A cnb mutant and its use

Abstract

This invention provides a calcineurin B subunit mutant and its application in the preparation of universal immune effector cells. The CNB mutant of this invention can antagonize the blocking of the NFAT signaling pathway by CsA, FK506, or voclosporin; and simultaneously exhibits resistance to CsA, FK506, and voclosporin. Expressing the CNB mutant of this invention into universal immune effector cells, when combined with immunosuppressive drugs to treat tumors or autoimmune diseases, can reduce the activity of the patient's own T cells, thereby avoiding the patient's rejection of heterologous universal immune effector cells and improving the colonization and therapeutic effect of universal immune effector cells in the patient's body. Furthermore, after treatment, the universal immune effector cells can be rejected after the patient's immune system recovers, making the strategy of discontinuing immunotherapy after treatment possible, which can greatly improve the safety of immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a CNB (calcineurin B subunit) mutant and its application in the preparation of universal immune cells (such as universal T cells). Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) immunotherapy is a promising immunotherapy approach that has shown higher survival rates in many phase III clinical trials for advanced cancers.

[0003] The basic structure of a CAR typically includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. CARs can directly target tumor surface antigens, activate CAR-T cells, and kill tumor cells. Currently, CAR-T therapy T cells are mostly obtained from the patient's own peripheral blood. CARs are expressed on the surface of T cells in vitro using viral or non-viral techniques and then expanded in large quantities. The CAR-T cells are then re-injected into the patient to treat the disease. Although autologous CAR-T therapy has achieved great success, its application is very limited. First, this "personalized" treatment 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 experience a severe deterioration or even death during the preparation process. Third, due to the disease or previous treatments (such as radiotherapy and chemotherapy), the number of T cells in the patient's body may be low, or the quality of the T cells may be poor, making it difficult to meet the requirements for T cells in preparation. Currently, the efficacy of autologous CAR-T therapy may vary from person to person and is difficult to assess.

[0004] Therefore, the need for universal CAR-T therapy has emerged. Universal CAR-T cells are derived from healthy human T cells, ensuring their quality and quantity. CAR-T preparation can be scaled up and standardized, significantly reducing preparation costs. More importantly, universal CAR-T cells can be prepared in advance, cryopreserved, and used at any time, making CAR-T therapy more immediate. However, because universal CAR-T therapy is based on healthy individuals rather than autologous T cells, this therapy inevitably faces two major problems associated with allogeneic cell transplantation: graft-versus-host disease (GVHD) and host-versus-graft rejection (HVGR). The former, caused by the donor cells attacking the recipient cells, can lead to serious and even life-threatening complications. The latter, caused by the recipient cells rejecting the donor cells, makes it difficult for CAR-T cells to persist and proliferate in the patient's body long-term, reducing tumor-killing effects and resulting in the loss of subsequent tumor surveillance.

[0005] The main solution for GVHD in universal CAR-T cells is the genetic modification of T cells. Since GVHD is primarily related to the αβ T cell surface receptor (TCR), current research focuses on knocking out the αβ TCR on T cells using various gene tools such as TALENs and CRISPR-Cas9. In recent years, researchers have been able to efficiently knock out the αβ TCR and precisely introduce CAR at specific sites. This "TCR knockout + CAR knock-in" method reduces CAR self-activation signals, internalizes CAR expression, slows down effector T cell differentiation and exhaustion, and ultimately enhances the anti-tumor ability of CAR-T cells. It is one of the mainstream methods for preparing universal CAR-T cells today.

[0006] Another significant problem with universal CAR-T cells is the occurrence of HVGR, which affects the proliferation and anti-tumor efficacy of donor CAR-T cells in vivo. Currently, there are two main approaches to addressing HVGR. The first is to make the HLA of CAR-T cells "invisible" relative to the recipient's immune system, for example, by knocking out β2 microglobulin to construct HLA-I-deficient CAR-T cells to overcome rejection. However, the absence of HLA-I makes them more vulnerable to attack by recipient NK cells. Addressing rejection from an HLA perspective would require more complex gene editing to evade NK cell-mediated rejection. However, excessive gene editing carries significant safety risks, such as greater off-target effects and cytotoxicity. Another approach focuses on suppressing the recipient's immune system, especially T cells. For example, expressing certain molecules on CAR-T cells that can bind to markers of activated lymphocytes can inhibit the rejection of CAR-T cells by recipient-activated lymphocytes, thus evading recipient rejection. However, because such CAR-T cells can indiscriminately recognize markers of activated lymphocytes, they can continuously inhibit the normal functioning of the recipient's lymphatic system, raising safety concerns.

[0007] Calcineurin is a heterodimer composed of the 61kD calmodulin-binding catalytic subunit calmodulin-binding catalytic subunit calmodulin-binding catalytic subunit calmodulin-binding regulatory subunit calmodulin-binding catalytic ... Calcineurin is the target of a class of drugs called calcineurin inhibitors, including immunosuppressive drugs such as cyclosporine A (CsA), voclosporin, pimecrolimus, and tacrolimus (FK506).

[0008] Following the approach of suppressing the recipient's immune system, the use of immunosuppressive drugs is becoming a new method for addressing HVGR. For example, CsA or FK506 is commonly used in the treatment of diseases requiring stem cell or organ transplantation. Their mechanism primarily involves binding to cyclin to form a complex, inhibiting calcineurin phosphorylation, suppressing its nuclear transport process, and subsequently blocking the activation of the NFAT signaling pathway, thereby inhibiting T cell activation, proliferation, and the transcriptional secretion of cytokines (such as IL-2 and IFN-γ). Therefore, if CsA / FK506 can be used to suppress recipient T cells, it will significantly reduce the recipient T cell's HVGR response to donor CAR-T cells. Moreover, CsA / FK506 is a first-line drug for preventing organ transplant rejection, can be used lifelong, and its safety has been thoroughly verified. Because CsA / FK506 has a general inhibitory effect on T cells, CAR-T cells need to be modified to tolerate the inhibitory effect of CsA / FK506, thus enabling CAR-T cells to perform their normal killing function. However, the long-term presence of modified CAR-T cells may lead to tumor development and off-target effects. Therefore, there is a need for a universal CAR-T cell that can function normally when the recipient's immune system is suppressed, and which can be withdrawn after treatment by restarting HVGR, allowing the allogeneic universal CAR-T cells to be cleared by the recipient.

[0009] Currently, there are various methods to induce T cell tolerance to CsA / FK506, including gene editing techniques such as knocking out CyPA, FKBP12, and expressing mutated CNA or CNB. However, these methods have drawbacks, such as only being able to antagonize CsA or FK506, not being able to simultaneously antagonize two or more immunosuppressants (e.g., knocking out CyPA or FKBP12), or having very poor antagonistic efficiency (e.g., antagonistic efficiency of mutated CNA, CNB, and FK506 <60%). This hinders the flexible clinical application of immunosuppressants. Therefore, there is a need to enable T cells to tolerate immunosuppressants more effectively, or even further, to tolerate multiple immunosuppressants simultaneously (e.g., simultaneously tolerating CsA and FK506, or simultaneously tolerating CsA, FK506, and voclosporin). Summary of the Invention

[0010] This invention provides a calcineurin B subunit mutant that is resistant to the inhibitory effects of calcineurin inhibitors. That is, even in the presence of calcineurin inhibitors, NFAT can still be dephosphorylated and activated. Therefore, the calcineurin B subunit mutant provided by this invention can at least be used for the preparation of universal immune cells.

[0011] The amino acid sequence of the calcineurin B subunit (CNB) mutant of the present invention starts with SEQ ID NO.49, and inserts three amino acids X1X2X3 or two amino acids X4X5 between positions 125 and 126. X1, X2, X4, and X5 are not acidic amino acids, wherein: X1 is a neutral or basic amino acid, X2 is a neutral, acidic, or basic amino acid, X3 is a neutral or 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, a T129K substitution can be further added 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 present 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 neutral amino acids 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 neutral amino acids 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, X4 and X5 are KI or HV.

[0015] Preferably, the amino acid sequence of the CNB mutant is based on SEQ ID NO.49, with three amino acids X1X2X3 or two amino acids X4X5 inserted only between positions 125 and 126. When two amino acids X4X5 are inserted, a T129K substitution can be further performed 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, acidic amino acid, or basic amino acid; and X3 is a neutral amino acid or basic amino acid. In this preferred embodiment, X2 can 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 can 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, X2 is preferably S, R, T, G, K, P, A, V, C, H, N, E, I, W, or M, and X3 is preferably V, A, R, C, L, Q, G, Y, T, P, K, M, F, I, N, S, or W. In this preferred embodiment, it is even more preferable that X2 is S, R, T, G, P, A, V, N, E, or W, and X3 is preferably V, A, R, C, L, Q, G, Y, T, K, M, F, N, S, or W. In this preferred embodiment, X1, X2, and X3 are selected from REQ, RGN, RPK, RRG, RRL, RRW, RSA, RST, RSY, RTG, RTT, RVF, KAS, KGA, KNA, KSC, KSM, KSR, and KWV.

[0018] In one preferred embodiment, X1 is selected from R; X2 is selected from S, R, T, G, P, V, or E; and 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, or RVF. More preferably, X1 is selected from R; X2 is selected from R, T, G, or V; and X3 is selected from G, L, T, N, W, or F. For example, X1X2X3 is selected from RGN, RRL, RRW, RTG, RTT, or RVF. More preferably, X1 is selected from R; X2 is selected from R or T; and X3 is selected from G, L, or W. For example, X1X2X3 is selected from RRL, RRW, or RTG.

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

[0020] 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.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; and 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, X2 is preferably S, R, T, G, K, P, A, C, H, or M, and X3 is preferably V, A, R, C, L, Q, G, Y, P, or I. In this preferred embodiment, X2 is more preferably R, K, or S, and X3 is preferably 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, and YSP.

[0022] In one preferred embodiment, X1 is selected from T; X2 is selected from R, A, or P; and 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 or basic amino acid; and X3 is a neutral or 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, X1 is preferably L, C, Q, R, S, T, G, A, F, N, or I, and X3 is preferably 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 preferred embodiment, X2 is selected from R; X1 is selected from R, S, T, G, A, F, N, or I; and 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. More preferably, X2 is selected from R; X1 is selected from R, S, T, G, A, N, or I; and 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; and 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 as 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 or basic amino acid; and X3 is a neutral or basic amino acid. In this preferred embodiment, X1 may preferably be K, T, S, G, K, R, L, A, F, P, or Y. In this preferred embodiment, X3 may preferably be S, V, R, C, A, N, K, I, Q, M, Y, T, P, or F. In this preferred embodiment, X1, X2, and X3 are 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, and KWV.

[0028] In one preferred embodiment, X2 is selected from S; X1 is selected from R, K, L, A, F, G, P, or Y; and 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; and 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; and 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 as any one of SEQ ID NO. 2-6, 9, 12, 14-15, 17-19, 23, 25-30, 32-36, 40-42, 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 pronounced 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; and 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; more preferably, X1X2X3 are 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 are 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. 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, 25, 41, 42, 15, 45, 18, 27, 33; 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, 16, 17, 2, 32, 11, 9, 37, 40, 29, 24, 20; more preferably, the amino acid sequence of the CNB mutant is shown in SEQ ID NO. 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 pronounced 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; and 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 are 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 are 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 are 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. 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, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34; 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, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24; more preferably, the amino acid sequence of the CNB mutant is shown in SEQ ID NO. 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, 32, 2, 45, 26, 16, 20, 39; 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 pronounced 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 are 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; More 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, or 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, X1, X2, and X3 are 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. 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, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18, 40, 15, 5, 31, 26, 9, 34, 39, 33; more 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, 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 shown in SEQ ID NO. 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, 20, 25, 14, 11, 24, 2, 4, 29, 41, 22, 23, 19, 18; 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 pronounced 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, and RPK. KGA, LSA, LMC; preferably, X1X2X3 are 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; more preferably, X1X2X3 are 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 The following are also preferred: X1, X2, and X3 are selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, SCR, SRV, RTT, ARQ, GRV, LHA, or TAV; the most preferred is that X1, X2, and X3 are 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. 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, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24, 25, 15, 41, 37, 18, 34; 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, 32, 2, 45, 26, 16, 20, 39, 27, 42, 33, 24; more preferably, the amino acid sequence of the CNB mutant is shown in SEQ ID NO. 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, 32, 2, 45, 26, 16, 20, 39; 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 pronounced tolerance to FK506, CsA, and voclosporin, 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, R SY, SCR, RGN, ARQ, TAV, GRV, RVF, RTT, RRW, STC, TRG; more preferably, X1X2X3 are 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 are selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, TRL, IRV, KNA, LHA, SCR, RGN, ARQ, TAV; most preferably, X1X2X3 are 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; more preferably, the amino acid sequence of the CNB mutant is as shown in SEQ ID NO. 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, 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] This invention uses a random mutation library screening method commonly used in the art to obtain the CNB mutant of this invention. Experimental results show that the CNB mutant of this invention is resistant to calcineurin inhibitors. In cell experiments, after the CNB mutant of this invention is transformed into expression cells, in the presence of calcineurin inhibitors, the cell resistance to calcineurin inhibitors is at least 10%, for example 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 resistance is calculated as: (for the same clone cell, (a certain function of the cell after the addition of calcineurin inhibitor) / (a ​​certain function of the cell without calcineurin inhibitor) × 100%. In one embodiment of this invention, the amount of the cell expressing the signaling protein GFP is used as a representation of cell function. Those skilled in the art can prepare the CNB mutant of this invention using protein preparation methods 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, the tolerance of the cells to CsA in the presence of 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 transformed into cells, the cells have a tolerance to FK506 of at least 50% in the presence of FK506, 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 transformed into expression cells, the cells have a tolerance to voclosporin of at least 10% in the presence of voclosporin, preferably at least 30%, more preferably at least 50%, and most preferably at least 100%.

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

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

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

[0043] In some 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 exogenous protein 1 is the CNB mutant described above, and exogenous protein 2 recognizes tumor antigens, pathogen antigens, or autoantigens. Preferably, the T cell receptor (TCR) complex, T cell receptor α homeostasis protein, and / or T cell receptor β homeostasis protein are knocked out in the universal CAR T cell. The T cell may be CD4+. + T cells, CD8 + T cells, CD4 - CD8 - T cells or NKT cells (γδT cells are inserted into TRDCs).

[0044] In one embodiment of the present invention, the universal CAR-T cell is made by inserting CAR and the aforementioned CNB mutant of the present invention into the TCR TRAC site of the T cell.

[0045] According to the present invention, the 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 cells are knocked out. Preferably, the knockout comprises administering one or more substances selected from the group consisting of: antisense RNA, siRNA, shRNA, CRISPR / Cas system, RNA editing systems such as RNA adenosine deaminase (ADAR), RNA-directed endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, base editing, CRISPR interference, and transcriptional repression mediated by zinc finger protein (ZFN) gene repressors and / or transcription activator-like effector (TALE) gene repressors.

[0046] Preferably, CRISPR / Cas9 technology is used to administer a guide RNA (gRNA) targeting a nucleic acid molecule (TRAC) encoding the T cell receptor α constant region protein to T cells to knock out endogenous TCRs. 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, a pathogen antigen, or an autoantigen. The tumor antigen, pathogen antigen, or autoantigen may be selected, for example, from Claudin 18.2, Claudin 18.1, 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 includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

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

[0051] The intracellular signaling domain refers to the functional portion of the CAR that functions by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or by acting as an effector in response to such messengers. In some embodiments of the invention, the intracellular signaling domain contains a functional signaling domain derived from stimulatory molecules and / or co-stimulatory molecules as 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). In some embodiments of the invention, the stimulatory molecule is a ζ-chain that binds to the T-cell receptor complex.

[0052] In one embodiment of the 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 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 includes knocking out the endogenous T cell receptor complex, T cell receptor α homeostasis protein, and / or T cell receptor β homeostasis protein in the cells.

[0055] More preferably, the method further includes causing the cells to express a foreign protein 2 capable of recognizing tumor antigens or pathogen antigens, wherein, in one embodiment of the invention, the foreign 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 CNB mutant described above in the present invention.

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

[0058] More preferably, the method further includes enabling T cells to express chimeric antigen receptors capable of recognizing tumor antigens or pathogen antigens.

[0059] In some specific implementations, the method includes the following steps:

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

[0061] 2) Knock out TCRs in T cells;

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

[0063] 4) Universal CAR T cells expressing the CNB mutant and CAR of the present invention, and with downregulated or absent TCR expression, were isolated.

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

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

[0066] Preferably, step 3) includes constructing an IRU-CAR plasmid containing “left homologous arm-self-splitting sequence-CAR-self-splitting sequence-CNB mutant-right homologous arm”.

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

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

[0069] In some embodiments of the present invention, after the TRAC site is cleaved by Cas9 to create a gap, the DNA 350-450 bp to the left of the gap is the left homologous arm sequence, and the DNA 350-450 bp to the right of the gap is the right homologous arm sequence. In one embodiment of the present invention, the left homologous arm contains a nucleotide 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 nucleotide sequence shown in SEQ ID NO. 61; the right homologous arm contains a nucleotide 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 nucleotide sequence shown in SEQ ID NO. 62.

[0070] The CAR includes:

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

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

[0073] 3) Antibodies or their functional fragments 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, the antibody or its functional fragment that specifically binds to the target antigen, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, the co-stimulatory signaling domain of CD137, and CD3ζ are used.

[0075] In one embodiment of the 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.

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

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

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

[0079] Preferably, the transfection method is selected from electroporation or liposome transfection. More preferably, 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 cells described herein, and an immunosuppressant. In one embodiment of the present invention, the pharmaceutical composition comprises the universal T cells or universal CAR T cells described herein, and an immunosuppressant.

[0082] Preferably, the immunosuppressants include: calcineurin inhibitors, such as cyclosporine A, FK 506, voclosporin, and pimecrolimus; DMARDs, such as gold salts, sulfasalazine, antimalarial drugs, methotrexate, D-penicillamine, azathioprine, mycophenolate mofetil, tacrolimus, sirolimus, dimethylaminotetracycline, leflunomide, and glucocorticoids; lymphocyte recirculation regulators, such as FTY720 and FTY720 analogs; and mTOR inhibitors, such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, and ABT578. AP23573 or TAFA-93; ascomycins with immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; imidazolidin; mycophenolate mofetil; 15-deoxyguanidin or its immunosuppressive homologues, analogues, 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 cyclosporine A, voclosporin, pimecrolimus, or FK 506.

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

[0084] In a preferred embodiment of the invention, the use of the aforementioned universal T cells or universal CAR T cells in the preparation of medicaments for treating tumors or autoimmune diseases is provided.

[0085] In addition, the use of the aforementioned universal immune effector cells in combination with immunosuppressants in the preparation of medicaments for the treatment of tumors or autoimmune diseases is provided.

[0086] In a preferred embodiment of the present invention, the use of the universal T cell or universal CAR T cell combined with an immunosuppressant described herein in the preparation of a medicament for treating tumors or autoimmune diseases is provided.

[0087] In addition, the use of the aforementioned universal immune effector cells in the preparation of medicaments for use in combination with immunosuppressants against tumors or for the treatment of autoimmune diseases is provided.

[0088] In a preferred embodiment of the present invention, the use of the universal T cells or universal CAR T cells described herein in the preparation of medicaments for use in combination with immunosuppressants to treat tumors or autoimmune diseases is provided.

[0089] The tumors mentioned include, but are not limited to, leukemia (such as acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (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, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphangioendothelial sarcoma, synovial vioma, mesothelioma, Ewing's tumor, smooth muscle cell tumor). Tumors, 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, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, retinoblastoma, esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma.

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

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

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

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

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

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Terminology Explanation:

[0107] "And / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as 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" as 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] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0109] In this specification and claims, nucleotides are referred to by their generally accepted single-letter codes. Unless otherwise stated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are represented herein by generally known single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. Those skilled in the art will understand that the T base in the codons disclosed herein exists in DNA, while the T base is replaced by a U base in the corresponding RNA.

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

[0111] The term "amino acid" as used in this article refers to both natural and non-natural amino acids, with natural amino acids being preferred. These include: 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"), and 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 those whose amino and carboxyl groups are equal in number in the amino acid molecule. Their isoelectric point is generally 5.5 to 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 those in which the number of carboxyl groups in the molecule is greater than the number of amino groups. Their isoelectric point is generally 2.8–3.2. Among natural amino acids, aspartic acid (“Asp” or “D”) and glutamic acid (“Glu” or “E”) are acidic amino acids. Basic amino acids are those in which the number of amino groups in the molecule is greater than the number of carboxyl groups. Their isoelectric point is greater than 7, generally 7.6–10.8. 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 correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this disclosure, the term homology includes both identity and similarity.

[0114] In some embodiments, polymer molecules are considered “homological” 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 (completely identical monomers) or similar (conservative substitutions). The term “homological” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0115] Identity: As used herein, the term "identity" refers to the 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 the alignment of both protein and nucleotide sequences. For example, a suitable program for determining percentage sequence identity is Bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to compare 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, part of the bioinformatics EMBOSS program suite, and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or ClustalOmega), MUSCLE, etc. Attached Figure Description

[0116] Figure 1 Schematic diagram of the universal CAR T cell (Immunosuppressant Resistant Universal (IRU)-CAR-T) of this invention: Based on the traditional universal CAR-T (i.e., CAR-T with TCR knockout, hereinafter referred to as WT CAR-T), a calcineurin B subunit mutant is additionally introduced, which can greatly reduce or even avoid the rejection of CAR-T by the recipient immune T cells when used in combination with immunosuppressive drugs.

[0117] Figure 2Mutant CNB clones and WT CNB expression were compared in the Jurkat-NFAT-GFP reporter cell line. Cells were stimulated with PMA (50 ng / ml) / Ionomycin (1 μg / ml) for 16 hours 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 Functional experiments of IRU-CAR-T: IRU-CAR-T or WT CAR-T cells were prepared by inserting the hCD19scFv-CD28-CD3z-CNB44 or hCD19scFv-CD28-CD3z-WT-CNB sequence into the TRAC locus of T cells. Nalm6 cells expressing luficerase-GFP and CAR-T cells were cultured together at an E:T ratio of 1:5 (A, B) or different ratios (C) under different immunosuppressants (CsA (300 ng / ml), FK506 (5 ng / ml)) for 24 hours (A, C) or 72 hours (B). In A, Brefredin A was added 6 hours before the end of culture. In C, T cells without CAR gene expression were used as control T cells (CTRL-T). A shows the results of the cytokine assay; B shows the results of the proliferation assay; C shows the results of the killing assay. Detailed Implementation

[0119] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0120] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods described are conventional molecular biology methods in the art and can be performed with reference to molecular biology lab manuals 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 examples, this invention uses gene editing technology to introduce CAR and CNB mutants into T cells. Knockout of αβTCR will prevent GVHD. Based on this, this invention co-expresses the CNB mutant with CAR, combined with the use of CsA or FK506, to suppress recipient T cells and reduce their rejection of IRU-CAR-T cells. Simultaneously, the CNB mutant will help IRU-CAR-T cells avoid the inhibition of IRU-CAR-T cell activation, proliferation, and tumor killing by CsA / FK506, allowing them to function normally and kill tumor cells even when the recipient's immune system is suppressed. After treatment, discontinuation of CsA / FK506 will allow the recipient's immune system to recover function, triggering the recurrence of HVGR, and the recipient will reject and clear IRU-CAR-T cells, avoiding the potential for tumorigenesis caused by the long-term presence of IRU-CAR-T cells. Figure 1 ).

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

[0124] Nalm6 FFluc-GFP cells were purchased from Shanghai Model Biotechnology Co., Ltd.; Nalm6-FFluc-GFP-β2m KO The cells were constructed using lentiviruses generated from the plasmid lentiCRISPR v2 (addgene) containing the gRNA sequence 5'-AGCTCACATGGTTCACACGGCGTTT-3'. The Jurkat NFAT GFP cells were a donation from Professor Chen Wei of Zhejiang University. AAV-293 cells were purchased from the China National Experimental Cell Resource Sharing Platform.

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

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

[0127] Part 1: Procedure for Collecting Peripheral Blood from Volunteers

[0128] 1. Collect peripheral blood from volunteers.

[0129] 2. T cells were extracted using the RosetteSep™ human T cell enrichment kit and Ficoll lymphocyte separation medium.

[0130] 2.1 Add RosetteSep™ human T cellenrichment cocktail to an appropriate volume centrifuge tube at a concentration of 50 μl / ml of blood;

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

[0132] 2.3 Mix peripheral blood with sterile FACS solution (PBS containing 0.1% serum) at a 1:1 ratio thoroughly, and slowly add it to the lymphocyte separation medium along the tube wall using a Pasteur dropper. Be gentle and take care to maintain a clear liquid interface.

[0133] 2.4 Place the sample in a centrifuge, set the acceleration to 1 and the deceleration to 0, and centrifuge 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 layer 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 membrane layer.

[0135] 2.6 Carefully remove some of the supernatant, leaving about 1 ml. Use a pipette to insert into the white membrane layer and aspirate the lymphocytes. Place the lymphocytes in another 15 ml centrifuge tube, add more than 5 times the volume of FACS, and centrifuge at 400×g for 10 min.

[0136] 3. T cells were stimulated with Human T CD3 / CD28 beads and cultured in X-vivo liquid medium containing IL-7 and IL-15 at 37°C in a 5% CO2 incubator for 48 hours.

[0137] Part Two: 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, for lentiviruses / retroviruses, the fragment to be inserted needs to be inserted into the lentivirus / retrovirus backbone, while for AAV viruses, the fragment to be inserted needs 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: AAV virus backbone (serotype AAV6) is used, and the sequence "left homologous arm-P2A-CAR-T2A-CNB mutant-right homologous arm" is inserted at the multiple cloning site.

[0140] After the TRAC site is excised by Cas9, creating a gap, the approximately 400bp DNA to the left of the gap forms the left homologous arm sequence, and the approximately 400bp DNA to the right forms the right homologous arm sequence.

[0141] The left homologous arm sequence can be amplified using primers primer1 and primer2. The nucleotide sequences of primer1 and primer2 are shown in SEQ ID NO.57 and 58, respectively.

[0142] The right homologous arm sequence can be amplified using primers primer3 and primer4, whose nucleotide sequences are shown in SEQ ID NO.59 and 60, respectively.

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

[0144] The right homologous arm contains 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, the transmembrane region of CD28 or CD8, or CD3ζ;

[0147] 2) Antibodies or their functional fragments that specifically bind to tumor antigens or pathogen antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD28, and CD3ζ;

[0148] 3) Antibodies or their functional fragments 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 their functional fragments that specifically bind to the target antigen, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, the co-stimulatory 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, and in the following examples, the amino acid sequence of P2A is shown in SEQ ID NO.55.

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

[0153] The amino acid sequence of the CNB mutant can be any of SEQ ID NO.:1-47.

[0154] 1. Preparation of IRU CAR plasmid vector

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

[0156] 1.2 Fragment-skeleton reconstruction: Fragments can be linked to the skeleton using one-step cloning or T4 ligation methods;

[0157] 1.3 After plasmid construction, the sequence correctness was confirmed by methods such as Sanger sequencing.

[0158] 2. Preparation of IRU CAR virus

[0159] Similarly, the preparation of IRU CAR virus requires different strategies depending on the type of virus selected. The following uses AAV virus as an example to illustrate the preparation of IRU CAR virus.

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

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

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

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

[0164] 3.1 Preparation of Cas9-gRNA mixture ribonucleoprotein (RNP): Mix gRNA that can target the TRAC site with Cas9 protein evenly, and incubate at 37℃ for 15 min to form RNP;

[0165] The gRNA may be selected from any 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 step one, heat at 90×g for 10 minutes, and resuspend in electroporation buffer;

[0167] 3.3 Mix thoroughly with RNP and transfer to an electroporation cuvette. Electroporate RNP into T cells using a LONZA Nucleofector IIB machine U-014.

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

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

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

[0171] 3.7 Using Biolegend TM CD3 selection kit removes CD3 + The specific steps for obtaining IRU CAR-T cells include:

[0172] a) Cell counting: Take the required amount of cells (300×g), centrifuge 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 resuspending solution and incubate on ice for 15 min;

[0174] c) Add the 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 min to remove streptavidin beads;

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

[0177] Taking FMC63 CAR (targeting human CD19 protein) as an example, the TRAC site of the TCR was knocked out using CRISPR / Cas9 technology, and FMC63 CAR and calcineurin B subunit mutants (CNB1-CNB47, amino acid sequences as shown in SEQ ID NO. 1-47) were introduced into the TRAC site using AAV vectors. This method can generate the target IRU-CAR-T. Control CAR-Ts (WT CNB and CTRL CNB, amino acid sequences as shown in SEQ ID NO. 48-49) were prepared using the same method. It is worth noting that the IRU-CAR-T in this invention can be of various types of CARs, and is not limited to FMC63 CAR.

[0178] Example 3: Functional verification of calmodulin phosphatase B subunit mutant antagonizing CsA / FK506 / voclosporin

[0179] The mutant CNB clone-mCherry sequence was expressed in the Jurkat cell line carrying 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] Using the same clone of cells, the percentage of GFP expression was measured in the presence and absence of CsA (300 ng / ml), FK506 (5 ng / ml), or voclosporin (100 nM). Tolerance to CsA, FK506, or voclosporin was calculated using the following formula. Two replicates were performed for each inhibitor for each mutant, and the result is the average of the two replicates.

[0181] GFP expression % = (Number of cells expressing GFP after stimulation / Total number of cells) × 100%

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

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

[0184] Tolerance to voclosporin was calculated as follows: for the same clone, (GFP expression % after voclosporin addition) / (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 showed that the Jurkat-NFAT-GFP cell line carrying the CNB mutant of this invention could 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 this invention can tolerate the inhibition of the TCR signaling pathway by CsA, FK506, or voclosporin, which is higher than the tolerance of known CNB mutants 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 were completely inhibited and unable to express GFP. Figure 2 ).

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

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

[0194] Cytokine detection and proliferation assay:

[0195] 1×10 5Sorted CAR-T cells and 5-fold irradiated Nalm6-FFLuc-GFP cells were co-cultured for 24 hours in 96-well U-bottom tissue culture plates with / without CsA (300 ng / ml) or FK506 (5 ng / ml) in 200 μl X-vivo medium + 10% FBS. For cytokine assays, the Golgi Plug protein transport inhibitor Brefeldin A was added 6 hours before the end of culture. For proliferation, CAR-T cells were cultured for 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; and Aqua were used as live / dead staining agents. FcR blocking reagents were used to block Fc receptor staining. All extracellular and intracellular staining was performed using standard staining protocols.

[0196] Lethality experiments:

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

[0198] Cytokine assays confirmed that IRU-CAR-T cells can release cytokines in the presence of CsA / FK506. Figure 3 As shown in Figure A, IRU-CAR-T cells, after 24 hours of stimulation with Nalm-6 tumor cells, released NFAT pathway-related cytokines—IL-2, IFNγ, and TNFα—without significant difference in the presence or absence of CsA or FK506. However, WT-CAR-T cells, after 24 hours of stimulation with Nalm-6 tumor cells, showed a decreased ability to release cytokines in the presence of CsA or FK506, suggesting that they were unable to perform normal tumor-killing activities.

[0199] Similarly, CAR-T proliferation experiments verified that IRU-CAR-T cells could proliferate continuously upon stimulation with tumor antigens regardless of the presence or absence 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, on the other hand, could only proliferate in the absence of CsA or FK506. Figure 3 B).

[0200] In lethality experiments ( Figure 3 C), using the human Nalm-6 tumor cell line (CD19) + The target cells are shown in the figure. It can be seen that IRU-CAR-T cells showed no significant difference in killing efficiency at different effector cell:target cell (E / T) ratios, in the presence of CsA or FK506. In contrast, WT-CAR-T cells showed a significantly reduced killing efficiency in the presence of CsA / FK506 compared to the absence of 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 functional experiments together demonstrated that IRU-CAR-T cells carrying the CNB mutant of this invention can resist the inhibition of activation by CsA or FK506 and perform normal proliferation and tumor killing functions.

[0202] This invention proposes a CNB mutant and a universal CAR T cell (IRU-CAR-T) prepared using it. This invention utilizes gene editing methods, such as CRISPR / Cas9 and AAV site-specific knockout / knock-in techniques, to simply and efficiently introduce the CAR and the aforementioned CNB mutant, avoiding excessive gene editing. Through combination with CsA, FK506, or voclosporin, CsA / FK506 / voclosporin can reduce the activation of recipient immune T cells during treatment, reducing rejection of IRU-CAR-T cells. Simultaneously, due to the presence of the CNB mutant, IRU-CAR-T cells are protected from the inhibition of CsA / FK506 / voclosporin, thereby effectively reducing the HVGR phenomenon induced by the recipient's immune system against IRU-CAR-T cells during treatment. After treatment is completed, discontinuing CsA / FK506 / voclosporin will restart the rejection of IRU-CAR-T cells by the recipient's immune T cells, thereby clearing the allogeneic IRU-CAR-T cells and achieving withdrawal after tumor treatment, which greatly increases the safety of this immunotherapy.

[0203] Compared with other methods of 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 embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within 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, starting from SEQ ID NO.49, has three amino acids X1X2X3 inserted only between positions 125 and 126. 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.

2. The calcineurin B subunit mutant as described in claim 1, characterized in that, The amino acid sequence of the CNB mutant is shown in any one of SEQ ID NO. 1-20, 22-37, 39-42, or 44-47.

3. The calcineurin B subunit mutant as described in claim 1, characterized in that, The CNB mutant exhibits more pronounced FK506 tolerance, and 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, REQ, LSY, FSV, TPV, GGC, TRG, RPK, ASI, STC, LSA, KGA, or GSQ.

4. The calcineurin B subunit mutant as described in claim 3, characterized in that, 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, REQ, LSY, FSV, TPV, GGC, TRG, RPK, ASI or STC.

5. The calcineurin B subunit mutant as described in claim 3, characterized in that, 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.

6. The calcineurin B subunit mutant as described in claim 3, characterized in that, X1X2X3 is selected from RTG, YSP, RRL, NRA, KWV, RRW, TRL, IRV, TRV, GRV, KSC, KNA, QKR, RVF, PSR, SCR, ARQ, SRV, or TAV.

7. The calcineurin B subunit mutant as described in claim 1, characterized in that, The CNB mutant exhibits more pronounced CsA tolerance, and 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, and LMC.

8. The calcineurin B subunit mutant as described in claim 7, characterized in that, 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.

9. The calcineurin B subunit mutant as described in claim 7, characterized in that, 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.

10. The calcineurin B subunit mutant as described in claim 7, characterized in that, 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.

11. The calcineurin B subunit mutant as described in claim 7, characterized in that, 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.

12. The calcineurin B subunit mutant as described in claim 7, characterized in that, X1X2X3 is selected from YSP, RTG, RRL, NRA, TRL, KSC, TRV, IRV, KWV, RGN, KSR, QKR, KNA, PSR, or SCR.

13. The calcineurin B subunit mutant as described in claim 1, characterized in that, The CNB mutant exhibits more pronounced voclosporin resistance, and 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.

14. The calcineurin B subunit mutant as described in claim 13, characterized in that, 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.

15. The calcineurin B subunit mutant as described in claim 13, characterized in that, X1 X2 16. The calcineurin B subunit mutant as described in claim 13, characterized in that, 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.

17. The calcineurin B subunit mutant as described in claim 13, characterized in that, X1X2X3 is selected from YSP, KWV, NRA, KSC, ASI, RTG, TRV, SRV, QKR, PSR, RRL, RRG, KSR, RSA, TRL or IRV.

18. The calcineurin B subunit mutant as described in claim 1, characterized in that, The CNB mutant exhibits more pronounced resistance to FK506, CsA, and voclosporin, 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, and TRG.

19. The calcineurin B subunit mutant as described in claim 18, characterized in that, 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.

20. The calcineurin B subunit mutant as described in claim 18, characterized in that, 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.

21. The calcineurin B subunit mutant as described in claim 18, characterized in that, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, SRV, QKR, PSR, RRL, KSR, TRL, IRV, KNA, LHA, SCR, RGN, ARQ, TAV.

22. The calcineurin B subunit mutant as described in claim 18, characterized in that, X1X2X3 is selected from YSP, KWV, NRA, KSC, RTG, TRV, QKR, PSR, RRL, TRL, and IRV.

23. Use of the calcineurin B subunit mutant according to any one of claims 1-22 in the preparation of universal immune effector cells.

24. The use as described in claim 23, characterized in that, The immune effector cells are T cells and NK cells.

25. The use as described in claim 24, characterized in that, The T cells are CD4. + T cells, CD8 + T cells, CD4 - CD8 - T cells, γδT cells, or NKT cells.

26. A universal immune effector cell, characterized in that, The universal immune effector cells express exogenous protein 1, wherein exogenous protein 1 is a calcineurin B subunit mutant as described in any one of claims 1-22.

27. The universal immune effector cell as described in claim 26, characterized in that, The amino acid sequence of the calcineurin B subunit mutant is shown in any one of SEQ ID NO. 1-20, 22-37, 39-42, or 44-47.

28. The universal immune effector cell as described in claim 26 or 27, characterized in that, The universal immune effector cells further express exogenous protein 2, which is capable of recognizing tumor antigens, pathogen antigens, or self-antigens.

29. The universal immune effector cell as described in claim 28, characterized in that, The exogenous protein 2 is a chimeric antigen receptor.

30. The universal immune effector cell as described in claim 29, characterized in that, The chimeric antigen receptor includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain; the antigen-binding domain binds to tumor antigens, pathogen antigens, or autoantigens; and the intracellular signaling domain contains a functional signaling domain derived from stimulatory molecules and / or co-stimulatory molecules.

31. The universal immune effector cell as described in claim 29, characterized in that, The CAR contains the amino acid sequence shown in SEQ ID NO.

63.

32. The universal immune effector cell as described in claim 26 or 27, characterized in that, The T cell receptor complex, T cell receptor α homeostasis protein, and / or T cell receptor β homeostasis protein were knocked out in the general immune effector cells.

33. The universal immune effector cell as described in claim 26 or 27, characterized in that, The immune effector cells are T cells or NK cells.

34. The universal immune effector cell as described in claim 33, characterized in that, The T cells are CD4. + T cells, CD8 + T cells, CD4 - CD8 - T cells, γδT cells, or NKT cells.

35. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the universal immune effector cells as described in any one of claims 26-34, and an immunosuppressant.

36. The pharmaceutical composition of claim 35, characterized in that, The immunosuppressant is selected from cyclosporine A, FK506 and / or voclosporin.

37. The use of the universal immune effector cells of claim 31 in the preparation of medicaments for treating tumors or autoimmune diseases; or the use of the universal immune effector cells of claim 31 in combination with immunosuppressants in the preparation of medicaments for treating tumors or autoimmune diseases; or the use of the universal immune effector cells of claim 31 in the preparation of medicaments for treating tumors or autoimmune diseases in combination with immunosuppressants. The tumor is selected from leukemia and lymphoma; the autoimmune disease is selected from systemic lupus erythematosus, multiple sclerosis, and neuromyelitis optica.

38. The use as described in claim 37, characterized in that, The immunosuppressant is selected from cyclosporine A, FK 506 and / or voclosporin.

39. The use as described in claim 37 or 38, characterized in that, The leukemias mentioned are selected from: acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and polycythemia vera.

40. The use as described in claim 37 or 38, characterized in that, The lymphoma referred to is Hodgkin's disease or non-Hodgkin's disease.

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