CNB mutant and application thereof

Abstract

The invention provides a calmodulin B subunit mutant and application of the calmodulin B subunit mutant in preparation of universal immune effector cells. The CNB mutant disclosed by the invention can be used for antagonizing the blocking of CsA, FK506 or volocinorin on an NFAT signal channel; meanwhile, the compound has drug resistance to CsA, FK506 and volocinorin at the same time. The CNB mutant disclosed by the invention is expressed into a universal immune effector cell, and when the CNB mutant is combined with an immunosuppressive drug to treat tumors or autoimmune diseases, the activity of a T cell of a patient can be reduced, so that rejection reaction of the patient to the heterologous universal immune effector cell is avoided, and the curative effect of the heterologous universal immune effector cell is improved. Colonization of universal immune effector cells in the body of a patient is improved, and the treatment effect of the universal immune effector cells is achieved. Meanwhile, after the treatment is finished, the universal immune effector cells can be repelled after the immune system of the patient is recovered, so that the strategy that the immune cells are removed after the treatment becomes possible, and the safety of the immune cell therapy can be greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a CNB (calcineurin B subunit) mutant and its application in preparing universal immune cells (such as universal T cells). Background Art

[0002] Chimeric Antigen Receptor T-Cell (CAR-T) immunotherapy is a very promising immunotherapy method today, and has shown higher survival rates in many Phase III clinical trials for advanced cancers.

[0003] The basic structure of CAR usually includes an antigen binding region, a transmembrane region and an intracellular signal transduction region. CAR can directly target tumor surface antigens, activate CAR-T cells and kill tumor cells. At present, T cells for CAR-T therapy are mostly taken from the patient's own peripheral blood. CAR is expressed on the surface of T cells in vitro through viral or non-viral technology and amplified in vitro. Then CAR-T cells are re-injected into the patient to treat the disease. Although autologous CAR-T therapy has achieved great success, its application range is very limited. First, this "personalized" treatment plan is very expensive, far beyond the affordability of ordinary families. Second, the preparation of autologous CAR-T cells takes about three weeks, and some seriously ill patients may have extremely worsening conditions or even death during the preparation process. Third, due to the disease or previous treatment (such as radiotherapy and chemotherapy), the number of T cells in the patient's body may be small, or the quality of T cells is poor, making it difficult to meet the requirements for T cells in preparation. At this stage, the efficacy of autologous CAR-T therapy may vary from person to person and is difficult to evaluate.

[0004] Therefore, the demand for universal CAR-T therapy has emerged. Universal CAR-T cells are taken from healthy human T cells, and their quality and quantity are guaranteed. CAR-T preparation can be scaled and unified, greatly reducing the preparation cost. More importantly, universal CAR-T cells can be prepared in advance, frozen and used at any time, making CAR-T therapy more immediate. Since universal CAR-T therapy is based on healthy people rather than autologous T cells, this therapy is bound to face two major problems in allogeneic cell transplantation: graft-versus-host disease (GVHD) and host rejection of grafts (host-versus-graft rejection, HVGR). The former is caused by the attack of donor cells on recipient cells, which can cause serious or even life-threatening complications. The latter is caused by the rejection of donor cells by recipient cells, making it difficult for CAR-T cells to exist and proliferate in the patient's body for a long time, reducing the tumor killing effect and losing the subsequent tumor monitoring effect.

[0005] The main solution to universal CAR-T cell GVHD is genetic modification of T cells. Since the occurrence of GVHD is mainly related to the αβT cell surface receptor (T cell receptor, TCR), existing research is mostly devoted to knocking out the αβTCR on the surface of T cells through various genetic tools such as TALENs, CRISPR-Cas9, etc. In recent years, researchers have been able to efficiently knock out the αβTCR and accurately introduce CAR at specific sites. This one-step "knockout TCR + knock-in CAR" method reduces the CAR self-activation signal, internalizes the expression of CAR, slows down the differentiation and exhaustion of effector T cells, and finally enhances the anti-tumor ability of CAR-T cells. It is one of the mainstream methods for the preparation of universal CAR-T today.

[0006] Another important problem of universal CAR-T cells is the occurrence of HVGR, which affects the proliferation and anti-tumor effect of donor CAR-T cells in vivo. At present, there are two main directions for solving HVGR. The first is to make the HLA of CAR-T cells "invisible" relative to the recipient's immune system, such as by knocking out β2 microglobulin to construct CAR-T cells with HLA-I molecules missing to solve rejection. However, the lack of HLA-I makes it more vulnerable to the attack of the recipient's NK cells. If rejection is to be solved from the perspective of HLA, more complex gene editing may be required to escape the NK cell-mediated rejection reaction. However, excessive gene editing poses great safety risks, such as greater off-target effects and cytotoxicity. Another idea focuses on inhibiting the recipient's immune system, especially T cells. For example, expressing certain molecules that can bind to activated lymphocyte markers on CAR-T cells can inhibit the rejection of CAR-T cells by receptor-activated lymphocytes, thereby escaping receptor rejection. However, since such CAR-T cells can indiscriminately recognize activated lymphocyte markers, they will continue to inhibit the normal functioning of the recipient's lymphatic system, and their safety is questionable.

[0007] Calcineurin (Calcineurin) is a heterodimer composed of 61kD calmodulin binding catalytic subunit calcineurin A (CNA) and 19kD calcium ion binding regulatory subunit calcineurin B (CNB). There are three isozymes of catalytic subunit, each encoded by a separate gene (PPP3CA, PPP3CB and PPP3CC), and two isoforms of regulatory subunit are also encoded by separate genes (PPP3R1, PPP3R2). Calcineurin is a calcium and calmodulin dependent serine / threonine protein phosphatase that can activate the T cells of the immune system. Calcineurin activates NFAT by dephosphorylating activated T cell nuclear factor (NFAT). The activated NFAT is then transferred from the T cell cytoplasm to the nucleus, where it upregulates the expression of interleukin 2 (IL-2) and other proteins, stimulating T cell response. Calcineurin is the target of a class of drugs called calcineurin inhibitors, which include the immunosuppressive drugs cyclosporine A (CsA), voclosporin, pimecrolimus, and tacrolimus (FK506).

[0008] Following the idea of ​​suppressing the receptor immune system, the use of immunosuppressive drugs is becoming a new method to solve HVGR. For example, CsA or FK506 is often used in the treatment of diseases that require stem cell transplantation or organ transplantation. Its mechanism is mainly to bind to cyclophilin to form a complex, inhibit calcineurin phosphorylation, inhibit its nuclear transport process and then block the subsequent activation of the NFAT signaling pathway, thereby inhibiting T cell activation, proliferation and cytokine (such as IL-2 and IFN-γ) transcription and secretion. Therefore, if CsA / FK506 can be used to inhibit recipient T cells, the HVGR response of recipient T cells to donor CAR-T cells will be greatly reduced. Moreover, CsA / FK506 is a first-line drug to prevent rejection of organ transplantation. Patients can use it for life, and its safety has been fully verified. Since CsA / FK506 has a general inhibitory effect on T cells, CAR-T cells need to be modified to enable them to tolerate the inhibitory effect of CsA / FK506, so that CAR-T cells can perform normal killing functions. However, if the modified CAR-T cells exist for a long time, there will be the risk of tumor induction and off-target. Therefore, it is necessary to provide a universal CAR-T cell that can work normally when the receptor immune system is suppressed, which can restart HVGR after the treatment, so that the allogeneic universal CAR-T cells can be cleared by the receptor and evacuated after the treatment is completed.

[0009] There are currently a variety of methods to make T cells tolerant to CsA / FK506, including knocking out CyPA, FKBP12, expressing mutant CNA, CNB, etc. by gene editing. However, these methods have the disadvantages of only antagonizing CsA or FK506, not being able to antagonize two or more immunosuppressants at the same time (such as knocking out CyPA, FKBP12) or having very poor antagonistic efficiency (such as mutant CNA, CNB, FK506 antagonistic efficiency <60%), which brings inconvenience to the flexible clinical application of immunosuppressants. Therefore, there is currently a demand for making T cells more effectively tolerant to immunosuppressants, or further being able to tolerate multiple immunosuppressants at the same time (for example, tolerating CsA and FK506 at the same time, or tolerating CsA, FK506 and voclosporin at the same time). Summary of the invention

[0010] The present invention provides a calcineurin B subunit mutant, which can resist the inhibitory effect of calcineurin inhibitors, that is, in the presence of calcineurin inhibitors, NFAT can still be dephosphorylated and activated. Therefore, the calcineurin B subunit mutant provided by the present invention can at least be used for the preparation of universal immune cells.

[0011] The amino acid sequence of the calcineurin B subunit (CNB for short) mutant of the present invention is based on SEQ ID NO.49 as the starting sequence, and three amino acids X1X2X3 or two amino acids X4X5 are inserted between positions 125 and 126, and X1, X2, X4, and X5 are not acidic amino acids, wherein: X1 is a neutral amino acid or a basic amino acid, X2 is a neutral amino acid, an acidic amino acid or a basic amino acid, X3 is a neutral amino acid or a basic amino acid, X4 is a neutral amino acid and X5 is a basic amino acid, or X4 is a basic amino acid and X5 is a neutral amino acid; when two amino acids X4X5 are inserted, there may be a T129K substitution at position 129.

[0012] Preferably, X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S or W. In certain specific embodiments of the invention, X1X2X3 is selected from: QKR, RSY, KSC, SCR, LSA, PSR, RRL, TRL, RTT, NRA, LHA, YSP, SRV, KNA, GGC, CKL, KSR, RPK, KSM, REQ, ARQ, TAV, FRV, LSY, RVF, ASI, RTG, RGN, KWV, GRV, RSA, STC, KGA, KAS, RST, RRG, RRW, LMC, FSV, TPV, IRV, TRG, TRV or GSQ.

[0013] Preferably, when X4 is a neutral amino acid and X5 is a basic amino acid, X4 is selected from a neutral amino acid with an isoelectric point (PI) of 5.02-5.70, such as F, Y, Q, N, S, C, M or T, and X5 is R or K; preferably, X4 is selected from S or T, and X5 is R; more preferably, X4X5 is SR.

[0014] Preferably, when X4 is a basic amino acid and X5 is a neutral amino acid, X4 is selected from K, R or H, and X5 is selected from a neutral amino acid with an isoelectric point (PI) of 5.88-6.30, such as W, P, G, A, V, L or I; preferably, X4 is selected from K or H, and X5 is selected from G, A, V, L or I; more preferably, X4X5 is KI or HV.

[0015] Preferably, the amino acid sequence of the CNB mutant is based on SEQ ID NO.49, with only three amino acids X1X2X3 or two amino acids X4X5 inserted between positions 125 and 126. When two amino acids X4X5 are inserted, there may be a T129K substitution at position 129.

[0016] In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.1-47.

[0017] In a preferred embodiment of the present invention, in the amino acid sequence of the CNB mutant, X1 is a basic amino acid selected from R, K or H, preferably R or K; X2 is a neutral amino acid, an acidic amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid. In this preferred embodiment, X2 may preferably be S, R, T, G, K, P, A, V, C, H, N, E, W or M, more preferably S, R, T, G, P, A, V, N, E or W. In this preferred embodiment, X3 may preferably be V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S or W, more preferably V, A, R, C, L, Q, G, Y, T, K, M, F, N, S or W. In this preferred embodiment, preferably X2 is S, R, T, G, K, P, A, V, C, H, N, E, I, W or M, and X3 is V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S or W. In this preferred embodiment, more preferably X2 is S, R, T, G, P, A, V, N, E or W, and X3 is V, A, R, C, L, Q, G, Y, T, K, M, F, N, S or W. In this preferred embodiment, X1X2X3 is selected from REQ, RGN, RPK, RRG, RRL, RRW, RSA, RST, RSY, RTG, RTT, RVF, KAS, KGA, KNA, KSC, KSM, KSR, KWV.

[0018] In one form of this preferred embodiment, X1 is selected from R; X2 is selected from S, R, T, G, P, V or E; X3 is selected from A, Q, G, Y, L, T, K, N, W or F. For example: X1X2X3 is selected from REQ, RGN, RPK, RRG, RRL, RRW, RSA, RST, RSY, RTG, RTT, RVF. Preferably: X1 is selected from R; X2 is selected from R, T, G or V; X3 is selected from G, L, T, N, W or F. For example: X1X2X3 is selected from RGN, RRL, RRW, RTG, RTT, RVF. More preferably: X1 is selected from R; X2 is selected from R or T; X3 is selected from G, L or W. For example: X1X2X3 is selected from RRL, RRW, RTG.

[0019] In one form of this preferred embodiment, X1 is selected from K; X2 is selected from S, N, G, A or W; X3 is selected from A, C, R, M, V or S. For example: X1X2X3 is selected from KAS, KGA, KNA, KSC, KSM, KSR, KWV. Preferably: X1 is selected from K; X2 is selected from S, N, I or W; X3 is selected from A, C, R or V. For example: X1X2X3 is selected from KNA, KSC, KSR, KWV. More preferably: X1 is selected from K; X2 is selected from S, I or W; X3 is selected from C or V. For example: X1X2X3 is selected from KSC, KWV.

[0020] In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 2, 3, 7, 9, 14, 17-20, 26, 28-30, 32, 34-37, 39.

[0021] In a preferred embodiment of the present invention, in the amino acid sequence of the CNB mutant, X1 is a neutral amino acid selected from S, L, T, G, A, F, Q, P, N, Y, C or I, preferably S, L, T, G, A or F; X2 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid. In this preferred embodiment, X2 may preferably be S, R, T, G, K, P, A, C, H or M, more preferably R, K or S. In this preferred embodiment, X3 may preferably be V, A, R, C, L, Q, G, Y, P or I. In this preferred embodiment, preferably X2 is S, R, T, G, K, P, A, C, H or M, and X3 is V, A, R, C, L, Q, G, Y, P or I. In this preferred embodiment, more preferably X2 is R, K or S, and X3 is V, A, R, C, L, Q, G, Y, P or I. In this preferred embodiment, X1X2X3 is selected from ARQ, ASI, CKL, FRV, FSV, GGC, GRV, GSQ, IRV, LHA, LMC, LSA, LSY, NRA, PSR, QKR, SCR, SRV, STC, TAV, TPV, TRG, TRL, TRV, YSP.

[0022] In one form of this preferred embodiment, X1 is selected from T; X2 is selected from R, A or P; X3 is selected from G, L or V. For example: TAV, TPV, TRG, TRL, TRV.

[0023] In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.1, 4-6, 8, 10-13, 15-16, 22-25, 27, 31, 33, 40-42, 44-47.

[0024] In a preferred embodiment of the present invention, in the amino acid sequence of the CNB mutant, X2 is a basic amino acid selected from R, K or H, preferably R or K; X1 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid. In this preferred embodiment, X1 may preferably be L, C, Q, R, S, T, G, A, F, N or I. In this preferred embodiment, X3 may preferably be V, A, L, R, Q, G or W. In this preferred embodiment, preferably X1 is L, C, Q, R, S, T, G, A, F, N or I, and X3 is V, A, L, R, Q, G or W. In this preferred embodiment, X1X2X3 is selected from LHA, CKL, QKR, RRL, RRG, RRW, SRV, TRL, TRG, TRV, GRV, ARQ, FRV, NRA or IRV.

[0025] In one form of this preferred embodiment, X2 is selected from R; X1 is selected from R, S, T, G, A, F, N or I; X3 is selected from V, A, L, Q, G or W. For example: X1X2X3 is selected from RRL, RRG, RRW, SRV, TRL, TRG, TRV, GRV, ARQ, FRV, NRA or IRV. Preferably: X2 is selected from R; X1 is selected from R, S, T, G, A, N or I; X3 is selected from V, A, L, Q or W. For example: X1X2X3 is selected from RRL, RRW, SRV, TRL, TRV, GRV, ARQ, NRA or IRV. More preferably: X2 is selected from R; X1 is selected from R, T, N or I; X3 is selected from V, A, L or W. For example: X1X2X3 is selected from RRL, RRW, TRL, TRV, NRA or IRV.

[0026] In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.1, 7, 8, 10, 11, 13, 16, 22, 24, 31, 37, 39, 44-46.

[0027] In a preferred embodiment of the present invention, in the amino acid sequence of the CNB mutant, X2 is a neutral amino acid selected from A, C, G, M, N, P, S, T, V or W, preferably A, G, P, S, T or V; X1 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid. In this preferred embodiment, X1 may be preferably K, T, S, G, K, R, L, A, F, P or Y. In this preferred embodiment, X3 may be preferably S, V, R, C, A, N, K, I, Q, M, Y, T, P or F. In this preferred embodiment, X1X2X3 is selected from KAS, TAV, SCR, GGC, KGA, RGN, LMC, KNA, RPK, TPV, ASI, FSV, GSQ, KSC, KSM, KSR, LSA, LSY, PSR, RSA, RST, RSY, YSP, RTG, RTT, STC, RVF, KWV.

[0028] In one form of this preferred embodiment, X2 is selected from S; X1 is selected from R, K, L, A, F, G, P or Y; X3 is selected from V, A, R, C, Q, Y, T, P, M or I. For example: X1X2X3 is selected from RSY, RSA, RST, KSC, KSR, KSM, LSA, LSY, GSQ, ASI, FSV, PSR or YSP. Preferably: X2 is selected from S; X1 is selected from K, P or Y; X3 is selected from R, C or P. For example: X1X2X3 is selected from KSC, KSR, PSR or YSP. More preferably: X2 is selected from S; X1 is selected from K or Y; X3 is selected from C or P. For example: X1X2X3 is selected from KSC or YSP.

[0029] In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 2-6, 9, 12, 14-15, 17-19, 23, 25-30, 32-36, 40-42, and 47.

[0030] The calcineurin inhibitors include, but are not limited to, cyclosporine A (CsA), voclosporin, pimecrolimus and tacrolimus (FK506).

[0031] In a further preferred embodiment, the CNB mutant exhibits more prominent FK506 tolerance, wherein X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, F, I, N or W. For example, X1 PV, GGC, TRG, RPK, ASI, STC, LSA, KGA or GSQ; preferably, X1 A, RTT, RRG, LMC, RGN, FRV, REQ, LSY, FSV, TPV, GGC, TRG, RPK, ASI or STC; Further preferably, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SRV, TAV, CKL, KSR, RSY, RSA, LHA, RTT, RRG, LMC, RGN, FRV or REQ; More preferably, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SRV or TAV.In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.28, 12, 7, 10, 30, 39, 8, 44, 46, 31, 3, 14, 1, 26, 6, 4, 22, 13, 23, 16, 17, 2, 32, 11, 9, 37, 40, 29, 24, 20, 25, 41, 42, 15, 45, 18, 27, 33, 5, 34, 47; preferably, the amino acid sequence of the CNB mutant is as shown in SEQ ID NO.28, 12, 7, 10, 30, 39, 8, 44, 46, 31, 3, 14, 1, 26, 6, 4, 22, 13, 23, 16, 17, 2, 32, 11, 9, 37, 40, 29, 24, 20, 25, 41, 42, 15, 45, 18, 27, 33 as shown in any one; further preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.28, 12, 7, 10, 30, 39, 8, 44, 46, 31, 3, 14, 1, 26, 6, 4, 22, 13, 23, 16, 17, 2, 32, 11, 9, 37, 40, 29, 24, 20 as shown in any one; more preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.28, 12, 7, 10, 30, 39, 8, 44, 46, 31, 3, 14, 1, 26, 6, 4, 22, 13, 23.

[0032] In a further preferred embodiment, the CNB mutant exhibits more prominent CsA tolerance, wherein X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, F, I, N or W. For example, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK, KGA, LSA, LMC; preferably, X1X2X3 is selected from YSP, RTG, RR L, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK or KGA; further preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC or FRV; more preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ or RRW; more preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA or TAV; most preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR or SCR.In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34, 5, 40; preferably, the amino acid sequence of the CNB mutant is as shown in SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34 as shown in any one; further preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24; more preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39 as shown in any one; More preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4.

[0033] In a further preferred embodiment, the CNB mutant exhibits more prominent voclosporin resistance, wherein X1 is selected from Y, K, N, A, R, T, S, Q, P, I, L, F or G, X2 is selected from S, W, R, T, K, E, N, H, C, G, A, P, M or V, and X3 is selected from P, V, A, C, I, G, R, L, Q, Y, N, M, K, F, T or W. For example, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA , LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV, RVF, RTT, KGA, RRW, STC, TRG, RST or GS Q; preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV, RVF, RTT, KGA, RRW or STC; further preferably, X1 X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV or RVF; more preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI , RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM or RPK; most preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL or IRV.In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18, 40, 15, 5, 31, 26, 9, 34, 39, 33, 45, 36, 47; preferably, the amino acid sequence of the CNB mutant is as shown in SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18, 40, 15, 5, 31, 26, 9, 34, 39, 33 as shown in any one; further preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18, 40, 15, 5, 31, 26; more preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18 as shown in any one; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44.

[0034] In a further preferred embodiment, the CNB mutant exhibits more prominent FK506 and CsA tolerance, wherein X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK , KGA, LSA, LMC; preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK or KGA; further preferably, X1X2X3 is selected from YSP, RTG, RRL , NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW , ASI, TPV, STC or FRV; more preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ , GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ or RRW; more preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA or TAV; most preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, QKR, KNA, PSR or SCR.In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34, 5, 40; preferably, the amino acid sequence of the CNB mutant is as shown in SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34 as shown in any one; further preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24; more preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23, 32, 2, 45, 26, 16, 20, 39 as shown in any one; More preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4, 13, 9, 22, 31, 11, 23; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 28, 7, 10, 8, 3, 46, 44, 30, 29, 17, 1, 14, 6, 4.

[0035] In a further preferred embodiment, the CNB mutant exhibits more prominent FK506, CsA and voclosporin tolerance, wherein X1X2X3 is selected from the group consisting of YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, RPK, GGC, GRV, RVF, RTT, KGA, RRW, STC, TRG; preferably, X1X2X3 is selected from the group consisting of YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, KSR, RSA, TRL, IRV, REQ, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, RPK, GGC, GRV, RVF, RTT, KGA, RRW, STC, TRG. SY, SCR, RGN, ARQ, TAV, GRV, RVF, RTT, RRW, STC, TRG; further preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, RSA, TRL, IRV, REQ, KNA, LHA, RSY, SCR, RGN, ARQ, TAV, GRV, RVF; more preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, TRL, IRV, KNA, LHA, SCR, RGN, ARQ, TAV; most preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, QKR, PSR, RRL, TRL, IRV.In a specific embodiment of the present invention, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 37, 17, 32, 8, 44, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 18, 15, 31, 26, 9, 34, 39, 33, 45; preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 27, 28, 46, 13, 1, 6, 7, 17, 32, 8, 44, 20, 14, 11, 24, 2, 4, 29, 22, 23, 31, 26, 9, 39, 33, 45; further preferably, the amino acid sequence of the CNB mutant is as shown in any one of SEQ ID NO.12, 30, 10, 3, 28, 46, 13, 1, 6, 7, 17, 32, 8, 44, 20, 14, 11, 2, 4, 29, 22, 23, 31, 26; More preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 30, 10, 3, 28, 46, 13, 1, 6, 7, 17, 8, 44, 14, 11, 4, 29, 22, 23; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.12, 30, 10, 3, 28, 46, 1, 6, 7, 8, 44.

[0036] The present invention uses the random mutation library screening commonly used in the art to obtain the CNB mutant of the present invention, and the experimental results show that the CNB mutant of the present invention has tolerance to calcineurin inhibitors. In the cell experiment, after the CNB mutant of the present invention is transferred into the expression cell, in the presence of calcineurin inhibitors, the cell's tolerance to calcineurin inhibitors is at least 10%, such as 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, preferably at least 30%, more preferably at least 50%, and most preferably at least 100%. The calculation method of the tolerance is: for the same cloned cell, (a certain cell function after adding calcineurin inhibitor) / (a certain cell function without calcineurin inhibitor)×100%. In one embodiment of the present invention, the amount of cell expression signal protein GFP is used as a manifestation of cell function. Those skilled in the art can prepare the CNB mutant of the present invention using the protein preparation method commonly used in the art.

[0037] In some embodiments of the present invention, the CNB mutant of the present invention has tolerance to CsA. After the CNB mutant of the present invention is transferred into cells, in the presence of CsA, the tolerance of the cells to CsA is at least 30%, preferably at least 50%, more preferably at least 80%, and most preferably at least 100%.

[0038] In some embodiments of the present invention, the CNB mutant of the present invention has tolerance to FK506. After the CNB mutant of the present invention is transferred into cells, in the presence of FK506, the tolerance of the cells to FK506 is at least 50%, more preferably at least 80%, and most preferably at least 100%.

[0039] In some embodiments of the present invention, the CNB mutant of the present invention has tolerance to voclosporin. After the CNB mutant of the present invention is transferred into expression cells, in the presence of voclosporin, the tolerance of the cells to voclosporin is at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 100%.

[0040] The present invention also provides a universal immune effector cell, wherein the immune effector cell expresses an exogenous protein 1, wherein the exogenous protein 1 is the aforementioned calcineurin B subunit mutant of the present invention. The immune effector cell may be a T cell, a NK cell, etc., and the T cell may be a CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, γδT cells or NKT cells. Preferably, the immune effector cell further expresses exogenous protein 2, which can recognize tumor antigens or pathogen antigens or self-antigens, including CAR or TCR. In some embodiments of the present invention, the exogenous protein 2 is CAR (chimeric antigen receptor). The exogenous protein 1 and / or exogenous protein 2 can be inserted into any position of the immune effector cell genome using viruses (lentivirus, retrovirus, adeno-associated virus, etc.) or non-viral means (double-stranded DNA, single-stranded DNA, RNA, etc.).

[0041] In a preferred embodiment, in order to further avoid the immune effector cell from rejecting the host cell, it is preferred to interfere with the function of the TCR complex of the immune effector cell. Various methods known in the art can be used, including but not limited to knocking out the T cell receptor (TCR) complex in the cell, knocking out the T cell receptor α constant region protein in the cell, and / or knocking out the T cell receptor β constant region protein in the cell. In some embodiments of the present invention, by inserting the coding gene of exogenous protein 1 and / or exogenous protein 2 into the coding gene of any one or any two or more elements of the constituent elements of the TCR complex of the immune effector cell, to interfere with the expression or function of any one or any two or more elements of the constituent elements of the TCR complex, thereby interfering with the TCR complex function of the cell. In one embodiment of the present invention, CAR is inserted into the TCR TRAC site of the immune effector cell. In another embodiment of the present invention, the aforementioned CNB mutant of the present invention is inserted into the TCR TRAC site of the immune effector cell. In another embodiment of the present invention, CAR and the aforementioned CNB mutant of the present invention are inserted into the TCR TRAC site of the immune effector cell.

[0042] In a preferred embodiment of the present invention, a universal T cell is provided, wherein the universal T cell expresses an exogenous protein 1, wherein the exogenous protein 1 is the CNB mutant described above in the present invention. Preferably, the T cell receptor (TCR) complex, the T cell receptor α constant region protein, and / or the T cell receptor β constant region protein in the universal T cell are knocked out. The T cell may be a CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells or NKT cells (γδT cells are inserted into TRDC).

[0043] In other preferred embodiments of the present invention, a universal CAR T cell is provided, wherein the universal CAR T cell expresses exogenous protein 1 and exogenous protein 2, wherein the exogenous protein 1 is the CNB mutant described above in the present invention, and the exogenous protein 2 recognizes a tumor antigen, a pathogen antigen, or a self-antigen. Preferably, the T cell receptor (TCR) complex, the T cell receptor α constant region protein, and / or the T cell receptor β constant region protein in the universal CAR T cell are knocked out. The T cell may be a CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells or NKT cells (γδT cells are inserted into TRDC).

[0044] In one embodiment of the present invention, the universal CAR-T cell is a cell in which CAR and the aforementioned CNB mutant of the present invention are inserted into the TCR TRAC site of the T cell.

[0045] According to the present invention, endogenous T cell receptor (TCR) complex, T cell receptor α constant region protein, and / or T cell receptor β constant region protein in the T cell are knocked out. Preferably, the knockout includes applying one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR / Cas system, RNA editing system such as RNA adenosine deaminase (ADAR), RNA-guided endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, base editing, CRISPR interference, and, zinc finger protein (Zincfinger) gene repressor and / or transcription activator-like effector (TALE) gene repressor-mediated transcriptional inhibition.

[0046] Preferably, a guide RNA (gRNA) targeting a nucleic acid molecule (TRAC) encoding the T cell receptor α constant region protein is administered to T cells using CRISPR / Cas9 technology to knock out endogenous TCR. More preferably, the gRNA is selected from any of the sequences shown in SEQ ID NO.50-54.

[0047] According to the present invention, the exogenous protein 2 recognizes a tumor antigen or a pathogen antigen or an autoantigen. The tumor antigen or pathogen antigen or autoantigen can be selected from, for example, Claudin18.2, Claudin18.1, Claudin 6, vascular endothelial growth factor receptor, phosphatidylinositol proteoglycan-3 (GPC3), B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, CD19, CD20, CD22, etc. In one embodiment of the present invention, the tumor antigen is CD19.

[0048] According to the present invention, the exogenous protein 2 is a chimeric antigen receptor (CAR).

[0049] The CAR comprises: an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

[0050] The antigen binding domain binds to a tumor antigen or a pathogen antigen as described above. In one embodiment of the present invention, the tumor antigen is CD19.

[0051] The intracellular signaling domain refers to the functional part of CAR, which plays a role by transmitting information within the cell, so as to regulate cell activity through a determined signal transduction pathway by producing a second messenger or by responding to such messengers as effectors. In some embodiments of the present invention, the intracellular signaling domain contains a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule defined below. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Co-stimulatory molecules include but are not limited to OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137), etc. In some embodiments of the present invention, the stimulatory molecule is a ζ chain bound to a T cell receptor complex.

[0052] In one embodiment of the present invention, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO.63.

[0053] The present invention also provides a method for preparing the aforementioned universal immune effector cells, the method comprising: engineering the immune effector cells to obtain universal immune effector cells expressing exogenous protein 1, wherein the exogenous protein 1 is the aforementioned CNB mutant of the present invention.

[0054] Preferably, the method further comprises knocking out the endogenous T cell receptor complex, T cell receptor α constant region protein, and / or T cell receptor β constant region protein in the cell.

[0055] More preferably, the method further comprises causing the cell to express an exogenous protein 2 capable of recognizing a tumor antigen or a pathogen antigen. In one embodiment of the present invention, the exogenous protein 2 is a chimeric antigen receptor.

[0056] In a preferred embodiment of the present invention, a method for preparing universal T cells is provided, the method comprising: engineering T cells to obtain universal T cells expressing the aforementioned CNB mutant of the present invention.

[0057] Preferably, the method further comprises knocking out the endogenous T cell receptor complex, T cell receptor α constant region protein, and / or T cell receptor β constant region protein in the T cells.

[0058] More preferably, the method further comprises causing the T cells to express a chimeric antigen receptor capable of recognizing a tumor antigen or a pathogen antigen.

[0059] In some specific embodiments, the method comprises the following steps:

[0060] 1) Obtain and culture healthy human T cells;

[0061] 2) Knockout of TCR in T cells;

[0062] 3) transferring CAR and the CNB mutant of the present invention into T cells;

[0063] 4) Isolating and obtaining universal CAR T cells that express the CNB mutant and CAR of the present invention and have downregulated or no TCR expression.

[0064] Preferably, step 1) comprises: extracting T cells from peripheral blood mononuclear cells of healthy donors, stimulating them with Human TCD3 / CD28 beads and culturing them for 24-72 hours.

[0065] Preferably, step 2) uses a technology selected from CRISPR / Cas technology, artificial zinc finger nuclease (ZFN) technology, transcription activator-like effector (TALE) technology or CRISPR / Cas technology; more preferably, CRISPR / Cas technology is used; more preferably, gRNA is selected from the sequence shown in any one of SEQ ID NO.50-54.

[0066] Preferably, step 3) includes constructing an IRU-CAR plasmid comprising "left homologous arm-self-cleavage sequence-CAR-self-cleavage sequence-CNB mutant-right homologous arm".

[0067] The self-cleavage sequence may, for example, be selected from P2A, T2A, E2A and F2A.

[0068] In some embodiments of the present invention, step 3) includes constructing an IRU-CAR plasmid comprising "left homology arm-P2A-CAR-T2A-CNB mutant-right homology arm".

[0069] In some embodiments of the present invention, after the TRAC site is excised by Cas9 to produce a gap, the DNA 350-450bp to the left of the gap is the left homology arm sequence, and the DNA 350-450bp to the right of the gap is the right homology arm sequence; in one embodiment of the present invention, the left homology arm comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or approximately 100% identical to the nucleotide sequence shown in SEQ ID NO.61; the right homology arm comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or approximately 100% identical to the nucleotide sequence shown in SEQ ID NO.62.

[0070] The CAR comprises:

[0071] 1) Antibodies or their functional fragments that specifically bind to tumor antigens or pathogen antigens, transmembrane regions of CD28 or CD8, and CD3ζ;

[0072] 2) antibodies or functional fragments thereof that specifically bind to tumor antigens or pathogen antigens, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD28, and step CD3ζ;

[0073] 3) antibodies or functional fragments thereof that specifically bind to tumor antigens or pathogen antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD137, and CD3ζ; and / or

[0074] 4) Simultaneously, an antibody or a functional fragment thereof that specifically binds to the target antigen, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3ζ are introduced.

[0075] In one embodiment of the present invention, CAR comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO.63.

[0076] In one embodiment of the present invention, the amino acid sequence of P2A is shown as SEQ ID NO.55.

[0077] In one embodiment of the present invention, the amino acid sequence of T2A is shown as SEQ ID NO.56.

[0078] In one embodiment of the present invention, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.1-47.

[0079] Preferably, the transfection method is selected from electroporation and liposome transfection. More preferably, the T cells are cultured for 6-10 days after transfection.

[0080] Preferably, in step 4), biotin-CD3 and streptavidin beads are used to remove TCR + T cells.

[0081] The present invention also provides a pharmaceutical composition comprising the universal immune effector cell of the present invention and an immunosuppressant. In one embodiment of the present invention, the pharmaceutical composition comprises the universal T cell or universal CAR T cell of the present invention and an immunosuppressant.

[0082] Preferably, the immunosuppressant comprises: calcineurin inhibitors, such as cyclosporin A, FK 506, voclosporin, pimecrolimus; DMARDs, such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; regulators of lymphocyte recirculation, such as FTY720 and FTY720 analogs; mTOR inhibitors, such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; ascomycins with immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homologues, analogs or derivatives; immunosuppressive monoclonal antibodies, for example, monoclonal antibodies against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86 or their ligands. More preferably, the immunosuppressant is selected from calcineurin inhibitors, such as cyclosporin A, voclosporin, pimecrolimus or FK 506.

[0083] The present invention also provides the use of the universal immune effector cells in preparing drugs for fighting tumors or treating autoimmune diseases.

[0084] In a preferred embodiment of the present invention, there is provided use of the universal T cells or universal CAR T cells in the preparation of drugs for anti-tumor or treatment of autoimmune diseases.

[0085] Also, provided is the use of the universal immune effector cell combined with an immunosuppressant in the preparation of a drug for anti-tumor or treatment of autoimmune diseases.

[0086] In a preferred embodiment of the present invention, there is provided use of the universal T cells or universal CAR T cells combined with immunosuppressants described in the present invention in the preparation of drugs for anti-tumor or treatment of autoimmune diseases.

[0087] Also, provided is the use of the universal immune effector cells in the preparation of drugs for anti-tumor or autoimmune disease treatment in combination with immunosuppressants.

[0088] In a preferred embodiment of the present invention, there is provided use of the universal T cells or universal CAR T cells of the present invention in the preparation of an anti-tumor or autoimmune disease treating drug in combination with an immunosuppressant.

[0089] The tumors include, but are not limited to, leukemias (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelo-monocytic leukemia, acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, heavy chain disease, solid tumors such as sarcomas and cancers (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, synovial vioma, mesothelioma, Ewing's tumor, smooth muscle Tumor, rhabdomyosarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma.

[0090] The autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis / psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, Hashimoto's disease / hypothyroidism, Sjogren's syndrome, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, lupus nephritis, Goodpasture's syndrome, autoimmune oophoritis, autoimmune orchitis, etc.

[0091] In some specific embodiments, the combination of universal immune effector cells expressing CNB mutants and immunosuppressants may include the following combinations: when universal immune effector cells (e.g., universal T cells, universal CAR T cells) express CNB mutants with amino acid sequences as shown in any one of SEQ ID NO.1-47, the immunosuppressant may be selected from CsA, voclosporin or FK 506. In some preferred embodiments, when the immunosuppressant is CsA, the CNB mutant amino acid sequence is selected from the amino acid sequence shown in any one of SEQ ID NO.1, 3-4, 6-8, 10, 12, 14, 17, 21, 28-30, 44 or 46. In some preferred embodiments, when the immunosuppressant is FK 506, the CNB mutant amino acid sequence is selected from the amino acid sequence shown in any one of SEQ ID NO.1, 3-4, 6-8, 10, 12-14, 21-23, 26, 28, 30-31, 39, 44 or 46. In some preferred embodiments, when the immunosuppressant is voclosporin, the amino acid sequence of the CNB mutant is selected from the amino acid sequence shown in any one of SEQ ID NO. 1, 3, 6-8, 10, 12, 13, 17, 21, 27, 28, 30, 32, 37, 44, and 46.

[0092] The specific mutation sites of the calcineurin B subunit mutants are shown in Table 1.

[0093] Table 1 Mutation sites of calcineurin B subunit mutants

[0094]

[0095]

[0096] The present invention also provides the following method.

[0097] A method for treating cancer / tumor, the method comprising administering the universal immune effector cells (e.g., universal T cells or universal CAR T cells) and immunosuppressants described in the present invention to a subject in need, or administering the pharmaceutical composition described in the present invention to a subject in need.

[0098] A method for treating an autoimmune disease, the method comprising administering the universal immune effector cells (e.g., universal T cells or universal CAR T cells) and immunosuppressants described in the present invention to a subject in need, or administering the pharmaceutical composition described in the present invention to a subject in need.

[0099] Sequence list of the present invention (Table 2):

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Terminology Note:

[0107] “And / or” will be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” used in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0108] "Including" and "comprising" have the same meaning, are intended to be open ended and allow but not require the inclusion of additional elements or steps. When the terms "including" or "comprising" are used herein, the terms "consisting of" and / or "consisting essentially of" are also included and disclosed.

[0109] In this specification and claims, nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' direction. Nucleobases are represented herein by the commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. It will be understood by the technician that the T bases in the codons disclosed herein are present in DNA, while the T bases will be replaced by U bases in the corresponding RNA.

[0110] In this specification and claims, the conventional one-letter or three-letter codes for amino acid residues are used.Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxyl orientation.

[0111] As used herein, "amino acid" refers to natural and non-natural amino acids, preferably natural amino acids, including: alanine ("Ala" or "A"), arginine ("Arg" or "R"), asparagine ("Asn" or "N"), aspartic acid ("Asp" or "D"), cysteine ​​("Cys" or "C"), glutamine ("Gln" or "Q"), glutamic acid ("Glu" or "E"), glycine ("Gly" or "G"), histidine ("His" or "H"), ”), isoleucine (“Ile” or “I”), leucine (“Leu” or “L”), lysine (“Lys” or “K”), methionine (“Met” or “M”), phenylalanine (“Phe” or “F”), proline (“Pro” or “P”), serine (“Ser” or “S”), threonine (“Thr” or “T”), tryptophan (“Trp” or “W”), tyrosine (“Tyr” or “Y”), and valine (“Val” or “V”).

[0112] Neutral amino acids are amino acids with equal numbers of amino and carboxyl groups in the amino acid molecule, and their isoelectric points are generally 5.5-6.3. Among natural amino acids, alanine ("Ala" or "A"), asparagine ("Asn" or "N"), cysteine ​​("Cys" or "C"), glutamine ("Gln" or "Q"), glycine ("Gly" or "G"), isoleucine ("Ile" or "I"), leucine ("Leu" or "L"), methionine ("Met" or "M"), phenylalanine ("Phe" or "F"), proline ("Pro" or "P"), serine ("Ser" or "S"), threonine ("Thr" or "T"), tryptophan ("Trp" or "W"), tyrosine ("Tyr" or "Y"), and valine ("Val" or "V") are neutral amino acids. Acidic amino acids are amino acids with more carboxyl groups than amino groups in the amino acid molecule, and their isoelectric points are generally 2.8 to 3.2. Among natural amino acids, aspartic acid ("Asp" or "D") and glutamic acid ("Glu" or "E") are acidic amino acids. Basic amino acids are amino acids with more amino groups than carboxyl groups in the amino acid molecule, and their isoelectric points are greater than 7, generally 7.6 to 10.8. Among natural amino acids, arginine ("Arg" or "R"), lysine ("Lys" or "K"), and histidine ("His" or "H") are basic amino acids.

[0113] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Typically, the term "homology" means the evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present disclosure, the term homology includes identity and similarity.

[0114] In some embodiments, polymer molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the monomers in the molecule are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0115] Identity: As used herein, the term "identity" refers to overall monomer conservation between polymer molecules, such as between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Suitable software programs are available from various sources and are used for comparisons of both protein and nucleotide sequences. For example, a suitable program for determining percent sequence identity is Bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, parts of the EMBOSS program suite for bioinformatics, and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X or ClustalOmega), MUSCLE, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 : Schematic diagram of the universal CAR T cell (Immunosuppressant Resistant Universal (IRU)-CAR-T) of the present invention: On the basis of the traditional universal CAR-T (i.e., CAR-T with TCR knocked out, hereinafter referred to as WT CAR-T), a calcineurin B subunit mutant is additionally introduced, so that the rejection of CAR-T by the receptor immune T cells can be greatly reduced or even avoided when used in combination with immunosuppressive drugs.

[0117] Figure 2: Mutant CNB clones and WT CNB were expressed in Jurkat-NFAT-GFP reporter cell line. Cells were stimulated with PMA (50 ng / ml) / Ionomycin (1 μg / ml) for 16 h under different immunosuppressive conditions (CsA (300 ng / ml), FK506 (5 ng / ml), voclosporin (100 nM)), and the differences in GFP expression were compared.

[0118] Figure 3 :IRU-CAR-T functional experiment: hCD19scFv-CD28-CD3z-CNB44 or hCD19scFv-CD28-CD3z-WT-CNB sequence was inserted into the T cell TRAC locus to prepare IRU-CAR-T or WT CAR-T cells. Nalm6 cells expressing luficerase-GFP were mixed with CAR-T cells at E:T=1:5 ratio (A, B) or different ratios (C) under different immunosuppressive conditions (CsA (300ng / ml), FK506 (5ng / ml)) for 24 hours (A, C) or 72 hours (B), where A added Brefredin A 6 hours before the end of the culture, and C used T cells that did not express the CAR gene as control T cells (CTRL-T). A is the result of the cytokine detection experiment; B is the result of the proliferation experiment; C is the result of the killing experiment. DETAILED DESCRIPTION

[0119] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0120] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods are conventional molecular biology methods in the art and can be performed with reference to the instructions of molecular biology experimental manuals in the art or kit product instructions.

[0121] Example 1: Schematic diagram of the universal CAR T cell of the present invention (hereinafter referred to as "IRU-CAR-T")

[0122] Taking CsA and FK506 as an example, the present invention uses gene editing technology to introduce CAR and CNB mutants on T cells. Knockout of αβTCR will avoid the occurrence of GVHD. On this basis, the present invention co-expresses CNB mutants with CAR, combined with the use of CsA or FK506, to inhibit recipient T cells and reduce their rejection of IRU-CAR-T; at the same time, CNB mutants will help IRU-CAR-T cells from CsA / FK506's inhibition of IRU-CAR-T cell activation, proliferation, and tumor killing, so that it can still work normally and kill tumor cells when the recipient's immune system is suppressed. After the treatment, the cessation of CsA / FK506 will restore the function of the recipient's immune system, trigger the recurrence of HVGR, and the receptor will reject and eliminate IRU-CAR-T cells, avoiding the hidden dangers of tumorization caused by the long-term existence of IRU-CAR-T cells ( Figure 1 ).

[0123] The cell lines and their culture conditions in the following examples are:

[0124] Nalm6 FFluc-GFP cells were purchased from Shanghai Model Organisms; Nalm6-FFluc-GFP-β2m KO The lentivirus was constructed using the plasmid lentiCRISPR v2 (addgene) containing the gRNA sequence 5'-AGCTCACATGGTTCACACGGCGTTT-3'. Jurkat NFAT GFP cells were donated by Professor Chen Wei of Zhejiang University. AAV-293 cells were purchased from the China National Laboratory Cell Resource Sharing Platform.

[0125] Adherent cell lines (AAV-293) and suspension cell lines (Jurkat NFAT GFP, Nalm6 FFluc-GFP, and Nalm6-FFluc-GFP-β2m KO ) were cultured in DMEM or RPMI 1640 complete medium (containing 10% fetal bovine serum (FBS)) at 37°C and 5% CO2. All cell lines were routinely tested for mycoplasma and the results were negative.

[0126] Example 2: Preparation of IRU-CAR-T

[0127] Part I: Procedure for collecting peripheral blood from volunteers

[0128] 1. Collect peripheral blood from volunteers.

[0129] 2. Extract T cells using RosetteSepTM human T cell enrichment kit and Ficoll lymphocyte separation medium.

[0130] 2.1 Add RosetteSepTM human T cell enrichment cocktail at 50 μl / ml blood into a centrifuge tube of appropriate volume;

[0131] 2.2 Add the volume of Ficoll lymphocyte separation solution indicated in the instructions into a 15ml or 50ml centrifuge tube according to the blood volume;

[0132] 2.3 Mix the peripheral blood and sterile FACS solution (PBS containing 0.1% serum) in a ratio of 1:1 and slowly drip it onto the lymphocyte separation solution along the tube wall with a Pasteur pipette. Be gentle and pay attention to maintaining a clear liquid interface.

[0133] 2.4 Place the sample in a centrifuge, centrifuge at 1200×g, set the acceleration to 1, and the deceleration to 0 for 20 minutes;

[0134] 2.5 After centrifugation, the tube can be seen to be divided into three layers. The upper layer is plasma and FACS, the lower layer is mainly red blood cells and granulocytes, and the middle is lymphocyte separation fluid. At the middle interface, there is a narrow band of white cloud layer mainly composed of lymphocytes, which is the white film layer;

[0135] 2.6 Carefully remove part of the upper liquid, leaving about 1 ml, insert a pipette into the buffy coat layer, suck out the lymphocytes, place the lymphocytes in another 15 ml centrifuge tube, add 5 times the volume of FACS, put it into a centrifuge at 400×g, and centrifuge for 10 minutes;

[0136] 3. Stimulate with Human T CD3 / CD28 beads and culture T cells in X-vivo liquid culture medium containing IL-7 and IL-15 in a 37℃ 5% CO2 incubator for 48 hours.

[0137] Part II: IRU CAR-T cell preparation

[0138] The preparation of IRU-CAR plasmid vectors requires different strategies depending on the type of IRU CAR virus selected. For example, lentivirus / retrovirus requires the fragment to be inserted into the lentivirus / retrovirus backbone, while AAV virus requires the fragment to be inserted into the AAV virus backbone (e.g., serotype AAV6).

[0139] The following uses AAV virus as an example to illustrate the construction of IRU-CAR plasmid: using the AAV virus backbone (serotype AAV6), the "left homologous arm-P2A-CAR-T2A-CNB mutant-right homologous arm" sequence is inserted into the multiple cloning site.

[0140] After the TRAC site is cut out by Cas9 to produce a gap, the approximately 400 bp DNA on the left side of the gap is the left homology arm sequence, and the approximately 400 bp DNA on the right side of the gap is the right homology arm sequence.

[0141] The left homology arm sequence can be amplified by primers primer1 and primer2, and the nucleotide sequences of primer1 and primer2 are shown in SEQ ID NOs. 57 and 58, respectively.

[0142] The right homology arm sequence can be amplified by primers primer3 and primer4, and the nucleotide sequences of primer3 and primer4 are shown in SEQ ID NOs. 59 and 60, respectively.

[0143] The left homology arm comprises the nucleotide sequence shown in SEQ ID NO.61.

[0144] The right homology arm comprises the nucleotide sequence shown in SEQ ID NO.62.

[0145] CAR includes:

[0146] 1) Antibodies or their functional fragments that specifically bind to tumor antigens or pathogen antigens, transmembrane regions of CD28 or CD8, and CD3ζ;

[0147] 2) antibodies or functional fragments thereof that specifically bind to tumor antigens or pathogen antigens, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD28, and CD3ζ;

[0148] 3) antibodies or functional fragments thereof that specifically bind to tumor antigens or pathogen antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD137, and CD3ζ; and / or

[0149] 4) antibodies or functional fragments thereof that specifically bind to target antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3ζ;

[0150] The amino acid sequence of the FMC63 CAR used in the following examples is shown in SEQ ID NO.63.

[0151] The P2A is a self-splicing peptide. In the following examples, the amino acid sequence of P2A is shown as SEQ ID NO.55.

[0152] The T2A is a self-splicing peptide. In the following examples, the amino acid sequence of T2A is shown as SEQ ID NO.56.

[0153] The amino acid sequence of the CNB mutant may be shown in any one of SEQ ID NOs.: 1-47.

[0154] 1. Preparation of IRU CAR plasmid vector

[0155] 1.1 Fragment acquisition: The "left homology arm-P2A-CAR-T2A-CNB mutant-right homology arm" fragment can be obtained by PCR or DNA synthesis method;

[0156] 1.2 Fragment backbone recombination: One-step cloning or T4 ligation can be used to link the fragment to the backbone;

[0157] 1.3 After plasmid construction is completed, the sequence correctness is confirmed by Sanger sequencing and other methods.

[0158] 2.IRU CAR virus preparation

[0159] As above, the preparation of IRU CAR virus requires different strategies according to the selection of virus type. The preparation of IRU CAR virus is explained below using AAV virus as an example.

[0160] 2.1 AAV toxin-producing cell culture: AAV-293 cells were cultured in DMEM high-glucose complete medium (containing 10% FBS) at 37°C in a 5% CO2 incubator. Subculture every 2-3 days to keep them in the active logarithmic phase of growth;

[0161] 2.2 IRU CAR virus transfection: Using the PEI transfection method, the IRU CAR plasmid constructed in the previous step and the AAV helper plasmid pDP6 plasmid were co-transfected into AAV-293 cells;

[0162] 2.3 IRU CAR virus purification: 48 hours after transfection, AAV-293 cells were collected. The cells were repeatedly frozen and thawed to release the virus into the supernatant. Subsequently, IRU CAR virus was purified using iodixanol density gradient centrifugation.

[0163] 3. Preparation of IRU CAR-T cells

[0164] 3.1 Preparation of Cas9-gRNA mixture ribonucleoprotein (RNP): Evenly mix the gRNA that can target the TRAC site and the Cas9 protein, incubate at 37°C for 15 minutes to form RNP;

[0165] The gRNA can be selected from any one of the sequences shown in SEQ ID NO.50-54 below; 5'-CAGGGUUCUGGAUAUCUGU-3' (SEQ ID NO.52) is used in the following examples.

[0166] 3.2 Take an appropriate amount of T cells cultured for 48 hours in the first step, centrifuge at 90×g for 10 minutes, and resuspend in electroporation solution;

[0167] 3.3 Mix evenly with RNP and transfer to an electroporation cup, and use LONZA Nucleofector IIB machine U-014 to electroporate RNP into T cells;

[0168] 3.4 After electroporation, add 1 ml of preheated culture medium;

[0169] 3.5 After 20 minutes, centrifuge at 90×g for 10 minutes to remove the electroporation solution;

[0170] 3.6 Add IRU CAR AAV virus to the electroporated T cells in the previous step. Continue to culture T cells in X-vivo liquid culture medium containing IL-7 and IL-15 (50ng / ml) for six days;

[0171] 3.7 Using Biolegend TM CD3 selection kit removes CD3 + T cells, obtain IRU CAR-T cells, the specific steps include:

[0172] a) Count cells, centrifuge the required amount of cells at 300 × g for 5 min, and resuspend in FACS buffer at an appropriate concentration;

[0173] b) Add the corresponding CD3-biotin antibody to every 100 μl of cell resuspension and incubate on ice for 15 min;

[0174] c) Add corresponding streptavidin beads to every 100 μl of cell resuspension and incubate on ice for 15 min;

[0175] d) Add 2.5 ml of FACS buffer and place the centrifuge tube on a magnet for 5 minutes to remove streptavidin beads;

[0176] e) Harvesting TCR - IRU CAR-T cells.

[0177] Take FMC63 CAR (target is human CD19 protein) as an example. The TRAC site of TCR is knocked out using Crispr / Cas9 technology, and FMC63 CAR and calcineurin B subunit mutants (CNB1-CNB47, amino acid sequences are shown in SEQ ID NO.1-47 respectively) are introduced into the TRAC site using AAV as a vector. This method can produce target IRU-CAR-T; the same method is used to prepare control group CAR-T (WT CNB and CTRL CNB, amino acid sequences are shown in SEQ ID NO.48-49 respectively). It is worth noting that the IRU-CAR-T in the present invention can be various types of CAR, not just limited to FMC63 CAR.

[0178] Example 3: Functional verification of calcineurin B subunit mutants in antagonizing CsA / FK506 / voclosporin

[0179] The mutant CNB clone-mCherry sequence was expressed in a Jurkat cell line with NFAT-GFP (Jurkat-NFAT-GFP). The Jurkat-NFAT-GFP cell line was stimulated with PMA (50 ng / ml) / Ionomycin (1 μg / ml) for 16 hours, and the expression level of GFP was detected to reflect the strength of the TCR signaling pathway.

[0180] The same cloned cells were used to detect the expression of GFP in the presence and absence of CsA (300 ng / ml), FK506 (5 ng / ml) or voclosporin (100 nM), and the tolerance to CsA, FK506 or voclosporin was calculated according to the following formula. Two parallel samples were made for each inhibitor of each mutant, and the results are the average of the two parallel samples.

[0181] GFP expression % = number of cells expressing GFP after stimulation / total number of cells × 100%

[0182] The tolerance to FK506 was calculated as follows: for the same clone cells, (% GFP expression after addition of FK506) / (% GFP expression without drug) × 100%

[0183] The calculation of tolerance to CsA was as follows: for the same clone cells, (% GFP expression after addition of CsA) / (% GFP expression without drug) × 100%

[0184] Tolerance to voclosporin was calculated as follows: for the same clone, (% GFP expression after addition of voclosporin) / (% GFP expression without drug) × 100%

[0185] Tolerance is classified into the following levels:

[0186] Level Code Tolerance range Level Code Tolerance range Level Code Tolerance range A ≥10％～＜20％ B ≥20％～＜30％ C ≥30％～＜40％ D ≥40％～＜50％ E ≥50％～＜60％ F ≥60％～＜70％ G ≥70％～＜80％ H ≥80％～＜90％ I ≥90％～＜100％ J ≥100％

[0187] The results are shown in Table 3 below.

[0188] Table 3:

[0189]

[0190]

[0191] The results show that the Jurkat-NFAT-GFP cell line carrying the CNB mutant of the present invention can highly express GFP in the presence of CsA (300 ng / ml), FK506 (5 ng / ml) or voclosporin (100 nM), indicating that the CNB mutant of the present invention can tolerate the inhibition of the TCR signaling pathway by CsA, FK506 or voclosporin, which is higher than the tolerance of the CNB mutant known in the prior art. It should be noted that at the above concentrations of CsA (300 ng / ml), FK506 (5 ng / ml) or voclosporin (100 nM), cells carrying wild-type CNB are completely inhibited and unable to express GFP ( Figure 2 ).

[0192] Example 4: Functional experiment of IRU-CAR-T

[0193] According to the method of Example 2, CNB mutant CNB44 containing an amino acid sequence as shown in SEQ ID NO.44 and IRU-CAR-T cells containing FMC63 CAR of anti-CD19 antibody were prepared, and CsA / FK506 was used as an immunosuppressant to perform in vitro functional experiments, which included three parts: cytokine detection experiment, proliferation experiment and killing experiment. Specifically, the hCD19scFv-CD28-CD3z-CNB44 or hCD19scFv-CD28-CD3z-WT-CNB sequence was inserted into the T cell TRAC locus to prepare IRU-CAR-T or WT CAR-T cells.

[0194] Cytokine detection, proliferation assay:

[0195] 1×10 5Sorted CAR-T cells were co-cultured with 5-fold irradiated Nalm6-FFLuc-GFP cells in 200 μl X-vivo medium + 10% FBS in U-bottom 96-well tissue culture plates in the presence / absence of CsA (300 ng / ml) or FK506 (5 ng / ml) for 24 hours. For cytokine detection, Brefeldin A, a Golgi Plug protein transport inhibitor, was added 6 hours before the end of the culture. For proliferation, CAR-T cells were cultured for up to 72 hours. Cells were stained with the following flow cytometry antibodies: Alexa Fluor 647-rabbit anti-mouse FMC63 scFv polyclonal antibody; Brilliant Violet 510-CD3e; PE / Cyanine7-CD4; APC / Cyanine7-CD8; Pacific Blue-IL-2; PE-IFNγ; PerCP / Cyanine5.5-TNFα, PE-Ki67, Aqua was used as a dead-live dye. FcR blocking reagent was used to block Fc receptor staining. All extracellular and intracellular staining were performed using standard staining protocols.

[0196] Killing experiment:

[0197] The sorted CAR-T cells were mixed with 5×10 3 Nalm6-FFLuc-GFP cells were co-cultured in 1 ml X-vivo medium + 10% FBS at 37°C in U-bottom deep well multiwell plates at the indicated E:T ratios. The same density of target cells was also cultured alone to determine the maximum luciferase expression (relative light units; RLU) as a control. For short-term cytotoxicity assays, all cells were harvested after 24 hours and the target cells were quantified using the standard protocol of the Bright-Lite luciferase assay system (Vazyme). Emitted light was detected in a luminescent plate reader (Molecular Devices iD5). Lysis was determined as (1-(RLUsample) / (RLUmax))×100.

[0198] Cytokine experiments verified that IRU-CAR-T can release cytokines in the presence of CsA / FK506. Figure 3 As shown in A, after 24 hours of stimulation with Nalm-6 tumor cells, IRU-CAR-T cells can release NFAT pathway-related killer cytokines: IL-2, IFNγ, and TNFα without significant difference regardless of the presence or absence of CsA or FK506. However, after 24 hours of stimulation with Nalm-6 tumor cells, the ability of WT-CAR-T cells to release cytokines decreased in the presence of CsA or FK506, indicating that they cannot carry out normal tumor killing activities.

[0199] Similarly, the CAR-T proliferation experiment verified that IRU-CAR-T cells can continue to proliferate under the stimulation of tumor antigens regardless of the presence of CsA or FK506, which may be an important reason why IRU-CAR-T cells can exert their anti-tumor function. WT-CAR-T cells can only proliferate in the absence of CsA or FK506 ( Figure 3 B).

[0200] In the killing experiment ( Figure 3 C), using the human Nalm-6 tumor cell line (CD19 + ) is the target cell. It can be seen that the killing efficiency of IRU-CAR-T in different effector cell:target cell (E / T) ratios in the presence of CsA or FK506 has no significant difference. In contrast, WT-CAR-T cells showed a significant decrease in killing efficiency in the presence of CsA / FK506 compared with those without CsA / FK506. CTRL-T cells refer to T cells that do not express CAR molecules and are used to detect background killing.

[0201] The three experiments in the functional experiment jointly proved that IRU-CAR-T cells carrying the CNB mutant of the present invention can resist the inhibition of their activation by CsA or FK506 and perform normal proliferation and tumor killing functions.

[0202] The present invention proposes a CNB mutant, and a universal CAR T cell (IRU-CAR-T) prepared therefrom. The present invention uses gene editing methods, such as CRISPR / Cas9 and AAV site-directed knockout knock-in technology, to simply and efficiently introduce CAR and the CNB mutant, avoiding excessive gene editing. By combining with CsA, FK506 or voclosporin, CsA / FK506 / voclosporin can reduce the activation of recipient immune T cells and reduce the rejection of IRU-CAR-T cells during treatment. At the same time, due to the presence of CNB mutants, IRU-CAR-T cells can be protected from the inhibition of CsA / FK506 / voclosporin, thereby effectively reducing the HVGR phenomenon caused by the recipient immune system to IRU-CAR-T during treatment. After completing the treatment, discontinuing the use of CsA / FK506 / voclosporin drugs will restart the rejection of IRU-CAR-T by the recipient immune T cells, resulting in the elimination of allogeneic IRU-CAR-T cells, achieving their withdrawal after tumor treatment, greatly increasing the safety of this immunotherapy.

[0203] Compared with other methods for antagonizing CsA / FK506, the CNB mutant of the present invention has the following advantages: 1. High tolerance to CsA, FK506 or voclosporin; 2. Simultaneous resistance to CsA, FK506 and voclosporin.

[0204] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A calcineurin B subunit mutant, characterized in that: The amino acid sequence of the calcineurin B subunit mutant is based on SEQ ID NO.49, and three amino acids X1X2X3 or two amino acids X4X5 are inserted between positions 125 and 126, wherein: X1 is a neutral amino acid or a basic amino acid, X2 is a neutral amino acid, an acidic amino acid or a basic amino acid, X3 is a neutral amino acid or a basic amino acid, X4 is a neutral amino acid and X5 is a basic amino acid, or X4 is a basic amino acid and X5 is a neutral amino acid; when two amino acids X4X5 are inserted, there may be a T129K substitution at position 129; Preferably, X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S or W; When X4 is a neutral amino acid and X5 is a basic amino acid, X4 is selected from F, Y, Q, N, S, C, M or T, and X5 is R or K; When X4 is a basic amino acid and X5 is a neutral amino acid, X4 is selected from K, R or H, and X5 is selected from W, P, G, A, V, L or I; Preferably, X1X2X3 is selected from: QKR, RSY, KSC, SCR, LSA, PSR, RRL, TRL, RTT, NRA, LHA, YSP, SRV, KNA, GGC, CKL, KSR, RPK, KSM, REQ, ARQ, TAV, FRV, LSY, RVF, ASI, RTG, RGN, KWV, GRV, RSA, STC, KGA, KAS, RST, RRG, RRW, LMC, FSV, TPV, IRV, TRG, TRV or GSQ; X4X5 is selected from SR, KI or HV.

2. The calcineurin B subunit mutant according to claim 1, characterized in that The amino acid sequence of the CNB mutant is based on SEQ ID NO. 49, and only three amino acids X1X2X3 or two amino acids X4X5 are inserted between positions 125 and 126. When two amino acids X4X5 are inserted, there may be a T129K substitution at position 129. Preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO.1-47.

3. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: X1 is a basic amino acid selected from R, K or H; X2 is a neutral amino acid, an acidic amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid; Preferably, X2 is S, R, T, G, K, P, A, V, C, H, N, E, W or M, and X3 is V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S or W; More preferably, X2 is S, R, T, G, P, A, V, N, E or W, and X3 is V, A, R, C, L, Q, G, Y, T, K, M, F, N, S or W; Further preferably, X1X2X3 is selected from REQ, RGN, RPK, RRG, RRL, RRW, RSA, RST, RSY, RTG, RTT, RVF, KAS, KGA, KNA, KSC, KSM, KSR, KWV; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 2, 3, 7, 9, 14, 17-20, 26, 28-30, 32, 34-37, 39.

4. The calcineurin B subunit mutant according to claim 3, characterized in that X1 is selected from R; X2 is selected from S, R, T, G, P, V or E; X3 is selected from A, Q, G, Y, L, T, K, N, W or F; Preferably: X1 is selected from R; X2 is selected from R, T, G or V; X3 is selected from G, L, T, N, W or F; More preferably: X1 is selected from R; X2 is selected from R or T; X3 is selected from G, L or W; Further preferably, X1X2X3 is selected from REQ, RGN, RPK, RRG, RRL, RRW, RSA, RST, RSY, RTG, RTT, RVF; or, X1X2X3 is selected from RGN, RRL, RRW, RTG, RTT, RVF; or, X1X2X3 is selected from RRL, RRW, RTG.

5. The calcineurin B subunit mutant according to claim 3, characterized in that: X1 is selected from K; X2 is selected from S, N, G, A or W; X3 is selected from A, C, R, M, V, or S; Preferably: X1 is selected from K; X2 is selected from S, N, I or W; X3 is selected from A, C, R or V; More preferably: X1 is selected from K; X2 is selected from S, I or W; X3 is selected from C or V; Further preferably, X1X2X3 is selected from KAS, KGA, KNA, KSC, KSM, KSR, KWV; or, X1X2X3 is selected from KNA, KSC, KSR, KWV; or, X1X2X3 is selected from KSC, KWV.

6. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: X1 is a neutral amino acid selected from S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid; Preferably, X1 is S, L, T, G, A or F; X2 is S, R, T, G, K, P, A, C, H or M, and X3 is V, A, R, C, L, Q, G, Y, P or I; More preferably, X1 is S, L, T, G, A or F; X2 is R, K or S, and X3 is V, A, R, C, L, Q, G, Y, P or I; Further preferably, X1X2X3 is selected from ARQ, ASI, CKL, FRV, FSV, GGC, GRV, GSQ, IRV, LHA, LMC, LSA, LSY, NRA, PSR, QKR, SCR, SRV, STC, TAV, TPV, TRG, TRL, TRV, YSP; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 1, 4-6, 8, 10-13, 15-16, 22-25, 27, 31, 33, 40-42, 44-47.

7. The calcineurin B subunit mutant according to claim 6, characterized in that: X1 is selected from T; X2 is selected from R, A or P; X3 is selected from G, L or V; Preferably, X1X2X3 is selected from TAV, TPV, TRG, TRL, TRV.

8. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: X2 is a basic amino acid selected from R, K or H; X1 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid; Preferably, X1 is L, C, Q, R, S, T, G, A, F, N or I, and X3 is V, A, L, R, Q, G or W; More preferably, X1X2X3 is selected from LHA, CKL, QKR, RRL, RRG, RRW, SRV, TRL, TRG, TRV, GRV, ARQ, FRV, NRA or IRV; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 1, 7, 8, 10, 11, 13, 16, 22, 24, 31, 37, 39, 44-46.

9. The calcineurin B subunit mutant according to claim 8, characterized in that X2 is R; X1 is selected from R, S, T, G, A, F, N or I; X3 is selected from V, A, L, Q, G or W; Preferably, X2 is R; X1 is selected from R, S, T, G, A, N or I; X3 is selected from V, A, L, Q or W; More preferably, X2 is R; X1 is selected from R, T, N or I; X3 is selected from V, A, L or W; Further preferably, X1X2X3 is selected from RRL, RRG, RRW, SRV, TRL, TRG, TRV, GRV, ARQ, FRV, NRA or IRV; or, X1X2X3 is selected from RRL, RRW, SRV, TRL, TRV, GRV, ARQ, NRA or IRV; or, X1X2X3 is selected from RRL, RRW, TRL, TRV, NRA or IRV.

10. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: X2 is a neutral amino acid selected from A, C, G, M, N, P, S, T, V or W, X1 is a neutral amino acid or a basic amino acid; X3 is a neutral amino acid or a basic amino acid; Preferably, X2 is A, G, P, S, T or V; X1 is K, T, S, G, K, R, L, A, F, P or Y; X3 is S, V, R, C, A, N, K, I, Q, M, Y, T, P or F; More preferably, X1X2X3 is selected from KAS, TAV, SCR, GGC, KGA, RGN, LMC, KNA, RPK, TPV, ASI, FSV, GSQ, KSC, KSM, KSR, LSA, LSY, PSR, RSA, RST, RSY, YSP, RTG, RTT, STC, RVF, KWV; Most preferably, the amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 2-6, 9, 12, 14-15, 17-19, 23, 25-30, 32-36, 40-42, 47.

11. The calcineurin B subunit mutant according to claim 10, characterized in that X2 is S; X1 is selected from R, K, L, A, F, G, P or Y; X3 is selected from V, A, R, C, Q, Y, T, P, M or I; Preferably, X2 is selected from S; X1 is selected from K, P or Y; X3 is selected from R, C or P; More preferably, X2 is selected from S; X1 is selected from K or Y; X3 is selected from C or P; Further preferably, X1X2X3 is selected from RSY, RSA, RST, KSC, KSR, KSM, LSA, LSY, GSQ, ASI, FSV, PSR or YSP; or, X1X2X3 is selected from KSC, KSR, PSR or YSP; or, X1X2X3 is selected from KSC or YSP.

12. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: The CNB mutant exhibits more prominent FK506 tolerance, wherein X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, F, I, N or W; Preferably, X1 CKL, KSR, RSY, RSA, LHA, RTT, RRG, LMC, RGN, FRV, REQ, LSY, FSV, TPV, GGC, TRG, RPK, ASI, STC, LSA, KGA or GSQ; Preferably, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SR V, TAV, CKL, KSR, RSY, RSA, LHA, RTT, RRG, LMC, RGN, FRV, REQ, LSY, FSV, TPV, GGC, TRG, RPK, ASI or STC; Further preferably, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SRV, TAV, CKL, KSR, RSY, RSA, LHA, RTT, RRG, LMC, RGN, FRV or REQ; More preferably, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SRV or TAV.

13. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: The CNB mutant exhibits more prominent CsA tolerance, wherein X1 is selected from R, K, S, L, T, G, A, F, Q, P, N, Y, C or I; X2 is selected from S, R, T, G, K, P, A, V, C, H, N, E, W or M; X3 is selected from V, A, R, C, L, Q, G, Y, T, P, K, F, I, N or W; Preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GR V, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK, KGA, LSA, LMC; Preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, AR Q, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC, FRV, LSY, GGC, FSV, RRG, RPK or KGA; Further preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ, RRW, ASI, TPV, STC or FRV; More preferably, XXXX is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, TAV, RSA, RSY, TRG, RVF, CKL, REQ or RRW; More preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA or TAV; Most preferably, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR or SCR.

14. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: The CNB mutant exhibits more prominent voclosporin tolerance, wherein X1 is selected from Y, K, N, A, R, T, S, Q, P, I, L, F or G, X2 is selected from S, W, R, T, K, E, N, H, C, G, A, P, M or V, and X3 is selected from P, V, A, C, I, G, R, L, Q, Y, N, M, K, F, T or W; preferably, X1X2X3 is selected from YSP, KWV, NRA , KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV, RVF, RTT, KGA, RRW, STC, TRG, RST or GSQ; Preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LS Y, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV, RVF, RTT, KGA, RRW or STC; Further preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM, RPK, LMC, GGC, LSA, GRV or RVF; More preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, KSM or RPK; most preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL or IRV.

15. The calcineurin B subunit mutant according to any one of claims 1 to 2, characterized in that: The CNB mutant exhibits more prominent FK506, CsA and voclosporin resistance, wherein X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL, IRV, REQ, LSY, KNA, LHA, FRV, RSY, SCR, RGN, FSV, ARQ, TAV, RPK, GGC, GRV, RVF, RTT, KGA, RRW, STC, TRG; Preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, KSR, RSA, TRL, IRV, REQ, KNA, LHA, FRV, RSY, SCR, RGN, ARQ, TAV, GRV, RVF, RTT, RRW, STC, TRG; Further preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, RSA, TRL, IRV, REQ, KNA, LHA, RSY, SCR, RGN, ARQ, TAV, GRV, RVF; More preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, TRL, IRV, KNA, LHA, SCR, RGN, ARQ, TAV; Most preferably, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, QKR, PSR, RRL, TRL, IRV.

16. Use of the calcineurin B subunit mutant according to any one of claims 1 to 15 in preparing universal immune effector cells; Preferably, the immune effector cells are T cells, NK cells; preferably, the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, γδ T cells or NKT cells.

17. A universal immune effector cell, characterized in that: The universal immune effector cell expresses exogenous protein 1, and the exogenous protein 1 is the calcineurin B subunit mutant according to any one of claims 1 to 15; Preferably, the amino acid sequence of the calcineurin B subunit mutant is shown in any one of SEQ ID NO.1-47; Preferably, the universal immune effector cell further expresses exogenous protein 2, and the exogenous protein 2 is capable of recognizing tumor antigens or pathogen antigens or self-antigens; Preferably, the exogenous protein 2 is a chimeric antigen receptor; Preferably, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain; the antigen binding domain binds to a tumor antigen or a pathogen antigen or a self-antigen; the intracellular signaling domain contains a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule; Preferably, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% identical to the amino acid sequence shown in SEQ ID NO.

63.

18. The universal immune effector cell according to claim 17, characterized in that In the universal immune effector cells, the T cell receptor complex, the T cell receptor α constant region protein, and / or the T cell receptor β constant region protein are knocked out.

19. The universal immune effector cell according to any one of claims 17 to 18, characterized in that The immune effector cells are T cells or NK cells; preferably, the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, γδ T cells or NKT cells.

20. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the universal immune effector cell according to any one of claims 17 to 19, and an immunosuppressant; Preferably, the immunosuppressant is selected from cyclosporin A, FK 506 and / or voclosporin.

21. Use of the universal immune effector cell according to any one of claims 17 to 19 in the preparation of a drug for anti-tumor or treatment of autoimmune diseases; or, use of the universal immune effector cell according to any one of claims 17 to 19 in combination with an immunosuppressant in the preparation of a drug for anti-tumor or treatment of autoimmune diseases; or, use of the universal immune effector cell according to any one of claims 17 to 19 in the preparation of a drug for anti-tumor or treatment of autoimmune diseases in combination with an immunosuppressant; Preferably, the immunosuppressant is selected from cyclosporin A, FK 506 and / or voclosporin; Preferably, the tumor is selected from leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulin blood disease, heavy chain disease, solid tumors such as sarcomas and carcinomas (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheliosarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendotheliosarcoma, synovial vioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, nipple cancer carcinoma, papillary adenocarcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma The autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, myasthenia gravis, polymyositis, psoriasis / psoriasis, pemphigus, vitiligo, multiple sclerosis, narcolepsy, neuromyelitis optica, type 1 diabetes, hyperthyroidism, Hashimoto's disease / hypothyroidism, Sjogren's syndrome, Crohn's disease, ulcerative colitis, celiac disease, autoimmune gastritis, primary biliary cholangitis, autoimmune hepatitis, lupus nephritis, Goodpasture's syndrome, autoimmune oophoritis, and autoimmune orchitis.

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