Chimeric antigen receptor targeting CD19 and / or CD20 and application thereof

Abstract

The invention provides a chimeric antigen receptor targeting CD19 and / or CD20 and application thereof, the chimeric antigen receptor comprises: an extracellular domain capable of binding to CD20 and / or CD19; the extracellular domain is connected with the amino acid sequence, the transmembrane domain is connected with the extracellular domain, the intracellular domain is connected with the transmembrane domain, and the intracellular domain is selected from all amino acid sequences or partial amino acid sequences of at least one of the following proteins: 2B4, DAP10 and CD3zeta. The chimeric antigen receptor disclosed by the invention has double-target recognition capability and can be simultaneously combined with CD19 and CD20, so that the chimeric antigen receptor can cover a full differentiation stage of pathogenic B cells in autoimmune diseases and block a production path of an autoantibody from a source of a pathological mechanism; meanwhile, based on the inherent regulation and control capability of NK cells on lesion T cells, pathogenic T cells are removed, and the removal effect of B cells is cooperated, so that bidirectional blocking treatment on autoimmune pathology is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular, the present application relates to a chimeric antigen receptor targeting CD19 and / or CD20 and application thereof. BACKGROUND

[0002] The core pathological mechanism of systemic lupus erythematosus (SLE) and rheumatoid arthritis and other autoimmune diseases lies in the abnormal activation of B cells and the persistence of autoreactive plasma cells, leading to the excessive production of pathogenic autoantibodies, and then triggering immune complex deposition and inflammatory damage in multiple organs of the body. The current clinical standard treatment regimen mainly relies on glucocorticoids (such as prednisone) and broad-spectrum immunosuppressants (such as cyclophosphamide, mycophenolate mofetil), which can control symptoms in the short term, but long-term use will cause serious treatment bottlenecks: first, extensive immunosuppression significantly increases the risk of opportunistic infections; second, drug cumulative organ toxicity causes irreversible damage; third, more than 60% of patients experience disease rebound after drug reduction, accompanied by progressive aggravation of kidney or nervous system damage. Although the application of biological agents in recent years has improved the prognosis of some patients, there are still 30-40% of refractory patients who respond insufficiently, highlighting the fundamental defects of existing therapies in terms of long-term remission and targeted precision.

[0003] With the development of cellular immunotherapy, chimeric antigen receptor (CAR) technology targeting B cell surface antigens provides a new idea for the treatment of autoimmune diseases. It is worth noting that CD19 single-target CAR-T therapy shows potential for inducing deep remission in refractory SLE in early clinical trials, but its single-target design has inherent antigen escape risk - CD19 expression is lost on terminally differentiated long-lived plasma cells, leading to the escape of these cells from clearance and the continuous secretion of autoantibodies, becoming a potential source of disease recurrence (clinical data show that the proportion of CD19-negative plasma cells remaining in relapsed patients is >65%).

[0004] Therefore, there is an urgent need to develop a chimeric antigen receptor structure that can cover the antigen expression profile of the entire stage of B cell differentiation to prevent escape, and enhance the survival and killing ability of cells in the suppressive microenvironment through synergistic signals. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. The present application proposes a chimeric antigen receptor targeting CD19 and / or CD20 and application thereof, which has dual-target recognition ability and can bind CD19 and CD20 at the same time - CD19 is expressed in early B cells and plasmablasts, and CD20 is highly expressed in mature B cells; thus, it can cover the entire differentiation stage of pathogenic B cells in autoimmune diseases, and block the production path of autoantibodies from the source of pathological mechanism.

[0006] The present application is based on the following findings of the inventor: Current treatments for autoimmune diseases such as SLE focus on B cells, but T lymphocytes also play an important role in the pathogenesis, pathogenesis and progression of autoimmune diseases such as SLE. They are involved in the regulation of immune tolerance, the promotion of inflammatory cytokine secretion, the differentiation of B lymphocytes and the production of autoantibodies. CD4 + T cells promote the activation and proliferation of cytotoxic T cells and macrophages by secreting IL-2, IL-12 and IFN-γ. Activated cytotoxic T cells directly infiltrate or activate and recruit macrophages and NKT cells, damaging renal parenchymal cells, leading to renal tubular cell dysfunction and fibrosis, and play a key role in the pathogenesis of lupus nephritis. CD4 + T cells secrete IL-17 and other cytokines to induce the expression of pro-inflammatory cytokines, chemokines and matrix metalloproteinases, causing inflammatory cell infiltration and tissue destruction. However, existing technologies, including current CAR-T cell therapies targeting B cells, can only eliminate abnormal B cells in SLE and other autoimmune patients, but cannot eliminate abnormally activated pathological T cells in SLE patients, resulting in significant short-term efficacy but not a cure, unsatisfactory efficacy maintenance, inability to avoid recurrence, and a low proportion of long-term remission.

[0007] Therefore, in a first aspect of the present application, a chimeric antigen receptor is provided. According to embodiments of the present application, the chimeric antigen receptor comprises an extracellular domain capable of binding to CD20 and / or CD19, a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain, wherein the intracellular domain is selected from all or part of the amino acid sequence of at least one of the following proteins: 2B4, DAP10 and CD3ζ. Thus, by using a tandem scFv structure, i.e., CD20 antibody light chain variable region (VL)-CD20 antibody heavy chain variable region (VH)-linker-CD19 antibody heavy chain variable region (VH)-CD19 antibody light chain variable region (VL) (CD20 VL-VH-linker-CD19 VH-VL), the chimeric antigen receptor structure (CAR structure) of the present application can have a double-target high coverage, while targeting CD19 expressed on early B cells and plasma cells and CD20 highly expressed on mature B cells. It can cover the antigen expression profile of the whole stage of B cell differentiation to prevent escape, and can enhance the survival and killing ability of cells in the suppressive microenvironment through synergistic signals to block the autoantibody production pathway from the source. In addition, the use of 2B4, DAP10 and CD3ζ fusion signal modules in the intracellular domain can significantly enhance the degranulation and cytokine secretion efficiency of immune cells, and adapt the natural killing properties of immune cells.

[0008] In a second aspect, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule comprises a first nucleic acid segment and, optionally, a second nucleic acid segment, which are connected; wherein the first nucleic acid segment encodes the chimeric antigen receptor of the first aspect; and the second nucleic acid segment is used to encode a fusion protein comprising IL-15 and IL-15Rα, which are connected. Thus, by integrating the membrane-anchored fusion protein at the C-terminal of the CAR structure, autocrine membrane-bound cytokine support can be achieved, solving the key bottleneck of insufficient in vivo expansion and short survival period of cells. In addition, based on the above nucleic acid molecule, the chimeric antigen receptor and the fusion protein can be efficiently expressed in vitro, simplifying the production process.

[0009] In a third aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule of the second aspect. Thus, after introducing the expression vector into a suitable recipient cell, the expression of the aforementioned nucleic acid molecule can be effectively realized under the mediation of the regulation system, and then the in vitro mass production of the nucleic acid molecule can be realized.

[0010] In a fourth aspect, the present application provides an immune cell. According to an embodiment of the present application, the immune cell carries the nucleic acid molecule of the second aspect or the expression vector of the third aspect or is capable of expressing the chimeric antigen receptor of the first aspect. Thus, the immune cell of the present application can highly express NKG2D, can recognize the corresponding ligand on the surface of the diseased B cells and T cells through NKG2D, can not only clear the abnormal B cells in the body of the self-immune patients such as SLE, but also can clear the abnormal activated pathological T cells in the body of the SLE patients, which is more conducive to fundamentally treating the disease, stabilizing the curative effect and promoting long-term remission.

[0011] In a fifth aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition contains one or more of the following: the chimeric antigen receptor of the first aspect; the nucleic acid molecule of the second aspect; the expression vector of the third aspect; and the immune cell of the fourth aspect. Thus, the pharmaceutical composition prepared based on the chimeric antigen receptor, the recombinant protein, the nucleic acid molecule, the expression vector or the immune cell can effectively target CD19 and CD20, can cover the antigen expression profile of the whole stage of B cell differentiation to prevent escape, and can enhance the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals, which can effectively treat B lymphoma and autoimmune diseases.

[0012] In a sixth aspect, the present application provides the use of the chimeric antigen receptor of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, the immune cell of the fourth aspect or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating and / or preventing B lymphoma, autoimmune diseases. Thus, the medicament prepared on the basis of the chimeric antigen receptor, the recombinant protein, the nucleic acid molecule, the expression vector or the immune cell can effectively target CD19 and CD20, cover the antigen expression profile of the whole stage of B cell differentiation to prevent escape, and enhance the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals, thereby effectively treating B lymphoma and autoimmune diseases.

[0013] Advantages of the present application: (1) The CAR structure of the present application adopts a tandem scFv structure (CD20 VL-VH-linker-CD19 VH-VL), has high coverage of double targets, can simultaneously target CD19 (expressed in early B cells and plasma cells) and CD20 (highly expressed in mature B cells), covers the whole differentiation stage of pathogenic B cells of SLE, and blocks the path of autoantibody production from the source; in addition, the membrane anchor type super IL-15 is integrated at the C-terminal of the CAR structure, which can realize self-secretion membrane-bound cytokine support, improve the expansion ability and survival time of NK cells in vivo, and significantly reduce the relapse rate of SLE; (2) The CAR-NK cell provided by the present application can highly express NKG2D, which recognizes the corresponding ligands on the surface of diseased B cells and T cells through NKG2D, not only to clear abnormal B cells in the body of patients with SLE and other autoimmune diseases, but also to clear abnormal activated pathological T cells in the body of SLE patients, which is more conducive to fundamentally treating diseases, stabilizing curative effects, and promoting long-term remission.

[0014] (3) The CAR-NK cell provided by the present application can stably express FcγRIIIa receptor (CD16), which has a stronger effect on the clearance of self-reactive B cells when combined with anti-CD19 or CD20 antibodies through ADCC effect, and has advantages for the treatment of SLE and other autoimmune diseases.

[0015] (4) Safety and process cost advantages: the NK cell of the present application has no HLA (human leukocyte antigen) restriction, avoiding the risk of GVHD (graft-versus-host disease); in addition, the present application does not require gene editing (such as TRAC / CIITA knockout), adopts an allogeneic NK cell expansion system, and the batch production cycle is shortened to 14-21 days, meeting the commercialization landing needs.

[0016] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the attendant drawings or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0017] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the accompanying drawings, wherein: Figure 1 is a schematic diagram of CAR structure according to embodiment 1 of the present application; Figure 2 is the expression of CD19 and CD20 of RAJI cells, Daudi cells, Farage cells and RPMI8226 cells according to embodiment 3 of the present application; Figure 3 is the killing rate test result of each experimental group on RAJI cells according to embodiment 4 of the present application; Figure 4 is the expression of respective receptors of PBMC-NK cells before expansion and NK cells after expansion according to embodiment 5 of the present application Figure 5 is the killing rate test result of CAR3-NK cells on B lymphoma cells RAJI, Daudi, Farage and human multiple myeloma cells RPMI8226 according to embodiment 5 of the present application; Figure 6 is the expression of CD19 and CD20 of SLE-PBMC-B cells and the killing rate test result of CAR3-NK cells on SLE-PBMC-B or PBMC-B cells according to embodiment 6 of the present application; Figure 7 is the in vivo clearance effect of CAR3-NK cells on B cells according to embodiment 7 of the present application; Figure 8 is the in vivo clearance effect of CAR3-NK cells on T cells according to embodiment 7 of the present application; Figure 9 is the sorting effect of T cells in SLE-PBMC and healthy donor PBMC according to embodiment 8 of the present application; Figure 10 is the expression of NKR related ligands of SLE-PBMC-T cells and PBMC-T cells according to embodiment 9 of the present application; Figure 11 is the killing rate test result of CAR3-NK cells on SLE-PBMC-T cells or PBMC-T cells according to embodiment 10 of the present application. DETAILED DESCRIPTION

[0018] Embodiments of the present application are described in detail below. The embodiments described below are exemplary in nature and are intended to explain the present application, and are not to be considered limiting on the present application.

[0019] It should be noted that the terms "first", "second" are used only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated, nor do they have any precedence or sequence. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0020] In the description of the present application, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0021] In order to more easily understand the present application, certain technical and scientific terms are defined below. Unless otherwise clearly defined in the present application elsewhere, all other technical and scientific terms used in the present application have the meanings generally understood by those skilled in the art to which the present application belongs.

[0022] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including what is indicated in the present application, but not excluding other aspects.

[0023] In the present application, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both cases where the event or condition occurs and cases where the event or condition does not occur.

[0024] In the present application, the term "chimeric antigen receptor" is abbreviated as CAR, which is an artificial receptor molecule manufactured by genetic engineering technology, including an extracellular region (extracellular domain), an intracellular region (intracellular domain) and a transmembrane region (transmembrane domain).

[0025] In the present application, the term "NKG2D ligand" refers to the state of cell surface expression of NKG2D ligand, which is a class of proteins capable of being recognized by NKG2D receptor, mainly expressed on the surface of stressed cells (such as tumor cells or virus-infected cells), is an important target for NK cells and some T cells to recognize and eliminate abnormal cells, and can provide a clear targeting target for CAR-immune cells, which helps to improve the specificity and effectiveness of treatment.

[0026] For nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions]. The number of nucleotides in the second sequence] minus [the total number of nucleotides in the first sequence], then multiplied by [100%], wherein the deletion, insertion, substitution or addition of each nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered as a difference in a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings.

[0027] For polypeptides, the terms "(substantial) homology", "identity" or "similarity" are used to describe or compare the degree of amino acid similarity of two or more polypeptides or specified sequences thereof when optimally aligned and compared (with appropriate insertion or deletion of nucleotides). The % homology between two sequences varies as the number of identical positions shared by these sequences when optimally aligned (i.e. % homology = number of identical positions / total number of positions x 100), wherein the optimal alignment is determined taking into account the number of gaps and the length of each gap that need to be introduced to achieve the optimal alignment of the two sequences. Sequence comparison and percentage identity determination between two sequences can be done using mathematical algorithms, as described in the following non-limiting examples.

[0028] In the present application, the term "expression vector" refers to a specially designed nucleic acid molecule tool, mainly used to achieve efficient transcription and translation of exogenous genes in host cells, thereby producing the desired protein or polypeptide. The expression vector usually contains a strong promoter (such as CMV or EF1a) to drive efficient transcription, a multiple cloning site for inserting the target gene, a terminator to ensure proper termination of transcription, and a selection marker gene (such as a puromycin resistance gene) and a reporter gene (such as GFP) for screening and monitoring gene expression. In addition, the expression vector can also contain an origin of replication to ensure stable replication in host cells, and optimized transcription and translation regulatory elements to improve expression efficiency. The expression vector not only can efficiently introduce exogenous genes into host cells, but also can achieve long-term stable gene expression in various cell types.

[0029] In the present application, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptoms thereof, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease; "treatment" used herein covers diseases of mammals, especially humans, including: (a) preventing the disease or condition from occurring in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, for example, arresting the development of the disease; or (c) relieving the disease, for example, reducing symptoms associated with the disease. "Treatment" used herein covers any administration of a drug or bispecific antibody to an individual to treat, cure, relieve, ameliorate, lessen or inhibit the disease of the individual, including but not limited to administration of a drug containing the drug described herein to an individual in need thereof.

[0030] In the present application, "immune cells" refer to cells from humans, more specifically, the immune cells in the present application can be derived from pluripotent stem cells through directed differentiation. According to the provision of different differentiation conditions, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present application can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be isolated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells; according to the provision of different differentiation conditions, these immune cells can be T cells, NK cells and macrophages; the immune cells in the present application can also be isolated from human blood, bone marrow, lymph or lymphoid organs or specific tissues (including but not limited to tumor tissues, peritoneal cavity, liver, lung, etc. various organs, muscle, etc.). For example, cells of innate or adaptive immunity, for example, bone marrow or lymphocytes, are usually T cells, NK cells and macrophages, etc.; preferably, according to the present application, the immune cells include NK cells; the immune cells of the present application can also be differentiated from immune cell progenitor cells, such as lymphoid progenitor cells.

[0031] In the present application, the term "therapeutically effective amount" refers to the amount of a compound that is sufficient to treat a disease or at least one clinical symptom of a disease when administered to an individual to treat the disease or the clinical symptom of the disease. The "therapeutically effective amount" can vary depending on, for example, the drug, the disease, and / or the symptom of the disease, the severity of the disease, and / or the symptom of the disease or condition, the age, body weight, and / or health status of the individual to be treated, and the judgment of the prescribing physician. An appropriate amount in any given case can be determined by one of skill in the art or can be determined by routine experimentation.

[0032] In the present application, the term "scFv structure" refers to a structure of an antibody or fragment in which an antibody heavy chain variable region and a light chain variable region are connected by a short peptide.

[0033] In the present application, "VL" refers to the variable region (V region) of the light chain (L chain) of a monoclonal antibody (abbreviated as mAb) having a relatively light molecular weight. "VH" refers to the variable region (V region) of the heavy chain (H chain) of a mAb having a relatively heavy molecular weight.

[0034] In the present application, "linker" refers to a connecting peptide.

[0035] In the case of a description of a time relationship, for example, in the case of a description of a time sequence using, for example, "after", "then", "before", and the like, unless "immediately" or "directly" is used, discontinuous cases are also included.

[0036] The present application provides a chimeric antigen receptor, a nucleic acid molecule, an expression vector, an immune cell, a pharmaceutical composition, and uses and methods thereof, which will be described in detail below.

[0037] Chimeric antigen receptor In a first aspect, the present application provides a chimeric antigen receptor. According to embodiments of the present application, the chimeric antigen receptor comprises an extracellular domain capable of binding to CD20 and / or CD19; a transmembrane domain connected to the extracellular domain, and an intracellular domain connected to the transmembrane domain, wherein the intracellular domain is selected from the entire amino acid sequence or a partial amino acid sequence of at least one of the following proteins: 2B4, DAP10 and CD3 zeta. Thus, by using a tandem scFv structure (CD20 VL-VH-linker-CD19 VH-VL), the chimeric antigen receptor structure (CAR structure) of the present application can have a double-targeting high coverage, while targeting CD19 expressed on early B cells and plasma blast cells and CD20 highly expressed on mature B cells, covering the antigen expression profile of the entire stage of B cell differentiation to prevent escape, and enhancing the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals to block the path of autoantibody production from the source. In addition, the use of 2B4, DAP10, and CD3 zeta fusion signal modules in the intracellular domain can significantly enhance the efficiency of immune cell degranulation and cytokine secretion, and adapt the natural killing properties of immune cells.

[0038] In some embodiments, the extracellular domain contains an antibody or antigen-binding fragment thereof targeting CD19 and / or contains an antibody or antigen-binding fragment thereof targeting CD20.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD19 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No: 1 or at least 90% identical thereto. In some embodiments, the antibody or antigen-binding fragment thereof targeting CD19 has a light chain variable region having an amino acid sequence as set forth in SEQ ID No: 2 or at least 90% identical thereto.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD20 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No: 3 or at least 90% identical thereto. In some embodiments, the antibody or antigen-binding fragment thereof targeting CD20 has a light chain variable region having an amino acid sequence as set forth in SEQ ID No: 4 or at least 90% identical thereto.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD19 is a first single-chain antibody having an amino acid sequence as set forth in SEQ ID No: 5. In some embodiments, the antibody or antigen-binding fragment thereof targeting CD20 is a second single-chain antibody having an amino acid sequence as set forth in SEQ ID No: 6. Thus, the CAR structure of the present application can better target CD19 and CD20, thereby better covering the entire differentiation stage of pathogenic B cells of SLE, and blocking the path of autoantibody production from the source.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD19 and the antibody or antigen-binding fragment thereof targeting CD20 are connected by a connecting peptide.

[0043] In some embodiments, the connecting peptide is selected from any one of a flexible Linker, a rigid Linker.

[0044] In some embodiments, the connecting peptide has an amino acid sequence as set forth in any one of SEQ ID Nos: 9-12.

[0045] In some embodiments, the intracellular domain of the chimeric antigen receptor is selected from all or part of the amino acid sequence of at least two of the following proteins: 2B4, DAP10, and CD3 zeta (or CD3Z).

[0046] In some embodiments, the intracellular domain of the chimeric antigen receptor is selected from any one of the following structures: (a): [CD3 zeta-ICD]-[2B4-ICD]; (b): [CD3 zeta-ICD]-[DAP10-ICD]-[2B4-ICD]; wherein, “[]” represents a domain, and “]-[” represents a connection between domains; the connection between the domains is a direct connection or a connection through a second connecting peptide; CD3 zeta-ICD, DAP10-ICD, and 2B4-ICD respectively represent the intracellular activation domains of CD3 zeta, DAP10, and 2B4 proteins.

[0047] Thus, using the 2B4, DAP10, and CD3 zeta fusion signal module (referred to as NCAD) for the intracellular domain can significantly enhance the NK cell degranulation and cytokine secretion efficiency, adapt the natural killing properties of NK cells, further prolong the survival time of NK cells in vivo, and improve the killing ability and long-term anti-autoimmune activity.

[0048] In some embodiments, the CD3 zeta-ICD has an amino acid sequence as set forth in SEQ ID No: 23.

[0049] In some embodiments, the 2B4-ICD has an amino acid sequence as set forth in SEQ ID No: 7.

[0050] In some embodiments, the DAP10-ICD has an amino acid sequence as set forth in SEQ ID No: 8.

[0051] In some embodiments, the transmembrane domain is derived from CD8a. In some embodiments, the transmembrane domain has an amino acid sequence as set forth in SEQ ID No: 13.

[0052] In some embodiments, the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID No: 22.

[0053] In some embodiments, the extracellular domain, the transmembrane domain, and the intracellular domain are directly connected or connected through a linker. In this way, the structural stability of the chimeric antigen receptor is optimized by direct connection or connection through a linker, reducing signal transduction disorders caused by improper connection of domains. This flexible connection mode enables the chimeric antigen receptor to be efficiently expressed in different immune cells and ensures efficient signal transduction, further improving the applicability of the chimeric antigen receptor for different application scenarios.

[0054] It should be noted that there is no particular limitation on the selection of the "linker" in the present application. All linker peptides known in the art that can be used to connect functional amino acids without affecting the function of the functional amino acids themselves are included in the protection scope of the present application and can be used as the linker in the present application.

[0055] Nucleic acid molecule In a second aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule comprises a first nucleic acid fragment and optionally a second nucleic acid fragment, the first nucleic acid fragment and the second nucleic acid fragment being connected; wherein the first nucleic acid fragment encodes the chimeric antigen receptor of the first aspect; and the second nucleic acid fragment is used to encode a fusion protein comprising IL-15 and IL-15R a, the IL-15 and the IL-15R a being connected. In this way, by integrating a membrane-anchored fusion protein at the C-terminal end of the CAR structure, autocrine membrane-bound cytokine support can be achieved, solving the key bottleneck of insufficient in vivo expansion and short survival period of cells. In addition, based on the above nucleic acid molecule, the chimeric antigen receptor and the fusion protein can be efficiently expressed in vitro, simplifying the production process.

[0056] In some embodiments, the nucleic acid molecule is DNA.

[0057] It should be noted that, for the nucleic acid molecules mentioned in the present specification and claims, the skilled person in the art should understand that, actually, either one of the complementary double strands, or both, are included. For the convenience, in the present specification and claims, although only one strand is given in most cases, actually, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in the present application include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0058] In some embodiments, the C-terminus of the IL-15 is connected to the N-terminus of the IL-15Ra, or the N-terminus of the IL-15 is connected to the C-terminus of the IL-15Ra. In some embodiments, the IL-15 has an amino acid sequence as shown in SEQ ID No: 14. In some embodiments, the IL-15Ra has an amino acid sequence as shown in SEQ ID No: 15.

[0059] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID No: 16. Thus, by integrating the fusion protein of the present application at the C-terminus of the CAR structure, a self-secreting membrane-bound cytokine support can be achieved, which solves the key bottleneck of insufficient in vivo expansion and short survival period of NK cells.

[0060] In some embodiments, the first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID No: 17. In some embodiments, the second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID No: 18. In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID No: 19.

[0061] Expression vector In the third aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector carries the nucleic acid molecule of the second aspect. Thus, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned nucleic acid molecule can be effectively realized under the mediation of the regulatory system, and then the in vitro mass production of the nucleic acid molecule can be realized.

[0062] It should be noted that, when the aforementioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control elements.

[0063] According to embodiments of the present application, the vector can refer to a cloning vector or an expression vector, which can be obtained by operably linking the nucleic acid to a commercially available vector such as a plasmid or a viral vector. The vector in the present application is not particularly limited, and common plasmids such as pSeTag2, PEE14, pMH3, etc. can be used.

[0064] In the present application, the term "operably linked" refers to the linkage of an exogenous gene to a vector, so that the control elements in the vector, such as transcription control amino acid sequences such as and translation control amino acid sequences such as etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Common vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, the expression of the nucleic acid molecule described above can be effectively realized under the mediation of the regulation system, and thus the in vitro mass acquisition of the protein encoded by the nucleic acid molecule can be realized.

[0065] In some embodiments, the expression vector is selected from a virus, a prokaryotic expression vector, or a eukaryotic expression vector.

[0066] In some embodiments, the vector comprises a virus selected from the group consisting of

[0067] Immune cells In a fourth aspect of the present application, an immune cell is provided. According to embodiments of the present application, the immune cell carries the nucleic acid molecule of the second aspect or the expression vector of the third aspect or is capable of expressing the chimeric antigen receptor of the first aspect. Thus, the immune cell of the present application can highly express NKG2D, recognize the corresponding ligand on the surface of the diseased B cells and T cells through NKG2D, not only clear the abnormal B cells in the body of the patients with autoimmune diseases such as SLE, but also clear the abnormal activated pathological T cells in the body of the patients with SLE, which is more conducive to the radical treatment of the disease, the stability of the therapeutic effect, and the promotion of long-term remission.

[0068] In some embodiments, the immune cell comprises at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδ T cells, and iPSC-derived immune cells. Thus, the chimeric antigen receptor design of the present application can select the most suitable cell platform according to different tumor types and treatment needs, further improving the flexibility and applicability of the treatment.

[0069] In some embodiments, the immune cell is an NK cell. The chimeric antigen receptor of the present application is mainly designed for NK cells, which has a better expression effect in NK cells, enhances the ability to treat autoimmune diseases, and further improves the safety and effectiveness when applied to treatment.

[0070] In some embodiments, the NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells in a culture medium containing interleukins and antibodies. In this way, the in vitro induced expansion of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0071] In some embodiments, the interleukins include at least one of IL-21, IL-2. In some embodiments, the final concentration of the interleukins in the culture medium is 1-100 ng / mL. For example, it can be 1 ng / mL, 3.5 ng / mL, 6.5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc., or it can be a range consisting of any of the above values.

[0072] In some embodiments, the antibodies include at least one of CD16 antibodies, CD3 antibodies. In some embodiments, the final concentration of the antibodies in the culture medium is 0.5 ng / mL-5 μg / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, etc., or it can be a range consisting of any of the above values.

[0073] In some embodiments, the in vitro induced expansion of peripheral blood mononuclear cells includes: separating and purifying first NK cells from the peripheral blood mononuclear cells; placing the first NK cells in a first culture system for first culture treatment; after 3-5 days of culture, adding a first cytokine to the first culture system for second culture treatment to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced with a second culture system. In this way, the method of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0074] In some embodiments, the first culture system includes a serum-free culture medium and at least one of the CD3 antibodies, the CD16 antibodies, and the IL-2. In this way, the use of the first culture system of the present application to culture cells can pre-activate the activation and proliferation signaling pathways of NK cells, make NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0075] In some embodiments, the concentration of the CD3 antibody in the first culture system is 0.5-5 ng / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0076] In some embodiments, the concentration of the CD16 antibody in the first culture system is 1-5 μg / mL. For example, it can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, etc., or a range consisting of any of the aforementioned values.

[0077] In some embodiments, the concentration of the IL-2 in the first culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0078] In some embodiments, the first cytokine is selected from the IL-21. In some embodiments of the present application, the concentration of the IL-21 in the first culture system is 20-100 ng / mL. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0079] In some embodiments, the second culture system comprises a serum-free medium and at least one of the IL-2, the IL-21. Thus, by changing to the second culture system, the activation and proliferation of the NK cells can be further maintained, and the cell aging signal during the cell expansion process can be prevented or delayed, so that the NK cells can maintain a persistent proliferation ability and an activated state.

[0080] In some embodiments, the concentration of the IL-2 in the second culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0081] In some embodiments, the IL-21 has a concentration of 20-100 ng / mL in the second culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0082] Pharmaceutical composition In a fifth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition contains one or more of the following: the chimeric antigen receptor of the first aspect; the nucleic acid molecule of the second aspect; the expression vector of the third aspect; the immune cell of the fourth aspect. Thus, the pharmaceutical composition prepared based on the chimeric antigen receptor, the recombinant protein, the nucleic acid molecule, the expression vector or the immune cell can effectively target CD19 and CD20, cover the antigen expression profile of the whole stage of B cell differentiation to prevent escape, and enhance the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals, thereby effectively treating B lymphoma and autoimmune diseases.

[0083] In some embodiments, the pharmaceutical composition further comprises a CD19 antibody and / or a CD20 antibody. In some embodiments, the CD19 antibody is selected from inebilizumab, tanibirumab. In some embodiments, the CD20 antibody is selected from rituximab, obinutuzumab, ofatumumab, ocrelizumab. The immune cells (CAR-NK cells) in the pharmaceutical composition of the present application highly express CD16 (FcγRIIIa), which can effectively bind the CD19 antibody and / or the CD20 antibody, enhance the antibody-dependent cell-mediated cytotoxicity (ADCC), not only more thoroughly clear the pathogenic B cells, but also effectively kill the pathological T cells, inhibit their excessive activation, thereby reducing the inflammatory response, preventing tissue damage, and maintaining immune homeostasis.

[0084] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0085] In some embodiments, the excipient comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0086] The pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and pharmaceutically acceptable adjuvants. Such adjuvants include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, liposome, lipid, protein, protein-antibody conjugate, peptide, cellulose, nanogel, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the drug of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by conventional methods with normal saline or aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions.

[0087] The effective amount of the active ingredient of the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials). The aforementioned factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be given daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0088] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the ingredients contained in the composition can be administered to the subject as a whole, or administered to the subject separately. When the ingredients contained in the pharmaceutical composition are administered to the subject separately, each ingredient can be administered to the subject simultaneously or sequentially.

[0089] Use In the sixth aspect of the present application, the present application provides the use of the chimeric antigen receptor of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, the immune cell of the fourth aspect or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating and / or preventing B lymphoma, autoimmune disease. Therefore, the medicament prepared on the basis of chimeric antigen receptor, recombinant protein, nucleic acid molecule, expression vector or immune cell can effectively target CD19 and CD20, not only covering the antigen expression profile of the whole stage of B cell differentiation to prevent escape, but also enhancing the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals, which can effectively treat B lymphoma and autoimmune disease.

[0090] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0091] In some embodiments, the drug has at least one of the following effects: eliminating abnormal B cells in the body of an autoimmune disease patient; eliminating abnormal T cells in the body of an autoimmune disease patient. The immune cells of the present application highly express NKG2D, and can recognize the corresponding ligands on the surface of diseased B cells and T cells through NKG2D. Therefore, the drug of the present application can not only eliminate abnormal B cells in the body of an SLE patient, but also can eliminate abnormally activated pathological T cells in the body of an SLE patient, which is more conducive to fundamentally treating the disease, stabilizing the therapeutic effect, and promoting long-term remission.

[0092] In a seventh aspect of the present application, the present application provides the use of the chimeric antigen receptor of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, the immune cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in treating and / or preventing B lymphoma and autoimmune diseases. Therefore, the chimeric antigen receptor of the present application can effectively target CD19 and CD20, covering the antigen expression profile of the whole stage of B cell differentiation to prevent escape, and enhancing the survival and killing ability of cells in the inhibitory microenvironment through synergistic signals, thereby effectively treating B lymphoma and autoimmune diseases. Therefore, the recombinant protein, nucleic acid molecule, expression vector, immune cell, or pharmaceutical composition prepared based on the chimeric antigen receptor have the same effects.

[0093] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0094] In an eighth aspect, the present application provides a method for treating and / or preventing B lymphoma, autoimmune disease. According to an embodiment of the present application, the method comprises administering to a subject a therapeutically effective amount of the chimeric antigen receptor of the first aspect, the isolated nucleic acid of the second aspect, the expression vector of the third aspect, the immune cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect. Thus, by using the method of the present application, B lymphoma, autoimmune disease can be effectively treated.

[0095] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0096] The sequence descriptions according to the present application are shown in Table A.

[0097] Table A. Amino acid / nucleotide sequence description table

[0098]

[0099]

[0100]

[0101] Embodiments of the present application are described in detail below. The embodiments described below are examples only and are not to be construed as limiting the present application. Unless otherwise defined, scientific and technical terms used in the embodiments are to be construed in accordance with the descriptions provided in the literature references cited above. When a product is not commercially available, it can be prepared according to the procedures described in the literature references cited above or according to the procedures described in the product manual.

[0102] Example 1: Novel CAR structure design targeting CD19 and CD20 and gene synthesis Based on previous research, in order to screen the intracellular segment more suitable for the signal transduction of NK cells, the inventors carried out multidimensional tests on signal structure domains such as chemokine receptors (such as CXCR family), transmembrane immune receptors (such as CD3 family, FcεRIγ), G protein-coupled receptor downstream elements (such as Gαi / o, Gαq) and lymphocyte linker proteins (such as SLAM family), and unexpectedly found that the tandem signal module composed of DAP10, 2B4 and CD3ζ has a significant synergistic activation effect, which shows obvious advantages in activating NK cells, that is, the signal transduction segments of these elements can be connected to form a new intracellular activation segment more suitable for NK cells, named NCAD (new cytoplasmic activation domain).

[0103] In order to realize the simultaneous targeting of CD19 and CD20, the present application designs a new CAR molecule (CAR3) based on NCAD, the structure of which is shown in detail in Figure 1 , and the amino acid sequence is SEQ ID No. 22. In addition, in order to study whether the NCAD loaded by CAR3 has stronger function, the present application also prepares two CAR molecules composed of commonly used intracellular segments (CAR1 and CAR2) as controls, and all CAR molecules express a fusion protein (mbIL15RF) composed of IL-15 and IL-15Rα in series, wherein the amino acid sequence of CAR1 is SEQ ID No. 20, and the amino acid sequence of CAR2 is SEQ ID No. 21. The difference between CAR1 and CAR3 is the composition of the intracellular segment.

[0104] Then, the inventors entrust GenScript to synthesize the sequences of CAR1 (SEQ ID No: 20), CAR2 (SEQ ID No: 21) and CAR3 (SEQ ID No: 22) and construct them into expression vectors, and three CAR molecule expression plasmids are constructed, that is, CAR1 plasmid, CAR2 plasmid and CAR3 plasmid are obtained.

[0105] Example 2: Preparation of a series of NK cells loaded with new CAR molecules The three expression plasmids in Example 1, i.e., the CAR1 plasmid, the CAR2 plasmid, and the CAR3 plasmid, and the empty expression plasmid were prepared into lentivirus, and viral concentration was performed to obtain lentivirus of the expression plasmid. The specific operation is as follows. 293T cells (purchased from Promocell) were expanded in advance into 10 cm culture dishes, and the expression plasmid 15 μg, PMD2G 5 μg, PSPAX2 7.5 μg (plasmid purchased from Genechem) were prepared into A liquid supplemented with 375 μL of opti-MEM (purchased from Gibco), and B liquid was prepared according to opti-MEM 345 μL, Transporter5 30 μL (purchased from Polysciences). The B liquid was slowly added to the A liquid, mixed, and incubated at room temperature for 20 min. The 293T cells to be transfected were taken out, and the cell growth was observed under a microscope. The incubated transfection complex was added dropwise to the cell culture dishes, gently shaken, and the culture was evenly distributed on the surface of the culture dish. After 24 h of culture at 37℃, 5% CO2 incubator, the liquid was changed. The culture solution was carefully aspirated, and 15 mL of preheated DMEM medium (purchased from HyClone) was added. The cells were returned to the 37℃, 5% CO2 incubator for further culture for 48 h. The virus liquid in the culture dish was aspirated with a 10 mL syringe, and filtered through a 0.45 μm filter (purchased from Millipore) into a 50 mL centrifuge tube, and a total of 12 mL of virus liquid was collected. According to the amount of 1 / 3 volume of each tube of virus liquid, 4× virus precipitation solution (purchased from Biyun Tian) was added, and placed in a 4℃ refrigerator overnight. The next day, 4℃, 3000×g centrifugation for 30 min. The supernatant was removed by a pipette pump, and a total of 500 μL of virus resuspension solution (purchased from Biyun Tian) was added. The precipitate was mixed and dispersed with a pipette to obtain concentrated lentivirus. The above lentivirus was added to 1×10 6 The NKG cell line (from the Institute of Immunology, School of Life Sciences, University of Science and Technology of China) was infected with the above lentivirus at a MOI of 20, and after 48 h of infection, puromycin was continuously added for screening and expansion to obtain CAR1-NK stable cell line, CAR2-NK stable cell line, CAR3-NK stable cell line, and empty expression plasmid NK stable cell line.

[0106] Example 3: Target cell screening for in vitro functional verification In order to screen the target cells to verify the in vitro killing effect of the CAR-NK stable cell line, the inventors detected and analyzed the expression of CD19, CD20 of human B lymphoma cells RAJI, Daudi, Farage, and human multiple myeloma peripheral blood B lymphocytes RPMI8226. Specifically, the tested cells were collected, washed twice with PBS, resuspended in FACS solution (containing 0.1% sodium azide and 0.4% BSA in PBS), counted, and the cell concentration was adjusted to 1×106 CD20 antibody and isotype control antibody, respectively, and incubated at 4°C for 30 minutes; after washing twice with PBS, resuspending in FACS fluid, analyzing cell fluorescence by flow cytometry, and analyzing the results by FlowJo software (Note: the antibody isotype control group is red, and the subsequent experiments are the same). Figure 2 As shown in FIG. 2, human B lymphoma cells RAJI, Daudi, and Farage highly express CD19 and CD20; human multiple myeloma RPMI8226 does not express CD19 and CD20.

[0107] Example 4: In vitro killing function verification of CAR-NK cells Next, the CAR-NK stable cell lines established in Example 2 and the empty plasmid NK stable cell lines were tested for in vitro killing activity by DAPI and CFSE staining. The suspended cells RAJI were stained with CFSE after being inoculated at a density of 1x10 5 The groups set are as follows: (1) Blank control group: naturally dead target cells (2) Traditional control group: killing of target cells by the empty plasmid NK stable cell line in Example 2 (3) Experimental group: killing of target cells by the three CAR-NK stable cell lines prepared in Example 2 The effector-to-target ratio of NK cells to target cells in the traditional control group and the experimental group was 3:1. After 24 hours of mixed culture, the supernatant was centrifuged, the cell pellet was washed, and then stained with DAPI. The stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. Figure 3 For the killing rate test results of RAJI cells as target cells, it can be seen that for RAJI cells which highly express CD19 and CD20, the three CAR-NK stable cell lines all have obvious killing effect (compared with the empty plasmid NK stable cell line), and the CAR3-NK stable cell line has the strongest killing ability.

[0108] Example 5: Expansion culture of CAR3-NK cells and further verification of in vitro killing function Because the NK cell lines used in the foregoing examples are derived from tumors, their biological characteristics and safety cannot meet the requirements of clinical application. In order to realize the clinical application value of NK cell drugs, the inventors selected healthy donor-derived NK cells as the basis for further preparation of CAR3-NK cells for subsequent research.

[0109] 1. Protein expression of allogeneic peripheral blood-derived NK cells before and after in vitro expansion The inventors studied the differences in the expression of important functional proteins of allogeneic peripheral blood-derived NK cells before or after in vitro expansion. The allogeneic peripheral blood NK cells were expanded using factor expansion method, and the specific process of expansion was as follows: Peripheral blood was collected from healthy donors to obtain peripheral blood mononuclear cells (PBMCs). Then, the NK cells were separated and purified using an NK cell purification kit (purchased from Meitianyida). The separated NK cells were placed in KBM581 serum-free medium containing 1 ng / mL CD3 antibody, 2 μg / mL CD16 antibody and 5 ng / mL IL-2 for initial culture. After 3-5 days of culture, 50 ng / mL IL-21 was added to the culture system for culture. During the culture process, the culture medium (KBM581 serum-free medium) was replaced regularly according to the cell growth state and fresh cytokines (5 ng / mL IL-2 and 50 ng / mL IL-21) were supplemented, and the culture was continued for 20-40 days. After the culture ended, the cell population was harvested, and finally high-purity, high-viability and enhanced cytotoxicity allogeneic NK cells were obtained. This method can achieve efficient expansion of allogeneic NK cells without feeder cells, and provides a stable and reliable cell source for the preparation of "off-the-shelf" NK cell immunotherapy products.

[0110] To verify the expression of important functional proteins such as receptors of allogeneic peripheral blood-derived NK cells expanded in vitro, the expression levels of representative molecules such as NCR receptor family (NKp30, NKp44), C agglutinin receptor family (CD69, NKG2D, NKG2C), activation-related receptor (CD25, DNAM-1), chemotaxis-related (CXCR3, LFA-1), proliferation-related (Ki67), killing-related (CD107a, Granzyme B, Perforin, IFN-γ, 4-1BB, TRAIL, CD16) were detected by flow cytometry. Specifically, the allogeneic peripheral blood-derived NK cells expanded in vitro and their corresponding PB-NK cells before expansion (i.e., the NK cells directly obtained after separation and purification, as a control group) were collected, washed twice with PBS, resuspended in FACS fluid (containing 0.1% sodium azide and 0.4% bovine serum albumin BSA in PBS), counted, and adjusted to a cell concentration of 1 × 10 6 Figure 4 ​As shown, compared with the PB-NK cell control, the allogeneic peripheral blood-derived in vitro expanded NK cells highly expressed NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, 4-1BB, suggesting that the in vitro expansion culture comprehensively enhanced the killing, activation, proliferation and chemotaxis ability of the NK cells. For the molecules beneficial to the NK cell function, such as NKG2C, TRAIL, CD16, DNAM-1, which were highly expressed in the PB-NK cells, the expanded NK cells also maintained a high expression level, proving that the allogeneic peripheral blood-derived in vitro expanded NK cells related to the present application have a unique functional related protein expression profile, meaning a substantial improvement in the NK cell function.

[0111] 2. Preparation of CAR3-NK cells from the expanded NK system The inventors used the lentivirus prepared by the CAR3 plasmid with the strongest killing ability in Example 4 to infect the PBMC-NK cells expanded in Step 1 of the present example, and then prepared the CAR3-NK cells of the PBMC-NK system. The specific process is as follows: The PBMC-derived NK cells were prepared by the method of Step 1 of the present example, and on the 7th day of culture, the NK cells were infected with lentivirus containing the CAR3 plasmid, and then expanded for 21 days to obtain the PBMC-derived CAR3-NK cells. The in vitro killing activity of the CAR3-NK cells was tested by DAPI and CFSE staining with 3 B lymphoma cells RAJI, Daudi and Farage and 1 human multiple myeloma RPMI8226 as target cells. After the tumor cells were stained with CFSE fluorescence, they were plated in the culture plate at a seeding density of 1x10 5 The groups were set as follows: (1) Blank control group: naturally dead target cells (2) Traditional control group: NK cells expanded in Example 5 killing target cells (3) Experimental group: CAR3-NK cells prepared in Example 5 killing target cells The effector-target ratio of the NK cells, CAR3-NK cells and target cells was 1:10 (except for RAJI, which was 1:1). After 4 hours of mixed culture, the supernatant was removed by centrifugation, and the cell precipitate was washed and then stained with DAPI. The stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. Figure 5For the killing rate test results of four kinds of cells as target cells, it can be seen that CAR3-NK has obvious killing effect on RAJI, Daudi and Farage cells with high expression of CD19 and CD20, and the killing efficiency is obviously improved compared with NK. While for RPMI8226 cells without expression of CD19 and CD20, the killing efficiency of NK and CAR3-NK has no obvious difference compared with the blank group.

[0112] Example 6: SLE-PBMC-B sorting for in vitro functional verification and CAR-NK in vitro killing SLE-PBMC-B functional verification In order to verify whether the CAR3-NK cells have killing effect on SLE-PBMC-B cells, the inventors sorted CD19 + and CD20 + B cells from the PBMC of SLE patients, and detected the joint killing effect. Specifically as follows: 1×10 8 SLE-PBMC frozen in liquid nitrogen was taken out, and after the liquid nitrogen volatilized, it was quickly placed in a 37-degree water bath for thawing; immediately after thawing, it was added to Buffer and transferred to a 15 mL centrifuge tube, 4°C, 300g centrifugation for 5 minutes; after removing the supernatant, 800 μL of Buffer (Buffer is commercial PBS+0.5% human serum albumin HSA) was used for resuspension.

[0113] 100 μL of CD19 + MACS microbeads and 100 μL of CD20 + MACS microbeads were mixed at 1:1, added to 800 μL of cell resuspension, mixed and incubated at 4°C for 15 minutes; then 10 mL of Buffer was added for resuspension, 4°C, 300g centrifugation for 5 minutes, the supernatant was removed, and finally 500 μL of Buffer was used to resuspend the cells; 3 mL of Buffer was used to rinse the LS column, and the resuspended cells were added to the LS column, and the eluate was collected, which was CD19 - and CD20 - cells; then the LS column was washed with 3 mL of Buffer for 3 times; finally, 2.5 mL of Buffer was added to wash out the CD19 + and CD20 + cells, and this step was repeated once. The collected CD19 + and CD20 + cells were counted, and the cell concentration was adjusted to 1×10 6PBMC were collected and resuspended in FACS buffer, and then added with PE-CD19, FITC-CD20 and isotype control antibody, respectively, and incubated at 4°C for 30 minutes. After washing twice with PBS, the cells were resuspended in FACS buffer, and the fluorescence of the cells was analyzed by flow cytometry, and the results were analyzed by FlowJo software. The results are shown in Figure 6. Figure 6 As shown on the left, the cells collected in the enrichment solution highly expressed CD19 and CD20, proving that the B cells expressing CD19 and CD20 were successfully sorted from the SLE-PBMC.

[0114] Next, the killing effect was detected in vitro. According to the killing detection experiment procedure and experimental grouping in Example 5, the effector cells and target cells were mixed at an effector-to-target ratio of 1:2 or 1:5 for 4 hours, and then centrifuged to remove the supernatant. After washing the cell precipitate, DAPI staining was performed, and the stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. Figure 6 As shown on the right, the killing rate test results for 20 hours of SLE-PBMC-B cells as target cells were obtained. It can be seen that compared with NK cells, CAR3-NK cells have obvious killing effect on SLE-PBMC-B.

[0115] Example 7: In vivo killing function verification of CAR-NK cells BRGSF mice are a severe immunodeficient model constructed by combining knockout of Rag2, Il2rg and Flk2 genes and introduction of SirpaNOD alleles. It is characterized by the simultaneous lack of T cells, B cells, NK cells and significantly reduced myeloid cells. This strain is superior to traditional models due to its extremely high degree of immunodeficiency and excellent transplantation compatibility of human cells / tissues, and it also has stronger irradiation tolerance due to the lack of PrkdcSCID mutation, which facilitates the construction of humanized models.

[0116] To verify whether CAR3-NK cells have killing effect on B cells in vivo, the inventors constructed a hPBMC humanized mouse model using SLE patient-derived PBMC on the basis of BRGSF female mice, and verified the killing effect of CAR3-NK cells on human SLE-B cells in vivo. The mice were administered via the tail vein 5 days after humanization reconstruction. The groups were set as follows: (1) Blank control group: injection of PBS (2) Traditional control group: injection of NK cells expanded in Example 5 (3) Experimental group: injection of CAR3-NK cells prepared in Example 5 The doses of NK cells and CAR3-NK cells were 5x10 6One day after drug administration, peripheral blood was collected from mice to determine the percentage of human immune cells. The preparation of mouse peripheral blood single-cell suspension was as follows: peripheral blood was collected from mice and heparin sodium was added for anticoagulation; centrifuged at 500 g for 5 minutes at 4°C, the supernatant was removed, and 100 μL of whole blood was added to 1 ml of erythrocyte lysis buffer for lysis in the dark for 5 minutes; centrifuged at 500 g for 5 minutes at 4°C to remove the supernatant; washed twice with PBS, resuspended in FACS solution, counted, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells were injected at a concentration of [number] cells / ml and blocked with mouse serum for 15 minutes. Then, antibodies against human AC7-CD45, PC5.5-CD3, BV785-CD56, BV510-CD19, FITC-CD4, BV605-CD8, and BV660-CD14 were added, and the cells were incubated at 4°C for 30 minutes. After washing the cell pellet, the cells were stained with DAPI, and stained cells were obtained by flow cytometry. The results were analyzed using FlowJo software. Results are as follows: Figure 7 As shown, CAR3-NK cells have a significant killing effect on human SLE-B cells in mice compared with NK cells. Figure 8 The percentage of other human immune cells in mice one day after drug administration shows the effect of CAR3-NK cells on CD4 cells in human SLE patients in mice. + T cells and CD8 + T cells all have a certain killing effect.

[0117] Example 8: SLE-PBMC-T sorting for in vitro functional validation To verify why CAR3-NK cells have a killing effect on SLE-PBMC-T cells, the inventors isolated CD4+ cells from PBMCs of SLE patients. + and CD8 + T cells are used for subsequent detection of kill-related ligands. The specific steps are as follows: using CD4... + MACS microbeads and CD8 + MACS microbead sorting of SLE-PBMC-T cells, and collection of CD4 cells + and CD8 + Cell count, adjust cell concentration to 1×10⁻⁶ 6 Cells / ml; FITC-CD4, BV605-CD8 antibodies and isotype control antibodies were added, and the cells were incubated at 4°C for 30 minutes; after washing twice with PBS, the cells were resuspended in FACS solution, and cell fluorescence was analyzed by flow cytometry. The results were analyzed using FlowJo software. Results are as follows: Figure 9 As shown, the cells collected in the enrichment medium highly expressed CD4 and CD8, proving that CD4 was successfully isolated from SLE-PBMCs. + and CD8 +T cells were also selected from CD4+ cells in healthy donor PBMCs. + and CD8 + T cells served as a control.

[0118] Example 9: Detection of SLE-PBMC-T related ligands To investigate the reasons for T cell cytotoxicity by NK cells in SLE, flow cytometry was used to directly compare the expression of NKR-related ligands between SLE-T cells and healthy donor PBMC-T cells. Next, the inventors examined the expression of ULBP1 / 2 / 3 / 4 / 5 / 6 and MICA / B-related ligands in SLE-PBMC-T cells and healthy donor PBMC-T cells. Specifically, cells were washed twice with PBS, resuspended in FACS solution, counted, and the cell concentration was adjusted to 1×10⁶. 6 Cells / ml; APC-ULBP1, Alexa Fluor 700-ULBP2 / 5 / 6, APC-ULBP3, PE-ULBP4, APC-MICA / B antibody and isotype control antibody were added respectively, and incubated at 4°C for 30 minutes; after washing twice with PBS, the cells were resuspended in FACS solution, and the cell fluorescence was analyzed by flow cytometry, and the results were analyzed using FlowJo software. Results are as follows: Figure 10 As shown, compared with healthy donor PBMC-T cells, SLE-PBMC-T cells highly express NKG2D ligands ULBP1, ULBP4, and MICA / B. This suggests that CAR-NK cells may recognize these NKG2D ligands expressed on the surface of pathogenic T cells from SLE patients through their inherently expressed NKG2D, thereby exerting a killing and clearance effect on pathogenic T cells.

[0119] Example 10: Validation of CAR-NK cell-mediated killing of SLE-PBMC-T cells in vitro Next, the in vitro killing effect of CAR3-NK cells on SLE patient PBMC-T cells and healthy donor PBMC-T cells was examined. CAR3-NK cells prepared in Example 5 were mixed with SLE-PBMC-T cells or PBMC-T cells at a 2:1 ratio and cultured for 4 hours. After centrifugation and removal of the supernatant, the cell pellet was washed and stained with DAPI. Stained cells were obtained using flow cytometry, and the results were analyzed using FlowJo software. The results are as follows: Figure 11 As shown, CAR3-NK cells significantly killed SLE-PBMC-T cells compared to healthy donor PBMC-T cells, which is consistent with the results of SLE-PBMC-T cells expressing high levels of NKG2D ligands ULBP1, ULBP4, and MICA / B.

[0120] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0121] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that, include: An extracellular domain that can bind to CD20 and / or CD19; A transmembrane domain, which is connected to the extracellular domain, and An intracellular domain, which is connected to the transmembrane domain. The intracellular domain is selected from the complete or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10 and CD3ζ.

2. The chimeric antigen receptor according to claim 1, characterized in that, The extracellular domain contains an antibody or antigen-binding fragment thereof targeting CD19 and / or contains an antibody or antigen-binding fragment thereof targeting CD20.

3. The chimeric antigen receptor according to claim 2, characterized in that, The antibody targeting CD19 or its antigen-binding fragment has a heavy chain variable region as shown in SEQ ID No: 1 or having at least 90% identity with it; Optionally, the antibody targeting CD19 or its antigen-binding fragment has a light chain variable region as shown in SEQ ID No: 2 or having at least 90% identity with it; Optionally, the antibody targeting CD20 or its antigen-binding fragment has a heavy chain variable region as shown in SEQ ID No: 3 or having at least 90% identity with it; Optionally, the antibody targeting CD20 or its antigen-binding fragment has a light chain variable region as shown in SEQ ID No: 4 or having at least 90% identity with it; Optionally, the antibody targeting CD19 or its antigen-binding fragment is a first single-chain antibody having the amino acid sequence shown in SEQ ID No: 5; Optionally, the antibody targeting CD20 or its antigen-binding fragment is a second single-chain antibody having the amino acid sequence shown in SEQ ID No:

6.

4. The chimeric antigen receptor according to claim 3, characterized in that, The antibody targeting CD19 or its antigen-binding fragment and the antibody targeting CD20 or its antigen-binding fragment are linked by a linker peptide; Optionally, the linker peptide is selected from either a flexible linker or a rigid linker; Optionally, the linker peptide has an amino acid sequence as shown in any one of SEQ ID No: 9 to 12.

5. The chimeric antigen receptor according to claim 4, characterized in that, The intracellular domain of the chimeric antigen receptor is selected from the complete or partial amino acid sequences of at least two of the following proteins: 2B4, DAP10, and CD3ζ; Optionally, the intracellular domain of the chimeric antigen receptor is selected from any of the following structures: (a): [CD3ζ-ICD]-[2B4-ICD]; (b): [CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]; Wherein, "[]" represents a structural domain, and "]-[" represents the connection between structural domains; The connections between the structural domains are either direct connections or connections via a second linker peptide; The CD3ζ-ICD, DAP10-ICD, and 2B4-ICD represent the intracellular activation domains of the CD3ζ, DAP10, and 2B4 proteins, respectively.

6. The chimeric antigen receptor according to claim 5, characterized in that, The CD3ζ-ICD has the amino acid sequence shown in SEQ ID No:23; Optionally, the 2B4-ICD has an amino acid sequence as shown in SEQ ID No: 7; Optionally, the DAP10-ICD has an amino acid sequence as shown in SEQ ID No:

8.

7. The chimeric antigen receptor according to claim 6, characterized in that, The transmembrane domain originates from CD8α; Optionally, the transmembrane domain has an amino acid sequence as shown in SEQ ID No:

13.

8. The chimeric antigen receptor according to any one of claims 1-7, characterized in that, The chimeric antigen receptor has the amino acid sequence shown in SEQ ID No:

22.

9. A nucleic acid molecule, characterized in that, It includes a first nucleic acid fragment and optionally a second nucleic acid fragment, wherein the first nucleic acid fragment and the second nucleic acid fragment are linked together; Wherein, the first nucleic acid fragment encodes the chimeric antigen receptor according to any one of claims 1-8; the second nucleic acid fragment is used to encode a fusion protein, the fusion protein comprising IL-15 and IL-15Rα, wherein IL-15 and IL-15Rα are linked together.

10. The nucleic acid molecule according to claim 9, characterized in that, The C-terminus of IL-15 is connected to the N-terminus of IL-15Rα, or the N-terminus of IL-15 is connected to the C-terminus of IL-15Rα. Optionally, the IL-15 has the amino acid sequence shown in SEQ ID No: 14; Optionally, the IL-15Rα has an amino acid sequence as shown in SEQ ID No: 15; Optionally, the fusion protein has the amino acid sequence shown in SEQ ID No: 16; Optionally, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID No: 17; Optionally, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID No:

18.

11. The nucleic acid molecule according to claim 10, characterized in that, The nucleic acid molecule has the nucleotide sequence shown in SEQ ID No:

19.

12. An expression carrier, characterized in that, Carrying the nucleic acid molecule according to any one of claims 9-11; Optionally, the expression vector is selected from viruses, prokaryotic expression vectors, or eukaryotic expression vectors; Optionally, the expression vector is selected from viruses.

13. An immune cell, characterized in that, The immune cells carry the nucleic acid molecule as described in any one of claims 9-11 or the expression vector as described in claim 12 or are capable of expressing the chimeric antigen receptor as described in any one of claims 1-8.

14. The immune cell according to claim 13, characterized in that, The immune cells include at least one of the above-mentioned immune cells derived from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδT cells, and iPSCs. Optionally, the immune cells are NK cells; Optionally, the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells in a culture medium containing interleukins and antibodies. Optionally, the interleukin includes at least one of IL-21 and IL-2; Optionally, the antibody includes at least one of CD16 antibody and CD3 antibody; Optionally, the in vitro induction and expansion of peripheral blood mononuclear cells includes: First NK cells were isolated and purified from the peripheral blood mononuclear cells; The first NK cell was placed in a first culture system for a first culture treatment; After culturing for 3-5 days, a first cytokine was added to the first culture system for a second culture treatment to obtain the NK cells; In the second culture process, when changing the culture system, the second culture system is used.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains one or more of the following: The chimeric antigen receptor according to any one of claims 1-8; The nucleic acid molecule according to any one of claims 9-11; The expression vector according to claim 12; The immune cells according to any one of claims 13-14.

16. The pharmaceutical composition according to claim 15, characterized in that, Further includes CD19 antibody and / or CD20 antibody; Optionally, the CD19 antibody is selected from inelizumab and tancituzumab; Optionally, the CD20 antibody is selected from rituximab, oxotuzumab, oxamumab, and oxorubicin.

17. Use of the chimeric antigen receptor according to any one of claims 1-8, the isolated nucleic acid according to any one of claims 9-11, the expression vector according to claim 12, the immune cell according to any one of claims 13-14, or the pharmaceutical composition according to any one of claims 15-16 in the preparation of a medicament, wherein the medicament is used to treat and / or prevent B-cell lymphoma and autoimmune diseases; Optionally, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

18. The use according to claim 17, characterized in that, The drug has at least one of the following effects: Clear abnormal B cells from the bodies of patients with autoimmune diseases; Eliminate abnormal T cells in patients with autoimmune diseases.

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