optionally, the DNA vector is a plasmid; Optionally, the RNA vector is a virus-derived vector; Optionally, the virus-derived vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxviral vector, or a herpesviral vector.

5. A genetically modified host cell, characterized in that, The genetically modified host cell comprises the nucleic acid molecule according to claim 3 or the expression vector according to claim 4; Optionally, the host cell is a mammalian cell; Optionally, the host cell is an immune cell; Optionally, 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; Optionally, the immune cells are T cells; Optionally, the genetically modified host cell is a T cell modified with the fusion protein according to claim 1 or 2.

6. A derivative, characterized in that, The derivatives are selected from: (1) The fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the genetically modified host cell according to claim 5, which contains a detectable label; (2) The fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the genetically modified host cell according to claim 5, which confers antibiotic resistance; or (3) The fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the genetically modified host cell according to claim 5, which is bound or conjugated to a therapeutic agent; Optionally, the detectable label is selected from fluorescent dyes, colloidal gold, chemiluminescent markers, or chemiluminescent catalysts; Optionally, the antibiotic resistance gene is selected from penicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, or kanamycin resistance gene; Optionally, the therapeutic agent is selected from radionuclides, cytokines, gold nanoparticles, virus particles, liposomes, magnetic nanoparticles, prodrug activating enzymes, or chemotherapeutic agents.

7. A pharmaceutical composition or biological preparation, characterized in that, The pharmaceutical composition comprises the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically modified host cell according to claim 5, and / or the derivative according to claim 6; Optionally, the biological preparation comprises the pharmaceutical composition; Optionally, the dosage form of the biological preparation is selected from freeze-dried powder injections, liquid suspensions, liposome preparations, microsphere preparations, or gel preparations.

8. A kit, characterized in that, The kit comprises the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically modified host cell according to claim 5, and / or the derivative according to claim 6.

9. Any of the following methods: (1) A method for preparing the genetically modified host cell according to claim 5, characterized in that, The method comprises the following steps: introducing the nucleic acid molecule according to claim 3 or the expression vector according to claim 4 into a host cell; Optionally, the introducing method is selected from transfection, microinjection, electroporation, DNA vectors, retroviral vectors, lentiviral vectors, poxviral vectors, herpes simplex virus vectors, adenoviral vectors, or adeno-associated viral vectors; (2) A method for non-therapeutically inhibiting the activity of CD19 and / or CD20 proteins in vitro, characterized in that the method comprises the following steps: contacting the genetically modified host cell according to claim 5, the derivative according to claim 6, the pharmaceutical composition or biological preparation according to claim 7 with somatic cells of an organism.

10. Any of the following applications: (1) Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically modified host cell according to claim 5, the derivative according to claim 6, and / or the kit according to claim 8 in the preparation of a medicament for treating and / or preventing CD19- and / or CD20-related diseases; (2) Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically modified host cell according to claim 5, the derivative according to claim 6, and / or the kit according to claim 8 in the preparation of a biological agent for treating and / or preventing CD19- and / or CD20-related diseases; (3) Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the genetically modified host cell according to claim 5, and / or the derivative according to claim 6 in the preparation of a kit for preparing fusion protein-modified immune cells for treating and / or preventing CD19- and / or CD20-related diseases; (4) Use of the kit according to claim 8 in the preparation of fusion protein-modified immune cells for treating and / or preventing CD19- and / or CD20-related diseases; (5) Use of the fusion protein according to claim 1 or 2 in the preparation of fusion protein-modified immune cells for treating and / or preventing CD19- and / or CD20-related diseases; Optionally, the CD19- and / or CD20-related diseases are non-Hodgkin lymphoma, chronic myelogenous leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, megakaryocytic leukemia, Burkitt 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.

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