Drug, method, and use for treating diseases caused by autoantibody production resulting from excessive proliferation of b cells

Abstract

Provided are a drug, a method, and use for treating diseases caused by autoantibody production resulting from excessive proliferation of B cells. The drug for treating diseases caused by autoantibody production resulting from excessive proliferation of B cells comprises: an mRNA encoding a chimeric antigen receptor molecule and a T cell targeting lipid nanoparticle. The drug can produce a chimeric antigen receptor T cell in vivo. The lipid nanoparticle comprises: a cationic lipid, a phospholipid, cholesterol, a PEG lipid, and a targeting molecule. The targeting molecule mediates T cell targeting. The lipid nanoparticle encapsulating the mRNA of the chimeric antigen receptor molecule (mRNA-LNP) is used to deliver the mRNA into a T cell in vivo to produce the chimeric antigen receptor T cell, and pathological autoreactive B cells are eliminated to achieve the treatment of diseases caused by autoantibody production resulting from excessive proliferation of B cells.

Description

A drug, method, and application for treating diseases caused by excessive B cell proliferation and the production of autoantibodies.

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024109165802, filed July 9, 2024; Chinese Patent Application No. 202411637380X, filed November 15, 2024; and Chinese Patent Application No. 2025105988627, filed May 9, 2025, entitled "A drug, method and application for treating diseases caused by excessive proliferation of B cells producing autoantibodies", the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention relates to the field of pharmaceutical technology, and more specifically, to a drug, method, and application for treating diseases caused by excessive proliferation of B cells producing autoantibodies. Background Technology

[0004] The pathogenesis of autoimmune diseases is complex, commonly characterized by chronic inflammatory damage to organs and tissues. The etiology and pathogenesis are not fully understood, but it is currently believed that they may be caused by a combination of factors including genetics, inflammation, hormones, and the environment, leading to an imbalance in the body's immune homeostasis, regulatory disorders, and abnormal function of autoreactive B cells and T cells, resulting in damage to tissues and organs. Diseases caused by excessive B cell proliferation producing autoantibodies include systemic lupus erythematosus, myositis, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, sensitized kidney transplantation, autoimmune encephalitis, immune-associated necrotizing myositis, refractory rheumatoid arthritis, systemic sclerosis, stiff-person syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Lambert-Eaton myasthenia gravis, and DAGLA encephalitis.

[0005] Conventional treatments for autoimmune diseases include the use of glucocorticoids and immunosuppressants. In recent years, biological antibody drugs have also entered clinical practice, but their efficacy remains limited. Furthermore, the therapeutic effect of monoclonal antibody drugs is limited by their inability to penetrate tissues. Since autoimmune disease patients have a large number of pathologically reactive B cells in their lymphatic tissues, monoclonal antibody drugs cannot enter the tissues to act on these pathological B cells. Therefore, conventional biological therapies are also less effective, and patients with autoimmune diseases still experience recurrent relapses.

[0006] In the first clinical case of CAR T-cell therapy for systemic lupus erythematosus (SLE), the patient presented with active lupus nephritis, nephrotic syndrome, pericarditis, pleurisy, rash, arthritis, and endocarditis prior to CAR T-cell therapy. The patient had previously received hydroxychloroquine, high-dose corticosteroids, immunosuppressants (cyclophosphamide, mycophenolate mofetil, tacrolimus), and belimumab and rituximab, but all treatments failed to control symptoms, resulting in persistent relapses. After pretreatment with fludarabine and cyclophosphamide (lymph node clearing), the patient received CD19 CAR T-cell therapy. Following CAR T-cell infusion, the number of cells rapidly increased in the patient, and no adverse events related to CAR T-cell therapy occurred. The patient's clinical condition and serological markers simultaneously improved, achieving complete remission without medication. The patient has remained relapse-free to date, demonstrating the effectiveness and durability of CAR T-cell therapy in SLE treatment. To date, clinical data from 119 cases of CAR T-cell therapy for autoimmune diseases have been published, all achieving clinical remission and meeting the clinical observation endpoint. Therefore, the efficacy of CAR T-cell therapy is undeniable. However, CAR T-cell therapy is expensive to produce and has a long production cycle. A lymphoma clearance procedure is required before treatment, and the risk of cytokine storm after CAR T-cell therapy is high. Furthermore, the current mainstream technology for CAR T-cell therapy involves viral integration and reprogramming of T-cell genes, which carries a risk of tumorigenesis for patients. Additionally, because CAR T-cell therapy integrates CAR molecules into the genome, the dynamics of CAR T cells in the human body are unpredictable. These factors compel the vast patient population requiring CAR T-cell therapy to urgently seek a more accessible, effective, and safer treatment method. Summary of the Invention

[0007] One of the objectives of this invention is to provide a new, safe and effective CAR T-cell replacement therapy for treating immune system diseases such as systemic lupus erythematosus, in order to improve the limitations of traditional CAR T-cell therapy.

[0008] Specifically, the present invention provides a composition comprising: mRNA encoding a chimeric antigen receptor molecule and T cell-targeting lipid nanoparticles;

[0009] The composition can generate chimeric antigen receptor T cells in vivo and clear autologous B cells;

[0010] The T-cell-targeting lipid nanoparticles comprise: cationic lipids, phospholipids, cholesterol, PEG lipids, and targeting molecules, wherein the targeting molecules mediate T-cell targeting.

[0011] In the compositions of the present invention, the mRNA may encode CD19 CAR, CD20 CAR, BCMACAR, CD19 and CD20 dual CAR, CD19 and BCMA dual CAR or CD20 and BCMA dual CAR, preferably the mRNA may encode CD19 CAR, CD20 CAR and / or BCMACAR, more preferably the mRNA may encode CD19 CAR.

[0012] In the composition of the present invention, the surface antigens of T cells recognized by the T cell-targeting lipid nanoparticles include one or more of CD3, CD4, CD5, CD7, and CD8;

[0013] Preferably, the surface antigen of the T cell recognized by the T cell-targeting lipid nanoparticles is one or more of CD3, CD8, CD7 or CD5, more preferably CD5 or CD8.

[0014] In the composition of the present invention, the targeting molecule of the T cell-targeting lipid nanoparticles comprises: an antibody that recognizes a T cell surface antigen, a linker, and a hydrophobic tail; the T cell surface antigen includes one or more of CD8, CD4, CD5, CD7, or CD3; the linker comprises PEG or a PEG derivative, and the hydrophobic tail comprises one or more substituted or unsubstituted C-terminals. 12 -C 18 Alkyl group, wherein the antibody and the linker are connected by an amino acid containing a reaction site, wherein the amino acid includes, but is not limited to, cysteine ​​and lysine;

[0015] Preferably, the antibody that recognizes T cell surface antigens includes nanobodies;

[0016] More preferably, the nanobody shown includes an antigen-binding domain that is specific to the antigen, wherein the antigen is a membrane protein molecule;

[0017] More preferably, the membrane protein molecule is an immune cell or immune-related cell membrane protein molecule.

[0018] For information on targeting molecules, antibodies that recognize cell surface antigens, linkers, and hydrophobic tails, refer to patent applications CN119751652A and WO2025113580A1, the contents of which are incorporated herein by reference.

[0019] In the composition of the present invention, the amino acid sequences of the antibody recognizing T cell surface antigens, CDR1, CDR2, and CDR3, are respectively shown in any one of the following groups:

[0020] (1) SEQ ID NO: 114; SEQ ID NO: 160; SEQ ID NO: 206;

[0021] (2)SEQ ID NO:115;SEQ ID NO:161;SEQ ID NO:207;

[0022] (3)SEQ ID NO:116;SEQ ID NO:162;SEQ ID NO:208;

[0023] (4)SEQ ID NO:117;SEQ ID NO:163;SEQ ID NO:209;

[0024] (5)SEQ ID NO:118;SEQ ID NO:164;SEQ ID NO:210;

[0025] (6)SEQ ID NO:119;SEQ ID NO:165;SEQ ID NO:211;

[0026] (7)SEQ ID NO:120;SEQ ID NO:166;SEQ ID NO:212;

[0027] (8)SEQ ID NO:121;SEQ ID NO:167;SEQ ID NO:213;

[0028] (9)SEQ ID NO:122;SEQ ID NO:168;SEQ ID NO:214;

[0029] (10)SEQ ID NO:123;SEQ ID NO:169;SEQ ID NO:215;

[0030] (11)SEQ ID NO:124;SEQ ID NO:170;SEQ ID NO:216;

[0031] (12)SEQ ID NO:125;SEQ ID NO:171;SEQ ID NO:217;

[0032] (13)SEQ ID NO:126;SEQ ID NO:172;SEQ ID NO:218;

[0033] (14)SEQ ID NO:127;SEQ ID NO:173;SEQ ID NO:219;

[0034] (15)SEQ ID NO:128;SEQ ID NO:174;SEQ ID NO:220;

[0035] (16)SEQ ID NO:129;SEQ ID NO:175;SEQ ID NO:221;

[0036] (17)SEQ ID NO:130;SEQ ID NO:176;SEQ ID NO:222;

[0037] (18)SEQ ID NO:131;SEQ ID NO:177;SEQ ID NO:223;

[0038] (19)SEQ ID NO:132;SEQ ID NO:178;SEQ ID NO:224;

[0039] (20)SEQ ID NO:133;SEQ ID NO:179;SEQ ID NO:225;

[0040] (21)SEQ ID NO:134;SEQ ID NO:180;SEQ ID NO:226;

[0041] (22)SEQ ID NO:135;SEQ ID NO:181;SEQ ID NO:227;

[0042] (23)SEQ ID NO:136;SEQ ID NO:182;SEQ ID NO:228;

[0043] (24)SEQ ID NO:137;SEQ ID NO:183;SEQ ID NO:229;

[0044] (25)SEQ ID NO:138;SEQ ID NO:184;SEQ ID NO:230;

[0045] (26)SEQ ID NO:139;SEQ ID NO:185;SEQ ID NO:231;

[0046] (27)SEQ ID NO:140;SEQ ID NO:186;SEQ ID NO:232;

[0047] (28)SEQ ID NO:141;SEQ ID NO:187;SEQ ID NO:233;

[0048] (29)SEQ ID NO:142;SEQ ID NO:188;SEQ ID NO:234;

[0049] (30)SEQ ID NO:143;SEQ ID NO:189;SEQ ID NO:235;

[0050] (31)SEQ ID NO:144;SEQ ID NO:190;SEQ ID NO:236;

[0051] (32)SEQ ID NO:145;SEQ ID NO:191;SEQ ID NO:237;

[0052] (33)SEQ ID NO:146;SEQ ID NO:192;SEQ ID NO:238;

[0053] (34)SEQ ID NO:147;SEQ ID NO:193;SEQ ID NO:239;

[0054] (35)SEQ ID NO:148;SEQ ID NO:194;SEQ ID NO:240;

[0055] (36)SEQ ID NO:149;SEQ ID NO:195;SEQ ID NO:241;

[0056] (37)SEQ ID NO:150;SEQ ID NO:196;SEQ ID NO:242;

[0057] (38)SEQ ID NO:151;SEQ ID NO:197;SEQ ID NO:243;

[0058] (39)SEQ ID NO:152;SEQ ID NO:198;SEQ ID NO:244;

[0059] (40)SEQ ID NO:153;SEQ ID NO:199;SEQ ID NO:245;

[0060] (41)SEQ ID NO:154;SEQ ID NO:200;SEQ ID NO:246;

[0061] (42)SEQ ID NO:155;SEQ ID NO:201;SEQ ID NO:247;

[0062] (43)SEQ ID NO:156;SEQ ID NO:202;SEQ ID NO:248;

[0063] (44)SEQ ID NO:157;SEQ ID NO:203;SEQ ID NO:249;

[0064] (45)SEQ ID NO:158;SEQ ID NO:204;SEQ ID NO:250;

[0065] (46)SEQ ID NO:159;SEQ ID NO:205;SEQ ID NO:251;

[0066] (47)SEQ ID NO:252;SEQ ID NO:275;SEQ ID NO:298;

[0067] (48)SEQ ID NO:253;SEQ ID NO:276;SEQ ID NO:299;

[0068] (49)SEQ ID NO:254;SEQ ID NO:277;SEQ ID NO:300;

[0069] (50)SEQ ID NO:255;SEQ ID NO:278;SEQ ID NO:301;

[0070] (51)SEQ ID NO:256;SEQ ID NO:279;SEQ ID NO:302;

[0071] (52)SEQ ID NO:257;SEQ ID NO:280;SEQ ID NO:303;

[0072] (53)SEQ ID NO:258;SEQ ID NO:281;SEQ ID NO:304;

[0073] (54)SEQ ID NO:259;SEQ ID NO:282;SEQ ID NO:305;

[0074] (55)SEQ ID NO:260;SEQ ID NO:283;SEQ ID NO:306;

[0075] (56)SEQ ID NO:261;SEQ ID NO:284;SEQ ID NO:307;

[0076] (57)SEQ ID NO:262;SEQ ID NO:285;SEQ ID NO:308;

[0077] (58)SEQ ID NO:263;SEQ ID NO:286;SEQ ID NO:309;

[0078] (59)SEQ ID NO:264;SEQ ID NO:287;SEQ ID NO:310;

[0079] (60)SEQ ID NO:265;SEQ ID NO:288;SEQ ID NO:311;

[0080] (61)SEQ ID NO:266;SEQ ID NO:289;SEQ ID NO:312;

[0081] (62)SEQ ID NO:267;SEQ ID NO:290;SEQ ID NO:313;

[0082] (63)SEQ ID NO:268;SEQ ID NO:291;SEQ ID NO:314;

[0083] (64)SEQ ID NO:269;SEQ ID NO:292;SEQ ID NO:315;

[0084] (65)SEQ ID NO:270;SEQ ID NO:293;SEQ ID NO:316;

[0085] (66)SEQ ID NO:271;SEQ ID NO:294;SEQ ID NO:317;

[0086] (67)SEQ ID NO:272;SEQ ID NO:295;SEQ ID NO:318;

[0087] (68)SEQ ID NO:273;SEQ ID NO:296;SEQ ID NO:319;

[0088] (69)SEQ ID NO:274;SEQ ID NO:297;SEQ ID NO:320;

[0089] Preferably, the amino acid sequence of the antibody recognizing T cell surface antigen is as shown in any one of SEQ ID NO:1-46, SEQ ID NO:85-107, and SEQ ID NO:331-355, or as shown in a sequence with ≥80% homology (e.g., 80%, 85%, 90%, 95%, 97%) to any one of SEQ ID NO:1-46, SEQ ID NO:85-107, and SEQ ID NO:331-355.

[0090] Preferably, the surface antigen of the T cells corresponding to SEQ ID NO:1-20, SEQ ID NO:44-45, SEQ ID NO:85-107 or SEQ ID NO:331-355 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:21-43 or SEQ ID NO:46 is CD5;

[0091] More preferably, the surface antigen of the T cells corresponding to SEQ ID NO:98-105 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:46 is CD5.

[0092] In the composition of the present invention, the molar ratio of the cationic lipid, phospholipid, cholesterol, PEG lipid and the targeting molecule is 30-70:5-30:0-65:0.2-5:0.005-1; preferably 40-60:10-20:30-60:0.5-2:0.01-0.5; more preferably 45-55:10-20:30-40:0.75-1.5:0.05-0.25;

[0093] A further preferred ratio is 47-52:12-18:32-38:1-1.2:0.05-0.20;

[0094] A further preferred ratio is 47-50:13-16:34-36:1.0-1.1:0.05-0.15;

[0095] Further preferred ratios are 47.5:15:36:1.0:0.05 or 47.5:15:36:1.0:0.1.

[0096] The cationic lipids include SM102, Alc0315, and have the structure shown in general formula (I), or isomers of the structure shown in general formula (I), pharmaceutically acceptable salts, prodrugs or solvates.

[0097] Wherein, G is selected from H, OR, CN, -C(=O)OR′, -OC(=O)R', -C(=O)NR'R”, -NR'C(=O)R”, NR'R” or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N;

[0098] Among them, R, R', and R" are the same or different from each other, and are each independently selected from H, C1-C 10 Alkyl, C3-C 10 cycloalkyl, C3-C 10 alkenyl or C3-C 10 Cycloalkenyl, C1-C terminally linked to a tertiary amine 10 alkyl groups, C3-C structures with tertiary amines at the end 10 Cycloalkyl groups, C3-C with terminal tertiary amines 10 Alkenyl or cyclic alkyl structure containing at least one heteroatom;

[0099] Preferably, R, R', and R'' are the same as or different from each other, and each is independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl, C3-C8 cycloalkenyl, and C1-C8 alkyl groups with a terminal tertiary amine. 10 alkyl groups, C3-C structures with tertiary amines at the end 10 Cycloalkyl groups, C3-C with terminal tertiary amines 10 Alkenyl or cyclic alkyl structure containing at least one heteroatom;

[0100] Wherein, the substituted carbon atoms or heteroatoms in the cyclic alkyl structure are not substituted or are substituted by one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups;

[0101] Preferably, the cyclic alkyl structure is not substituted or is substituted by one or more C1-C4 alkyl, C3-C8 cycloalkyl or hydroxyl groups;

[0102] More preferably, R′ and R” are the same or different from each other, and each is independently selected from H, C1-C4 alkyl groups, and C1-C4 groups terminally linked with tertiary amines. 10 alkyl groups, C3-C structures with tertiary amines at the end 10 A cycloalkyl or cycloalkyl structure containing at least one heteroatom; the cycloalkyl structure is not substituted or is substituted by one or more C1-C4 alkyl or C3-C8 cycloalkyl groups.

[0103] And / or, M1, M2, M3, and M4 may be the same as or different from each other, and each is independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 alkenyl or C3-C 24Cycloalkenyl; preferably, M1, M2, M3, and M4 may be the same as or different from each other, and M1 and M4 are each independently selected from C4-C containing branches. 22 Alkylene, branched C4-C 22 Cycloalkylene, branched C4-C 22 alkenyl or branched C4-C 22 The cycloene groups, M2 and M3, are each independently selected from C4-C6. 22 Alkylene, C4-C 22 Cycloalkylene, C4-C 22 alkenyl or C4-C 22 Cycloalkenyl group; preferably, M2 and M3 are the same and are C4-C. 22 Alkylene; M1 is the same as M4, and is a branched C4-C. 22 Alkylene;

[0104] And / or, R 1 R 2 They may be the same as or different from each other, and each is independently selected from H, C1-C. 24 Alkyl, C3-C 24 cycloalkyl, C2-C 24 alkenyl or C3-C 24 Cycloalkenyl; preferably, R 1 R 2 They are the same or different from each other, and each is independently selected from C4-C. 22 Alkyl or C4-C 22 alkenyl;

[0105] And / or, L1, L2, L3, and L4 may be the same as or different from each other, and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S-, or -SS-; preferably,

[0106] L1, L2, L3, and L4 may be the same as or different from each other, and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, or -SS-; when L1, L2, L3, and L4 are independently selected from -C(=O)NR- or -NRC(=O)-, R is independently preferred from H or C1-C 10 Alkyl; preferably, L1 and L4 are the same, and are -C(=O)O- or -OC(=O)-;

[0107] And / or, M5 is selected from single bonds, C1-C 16 Alkylene, C2-C 16The group consists of an alkenyl group, a C3-C8 cycloalkyl group, or a C3-C8 cycloalkyl group. Preferably, M5 is selected from single bonds, C2-C... 16 Alkylene, C2-C 16 alkenyl, C4-C8 cycloalkylene, or C3-C8 cycloalkylene; preferably, M5 is selected from single bonds, C2-C 16 Alkylene or C4-C6 cycloalkylene;

[0108] More preferably, M5 and G are connected to form Specifically, any one of A1 to A52 can be selected:

[0109] Among them, the Preferably, it is selected from any one of A1-A18, A22-A24, and A28-A52; more preferably, it is selected from any one of A15, A23, A29, A33, and A39-A52.

[0110] And / or, R 1 The segment -L1-M1-L2-M2- is R 1 -C(=O)O-M1-OC(=O)-M2-、R 1 -C(=O)NR-M1-OC(=O)-M2-or R 1 -C(=O)O-M1-SS-M2-;R 2 The L4-M4-L3-M3 segment is R. 2 -C(=O)O-M4-OC(=O)-M3-、R 2 -C(=O)NR-M4-OC(=O)-M3-or R 2 -C(=O)O-M4-SS-M3-;

[0111] More preferably, the general formula (I) structure is selected from any of the following structures:

[0112] For information on cationic lipids, phospholipids, cholesterol, PEG lipids and their preparation methods, please refer to patent applications CN118724740A and CN119751652A, WO2025113580A1 and patent WO2024198497A1, the contents of which are incorporated herein by reference.

[0113] Specifically, the cationic lipids, phospholipids, cholesterol, and PEG lipids are E12LA6B6O3, DSPC, CHOL, and DSPE-PEG2000, respectively.

[0114] In the composition of the present invention, the molar ratio of the cationic lipid to the mRNA is 4-8:1, preferably 5-7.5:1, more preferably 6-7.5:1, and even more preferably 6.5-7.5:1.

[0115] In the composition of the present invention, the mRNA comprises a coding region encoding a polypeptide, a 5' untranslated region and a 3' untranslated region, and a polyadenylated tail at the end of the 3' untranslated region;

[0116] Preferably, the 3' untranslated region includes at least one microRNA binding site for expression in a specific cell type, such that mRNA expression can be reduced, wherein the mRNA is targeted for degradation or reduced translation in the presence of the microRNA; the at least one microRNA binding site includes miR-122; the sequence of miR-122 is shown in SEQ ID NO:108; more preferably, the sequence of the 3' untranslated region is shown in any one of SEQ ID NO:110-112;

[0117] Preferably, the sequence of the 5' untranslated region is as shown in SEQ ID NO:109;

[0118] Preferably, the polyadenylate tail sequence is as shown in SEQ ID NO:113.

[0119] In the compositions of the present invention, the coding region sequence of the polypeptide encoding the mRNA is as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461, or as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, or SEQ ID NO:457-461. The sequence is a sequence with ≥80% homology to any one of the sequences shown in SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461; preferably ≥90%; more preferably ≥95%; and even more preferably ≥98%.

[0120] Preferably, the full-length sequence of the mRNA is as shown in any one of SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471, or as shown in any one of SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, or .... The sequence is a sequence having ≥80% homology to any one of the sequences shown in SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471; preferably ≥90%; more preferably ≥95%; even more preferably ≥98%.

[0121] Preferably, the nucleic acid sequence encoding the CD19 antigenic domain as shown in any one of SEQ ID NO:47-49, SEQ ID NO:54-56, SEQ ID NO:356-357, or SEQ ID NO:461; the nucleic acid sequence encoding the BCMA antigenic domain as shown in any one of SEQ ID NO:52-53, SEQ ID NO:63, SEQ ID NO:406-407, SEQ ID NO:412-413, or SEQ ID NO:418-419; the nucleic acid sequence encoding the CD20 antigenic domain as shown in any one of SEQ ID NO:50-51 or SEQ ID NO:57-60; the nucleic acid sequence encoding the BCMA-CD20 antigenic domain as shown in any one of SEQ ID NO:64-65; and the nucleic acid sequences encoding the BCMA-CD20 antigenic domain as shown in any one of SEQ ID NO:61-62, SEQ ID NO:324-326, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:461, SEQ ID NO:64-65, SEQ ID NO:379-381, SEQ ID NO:64-65, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:64-65 ... The nucleic acid sequence shown in any one of SEQ ID NO:388-390, SEQ ID NO:397-399 or SEQ ID NO:457-460 encodes the antigenic domain of CD19-BCMA;

[0122] Preferably, the amino acid sequences of the antigenic domain of CD19 encoded by nucleic acid sequences shown in SEQ ID NO:47-49, SEQ ID NO:54-56, SEQ ID NO:356-357, and SEQ ID NO:461 are as shown in SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444, and SEQ ID NO:476, respectively; the amino acid sequences of the antigenic domain of BCMA encoded by nucleic acid sequences shown in SEQ ID NO:52-53, SEQ ID NO:63, SEQ ID NO:406-407, SEQ ID NO:412-413, and SEQ ID NO:418-419 are as shown in SEQ ID NO:429-430, SEQ ID NO:440, and SEQ ID NO:451-453, respectively; and the amino acid sequences of the antigenic domain of CD20 encoded by nucleic acid sequences shown in SEQ ID NO:50-51 and SEQ ID NO:57-60 are as shown in SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444, and SEQ ID NO:476, respectively. The amino acid sequences of the antigenic domains of BCMA-CD20 encoded by the nucleic acid sequences shown in SEQ ID NO:427-428 and SEQ ID NO:434-437 are shown in SEQ ID NO:64-65, respectively; the amino acid sequences of the antigenic domains of CD19-BCMA encoded by the nucleic acid sequences shown in SEQ ID NO:61-62, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, and SEQ ID NO:457-460 are shown in SEQ ID NO:438-439, SEQ ID NO:445-450, and SEQ ID NO:472-475, respectively.

[0123] In the composition of the present invention, the mRNA is a modified mRNA, the modified mRNA comprising a substitute uridine, the substitute uridine being selected from pseudouridine or 1-methylpseudouridine.

[0124] For information on mRNA structure and preparation methods, please refer to patent application CN118910042A, the contents of which are incorporated herein by reference.

[0125] The present invention also provides the use of the above composition in reducing autoreactive pathological B cells in patients, improving one or more of the following: autoantibody levels, C-reactive protein levels, proteinuria levels, type I interferon levels, vascular inflammation, kidney involvement, lung involvement, and heart involvement.

[0126] The present invention also provides the use of the above composition in improving diseases caused by excessive proliferation of B cells producing autoantibodies in patients.

[0127] The present invention also provides a method for treating a disease caused by excessive proliferation of B cells producing autoantibodies, the method comprising administering the above-described composition.

[0128] In the application of this invention, the diseases caused by the excessive proliferation of B cells producing autoantibodies include systemic lupus erythematosus, myositis, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, sensitized kidney transplantation, autoimmune encephalitis, immune-associated necrotizing myositis, refractory rheumatoid arthritis, systemic sclerosis, stiff-person syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, Lambert-Eton myasthenia gravis, and DAGLA encephalitis;

[0129] Preferably, the disease caused by the excessive proliferation of B cells producing autoantibodies is myositis, systemic lupus erythematosus, systemic sclerosis, neuromyelitis optica, or myasthenia gravis; more preferably, it is systemic lupus erythematosus.

[0130] The present invention also provides the use of the above composition in the preparation of products that reduce the level of autoreactive pathological B cells in patients, improve the level of autoantibodies, C-reactive protein, proteinuria, type I interferon, vascular inflammation, kidney involvement, lung involvement, and heart involvement.

[0131] The present invention also provides the use of the above composition in the preparation of products that improve diseases caused by excessive proliferation of B cells in patients producing autoantibodies.

[0132] In the application of this invention, the diseases caused by the excessive proliferation of B cells producing autoantibodies include one or more of the following: systemic lupus erythematosus, myositis, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, sensitized kidney transplantation, autoimmune encephalitis, immune-associated necrotizing myositis, refractory rheumatoid arthritis, systemic sclerosis, stiff-person syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Lambert-Eaton myasthenia gravis, and DAGLA encephalitis.

[0133] Preferably, the disease caused by the excessive proliferation of B cells producing autoantibodies is myositis, systemic lupus erythematosus, systemic sclerosis, neuromyelitis optica, or myasthenia gravis; more preferably, it is systemic lupus erythematosus.

[0134] The present invention also provides a method for targeted delivery of mRNA to subject T cells for non-disease diagnosis or treatment purposes, the method comprising the step of contacting the above composition with subject T cells; wherein the mRNA can generate chimeric antigen receptors within the T cells.

[0135] The present invention also provides a method for expressing chimeric antigen receptor T cells in subject T cells for purposes other than disease diagnosis or treatment, the method comprising the step of contacting the above composition with subject T cells; wherein the mRNA can generate chimeric antigen receptors within the T cells.

[0136] The methods described above in this invention can be used for non-disease diagnosis or treatment purposes, such as compound efficacy evaluation and animal experiments.

[0137] The present invention also provides a method for treating and improving diseases caused by excessive proliferation of B cells producing autoantibodies, wherein the above-described composition is administered to a patient with a disease caused by excessive proliferation of B cells producing autoantibodies, so as to deliver mRNA capable of producing chimeric antigen receptors in vivo to the T cells of the patient with the disease caused by excessive proliferation of B cells producing autoantibodies.

[0138] In the method of this invention, the diseases caused by the excessive proliferation of B cells producing autoantibodies include systemic lupus erythematosus, myositis, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, sensitized kidney transplantation, autoimmune encephalitis, immune-associated necrotizing myositis, refractory rheumatoid arthritis, systemic sclerosis, stiff-person syndrome, chronic inflammatory demyelinating polyradiculopathy (CIDP), Lambert-Eaton myasthenia gravis, and DAGLA encephalitis.

[0139] Preferably, the disease caused by the excessive proliferation of B cells producing autoantibodies is myositis, systemic lupus erythematosus, systemic sclerosis, neuromyelitis optica, or myasthenia gravis; more preferably, it is systemic lupus erythematosus.

[0140] The present invention also provides a pharmaceutical composition comprising the above-described composition and a pharmaceutically acceptable carrier.

[0141] The present invention also provides an antibody that recognizes T cell surface antigens, wherein the amino acid sequences CDR1, CDR2, and CDR3 are respectively shown in any of the following groups:

[0142] (1) SEQ ID NO: 114; SEQ ID NO: 160; SEQ ID NO: 206;

[0143] (2)SEQ ID NO:115;SEQ ID NO:161;SEQ ID NO:207;

[0144] (3)SEQ ID NO:116;SEQ ID NO:162;SEQ ID NO:208;

[0145] (4)SEQ ID NO:117;SEQ ID NO:163;SEQ ID NO:209;

[0146] (5)SEQ ID NO:118;SEQ ID NO:164;SEQ ID NO:210;

[0147] (6)SEQ ID NO:119;SEQ ID NO:165;SEQ ID NO:211;

[0148] (7)SEQ ID NO:120;SEQ ID NO:166;SEQ ID NO:212;

[0149] (8)SEQ ID NO:121;SEQ ID NO:167;SEQ ID NO:213;

[0150] (9)SEQ ID NO:122;SEQ ID NO:168;SEQ ID NO:214;

[0151] (10)SEQ ID NO:123;SEQ ID NO:169;SEQ ID NO:215;

[0152] (11)SEQ ID NO:124;SEQ ID NO:170;SEQ ID NO:216;

[0153] (12)SEQ ID NO:125;SEQ ID NO:171;SEQ ID NO:217;

[0154] (13)SEQ ID NO:126;SEQ ID NO:172;SEQ ID NO:218;

[0155] (14)SEQ ID NO:127;SEQ ID NO:173;SEQ ID NO:219;

[0156] (15)SEQ ID NO:128;SEQ ID NO:174;SEQ ID NO:220;

[0157] (16)SEQ ID NO:129;SEQ ID NO:175;SEQ ID NO:221;

[0158] (17)SEQ ID NO:130;SEQ ID NO:176;SEQ ID NO:222;

[0159] (18)SEQ ID NO:131;SEQ ID NO:177;SEQ ID NO:223;

[0160] (19)SEQ ID NO:132;SEQ ID NO:178;SEQ ID NO:224;

[0161] (20)SEQ ID NO:133;SEQ ID NO:179;SEQ ID NO:225;

[0162] (21)SEQ ID NO:134;SEQ ID NO:180;SEQ ID NO:226;

[0163] (22)SEQ ID NO:135;SEQ ID NO:181;SEQ ID NO:227;

[0164] (23)SEQ ID NO:136;SEQ ID NO:182;SEQ ID NO:228;

[0165] (24)SEQ ID NO:137;SEQ ID NO:183;SEQ ID NO:229;

[0166] (25)SEQ ID NO:138;SEQ ID NO:184;SEQ ID NO:230;

[0167] (26)SEQ ID NO:139;SEQ ID NO:185;SEQ ID NO:231;

[0168] (27)SEQ ID NO:140;SEQ ID NO:186;SEQ ID NO:232;

[0169] (28)SEQ ID NO:141;SEQ ID NO:187;SEQ ID NO:233;

[0170] (29)SEQ ID NO:142;SEQ ID NO:188;SEQ ID NO:234;

[0171] (30)SEQ ID NO:143;SEQ ID NO:189;SEQ ID NO:235;

[0172] (31)SEQ ID NO:144;SEQ ID NO:190;SEQ ID NO:236;

[0173] (32)SEQ ID NO:145;SEQ ID NO:191;SEQ ID NO:237;

[0174] (33)SEQ ID NO:146;SEQ ID NO:192;SEQ ID NO:238;

[0175] (34)SEQ ID NO:147;SEQ ID NO:193;SEQ ID NO:239;

[0176] (35)SEQ ID NO:148;SEQ ID NO:194;SEQ ID NO:240;

[0177] (36)SEQ ID NO:149;SEQ ID NO:195;SEQ ID NO:241;

[0178] (37)SEQ ID NO:150;SEQ ID NO:196;SEQ ID NO:242;

[0179] (38)SEQ ID NO:151;SEQ ID NO:197;SEQ ID NO:243;

[0180] (39)SEQ ID NO:152;SEQ ID NO:198;SEQ ID NO:244;

[0181] (40)SEQ ID NO:153;SEQ ID NO:199;SEQ ID NO:245;

[0182] (41)SEQ ID NO:154;SEQ ID NO:200;SEQ ID NO:246;

[0183] (42)SEQ ID NO:155;SEQ ID NO:201;SEQ ID NO:247;

[0184] (43)SEQ ID NO:156;SEQ ID NO:202;SEQ ID NO:248;

[0185] (44)SEQ ID NO:157;SEQ ID NO:203;SEQ ID NO:249;

[0186] (45)SEQ ID NO:158;SEQ ID NO:204;SEQ ID NO:250;

[0187] (46)SEQ ID NO:159;SEQ ID NO:205;SEQ ID NO:251;

[0188] (47)SEQ ID NO:252;SEQ ID NO:275;SEQ ID NO:298;

[0189] (48)SEQ ID NO:253;SEQ ID NO:276;SEQ ID NO:299;

[0190] (49)SEQ ID NO:254;SEQ ID NO:277;SEQ ID NO:300;

[0191] (50)SEQ ID NO:255;SEQ ID NO:278;SEQ ID NO:301;

[0192] (51)SEQ ID NO:256;SEQ ID NO:279;SEQ ID NO:302;

[0193] (52)SEQ ID NO:257;SEQ ID NO:280;SEQ ID NO:303;

[0194] (53)SEQ ID NO:258;SEQ ID NO:281;SEQ ID NO:304;

[0195] (54)SEQ ID NO:259;SEQ ID NO:282;SEQ ID NO:305;

[0196] (55)SEQ ID NO:260;SEQ ID NO:283;SEQ ID NO:306;

[0197] (56)SEQ ID NO:261;SEQ ID NO:284;SEQ ID NO:307;

[0198] (57)SEQ ID NO:262;SEQ ID NO:285;SEQ ID NO:308;

[0199] (58)SEQ ID NO:263;SEQ ID NO:286;SEQ ID NO:309;

[0200] (59)SEQ ID NO:264;SEQ ID NO:287;SEQ ID NO:310;

[0201] (60)SEQ ID NO:265;SEQ ID NO:288;SEQ ID NO:311;

[0202] (61)SEQ ID NO:266;SEQ ID NO:289;SEQ ID NO:312;

[0203] (62)SEQ ID NO:267;SEQ ID NO:290;SEQ ID NO:313;

[0204] (63)SEQ ID NO:268;SEQ ID NO:291;SEQ ID NO:314;

[0205] (64)SEQ ID NO:269;SEQ ID NO:292;SEQ ID NO:315;

[0206] (65)SEQ ID NO:270;SEQ ID NO:293;SEQ ID NO:316;

[0207] (66)SEQ ID NO:271;SEQ ID NO:294;SEQ ID NO:317;

[0208] (67)SEQ ID NO:272;SEQ ID NO:295;SEQ ID NO:318;

[0209] (68)SEQ ID NO:273;SEQ ID NO:296;SEQ ID NO:319;

[0210] (69)SEQ ID NO:274;SEQ ID NO:297;SEQ ID NO:320;

[0211] Preferably, the amino acid sequence of the antibody recognizing T cell surface antigen is as shown in any one of SEQ ID NO:1-46, 85-107, 331-355 or as shown in any one of SEQ ID NO:1-46, 85-107, 331-355, having ≥80% homology with the sequence shown in any one of SEQ ID NO:1-46, 85-107, 331-355;

[0212] Preferably, the surface antigen of the T cells corresponding to SEQ ID NO:1-20, SEQ ID NO:44-45, SEQ ID NO:85-107 or SEQ ID NO:331-355 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:21-43 or SEQ ID NO:46 is CD5;

[0213] More preferably, the surface antigen of the T cells corresponding to SEQ ID NO:98-105 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:46 is CD5.

[0214] The present invention also provides a peptide chain encoded by any one of the mRNAs shown in SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419 or SEQ ID NO:457-461;

[0215] Alternatively, the mRNA encoded by a sequence having ≥80% homology to any one of the sequences shown in SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461; preferably ≥90%; more preferably ≥95%; even more preferably ≥98%;

[0216] Preferably, the mRNA is encoded by any one of the mRNAs shown in SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471;

[0217] Alternatively, the mRNA encoded by a sequence having ≥80% homology to any one of the sequences shown in SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471; preferably ≥90%; more preferably ≥95%; even more preferably ≥98%;

[0218] Preferably, as shown in any one of SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444, SEQ ID NO:476; or as shown in any one of SEQ ID NO:429-430, SEQ ID NO:440, SEQ ID NO:451-453; or as shown in any one of SEQ ID NO:427-428, SEQ ID NO:434-437; or as shown in any one of SEQ ID NO:441-442; or as shown in any one of SEQ ID NO:438-439, SEQ ID NO:445-450, SEQ ID NO:472-475.

[0219] The beneficial effects of this invention are at least as follows:

[0220] This invention utilizes a specific T-cell-targeting LNP to deliver CAR molecules to the body, generating CAR T cells in situ, thereby eliminating B cells to achieve a therapeutic effect without the need for prior lymphocyte clearance. Furthermore, the T-cell-targeting LNP used in this invention is a non-viral vector, exhibiting good safety. The delivered CAR molecule is mRNA, which transiently reprograms into T cells in vivo. The progeny T cells proliferating after killing B cells do not express the CAR molecule because they have not undergone gene integration. Therefore, the kinetic trajectory of the CAR T cells in vivo is very clear and transient, further suggesting a lower probability of inducing a cytokine storm compared to traditional CAR T therapy. This makes it an ideal, innovative, and effective alternative to CAR T therapy.

[0221] The newly developed antibodies of this invention are single-chain antibodies (such as VHH, with a molecular weight of approximately 12-15 kDa), which are smaller in size than other antibodies and employ site-directed conjugation. Therefore, the LNP preparation process is simple, and the resulting product is more stable and has better drug-like properties. For example, the LNPs modified with CD8 and CD5 antibodies developed in this invention can efficiently target T cells, significantly improve the reprogramming efficiency of CAR-T cells in vivo, and are easy to scale up. Currently, multiple batches with yields of over 500 mg have been produced, with stable and uniform quality between batches. Secondly, the nanobodies used in this invention lack the Fc segment, directly avoiding Fc-mediated complement activation and immune cell recognition, reducing non-specific immune responses. Thirdly, the antibodies of this invention have undergone humanization modification, reducing the risk of anti-drug antibodies (ADA) caused by "non-human epitopes," which can significantly reduce immunogenicity. Attached Figure Description

[0222] Figure 1 shows representative cryo-electron microscopy images of cLNP and CD8-TLNP1.

[0223] Figure 2 shows the statistical results of the expression ratio of EGFP encapsulated with different CD8-TLNPs. The figure shows the average value of three biological replicates.

[0224] Figure 3 shows the statistical results of the expression ratio data of different molecules encapsulated by CD8-TLNP. The figure shows the average value of two biological replicates.

[0225] Figure 4 shows the statistical results of changes in CAR molecule expression rate in vivo with different doses of CAR-CD5-TLNP. The horizontal axis is in days. The figure represents the average value of three biological replicates.

[0226] Figure 5 shows the statistical results of the changes in the proportion of B cells in mice in different CD8-TLNP1 groups. The figure represents the average value of three biological replicates.

[0227] Figure 6 shows the statistical results of the changes in the total number of B cells in mice in different CD8-TLNP1 groups. The figure shows the average value of three biological replicates.

[0228] Figure 7 shows the statistical results of changes in B cell count in mice with different doses of CD5-TLNP. The horizontal axis is in days. The figure represents the average value of three biological replicates.

[0229] Figure 8 is a gate analysis diagram for determining the purity of BMDC using CD11c.

[0230] Figure 9 shows the changes in the proportion of B cells in SLE mice treated with different CD5-TLNPs. The figure represents the average value of five biological replicates.

[0231] Figure 10 shows the statistical results of changes in the number of B cells in SLE mice treated with different CD5-TLNPs. The figure represents the average value of five biological replicates.

[0232] Figure 11 shows the statistical results of changes in dsDNA IgG antibody levels in SLE mice treated with different CD5-TLNPs. The figure represents the average value of five biological replicates.

[0233] Figure 12 shows the statistical results of dsDNA IgG1 antibody levels in SLE mice treated with different CD5-TLNPs. The figure represents the average of five biological replicates.

[0234] Figure 13 shows the statistical results of proteinuria levels in SLE mice treated with different CD5-TLNPs at 16 weeks. The figure represents the average of 5 biological replicates.

[0235] Figure 14 is a representative immunofluorescence image of the kidney C3 complement and IgG antibody levels in SLE mice treated with different CD5-TLNPs at 20 weeks.

[0236] Figure 15 is a schematic diagram of the mechanism of action of mRNA-LNP drugs that generate CAR T cells in vivo to treat autoimmune diseases.

[0237] Figure 16 shows the statistical results of the general performance scores of mice with different CD8-TLNP3 treatments in Example 9. The figure shows the average value of three biological replicates.

[0238] Figure 17 shows the statistical results of limb traction tests in mice with different CD8-TLNP3 treatments for myositis in Example 9. The figure represents the average value of three biological replicates.

[0239] Figure 18 shows the statistical results of plasma creatine kinase in mice with different CD8-TLNP3 treatments in Example 9. The figure represents the average value of three biological replicates.

[0240] Figure 19 shows the twist statistics of mouse muscles under the strongest current stimulation when TA was stimulated in mice with different CD8-TLNP3 treatments in Example 9. The figure is the average value of three biological replicates.

[0241] Figure 20 shows the tetanic statistics of mouse muscles under the strongest current stimulation when different CD8-TLNP3-treated myositis mice in Example 9 were stimulated with TA. The figure represents the average value of three biological replicates.

[0242] Figure 21 shows the statistical results of the behavioral disorder scores of NMOSD mice treated with different CD5-TLNPs in Example 10. The figure shows the average value of three biological replicates.

[0243] Figure 22 shows the statistical results of anti-AQP4 IgG antibodies in the serum of NMOSD mice treated with different CD5-TLNPs in Example 10. The figure shows the average value of three biological replicates.

[0244] Figure 23 shows the statistical results of the general performance score of EAMG mice with CD8-TLNP1 treatment for myasthenia gravis in Example 11. The figure shows the average value of three biological replicates.

[0245] Figure 24 shows the results of the suspension time statistics of EAMG mice with CD8-TLNP1 treatment for myasthenia gravis in Example 11. The figure is the average value of 3 biological replicates.

[0246] Figure 25 shows the results of anti-AChR IgG antibody in the serum of EAMG mice with myasthenia gravis treated with CD8-TLNP1 in Example 11. The figure represents the average value of three biological replicates.

[0247] Figure 26 shows the twitch statistics of the mouse muscles under the strongest current stimulation when CD8-TLNP1 was used to treat the EAMG mouse model of myasthenia gravis in Example 11. The figure is the average value of three biological replicates.

[0248] Figure 27 shows the statistical results of the proportion of surviving NALM-6 cells in different drug administration groups in Example 12. The figure is the average value of three biological replicates.

[0249] Figure 28 shows B cell clearance data after in vitro CD20 CAR mRNA CD8-TLNP7 treatment in non-human primates in Example 13. The figure represents the average of three biological replicates.

[0250] Figure 29 shows the B cell clearance data in healthy adults and SLE patients after in vitro treatment with CAR mRNA CD8-TLNP7 in Example 14. The number of healthy adults in the figure is 3, and the number of SLE patients is 21.

[0251] Figure 30 shows the statistical results of the proportion of B cells in PBMCs of different drug administration groups in Example 15. In the figure, the PBS and CAR4 groups are the average values ​​of two biological replicates, and the other groups are the average values ​​of four biological replicates.

[0252] Figure 31 shows the dynamic changes in the proportion of B cells in NHP in Example 16.

[0253] Figure 32 shows the changes in the number of B cells in Example 16.

[0254] Figure 33 shows the relative number of B cells in Example 16.

[0255] Figure 34 shows the expression of CAR mRNA in the peripheral blood of NHP in Example 17.

[0256] Figure 35 shows the relative number of B cells in Example 17.

[0257] Figure 36 shows the expression of CAR mRNA in the peripheral blood of NHP in Example 18.

[0258] Figure 37 shows the relative number of B cells in Example 18.

[0259] Figure 38 shows the effect of B cell clearance in peripheral blood of NHP in Example 19.

[0260] Figure 39 shows the effect of B cell clearance in the spleen in Example 19.

[0261] Figure 40 shows the effect of B cell clearance in bone marrow in Example 19.

[0262] Figure 41 shows the effect of B cell clearance in the inguinal lymph nodes in Example 19.

[0263] Figure 42 shows the mRNA expression results in the liver and spleen of NHP patients in Example 20, with and without miR-122-encapsulated CD8-TLNP. The left panel shows the expression results of CD8-TLNP-encapsulated miR-122-encapsulated mRNA in NHP liver, and the right panel shows the expression results of CD8-TLNP-encapsulated miR-122-encapsulated mRNA in both NHP liver and spleen. The figures represent the average values ​​of two biological replicates.

[0264] Figure 43 is a statistical chart of the expression ratio of CAR in PBMC in Example 21.

[0265] Figure 44 is a statistical chart of the clearance effect (in vitro killing) of B cells in PBMCs in Example 21.

[0266] Figure 45 is a statistical chart of the changes in the number of B cells in mice in Example 21.

[0267] Figure 46 shows the statistical graph of CAR CD19 expression in PBMCs of different drug administration groups in Example 22. The figure represents the average value of two biological replicates.

[0268] Figure 47 shows the statistical graph of CAR BCMA expression in PBMCs of different drug administration groups in Example 22. The figure represents the average value of two biological replicates.

[0269] Figure 48 is a statistical graph of B cell killing efficiency in PBMCs of different drug administration groups in Example 22. The figure shows the average value of two biological replicates.

[0270] Figure 49 shows the statistical graph of the killing efficiency of PBMCs in different drug administration groups in Example 22. The group with an effector-to-target ratio of 1:1 is the average of 3 biological replicates, and the group with an effector-to-target ratio of 2:1 is the average of 2 biological replicates.

[0271] Figure 50 is a statistical graph showing the B cell killing efficiency in the peripheral blood of HSC mice in different drug administration groups in Example 23. The figure shows the average value of two biological replicates.

[0272] Figure 51 is a statistical graph showing the B-cell killing efficiency of NHP in peripheral blood of different drug administration groups in Example 24. The figure shows the average value of two biological replicates.

[0273] Figure 52 is a statistical graph showing the B-cell killing efficiency in the bone marrow of NHP cells in different drug administration groups in Example 24. The figure shows the average value of two biological replicates.

[0274] Figure 53 is a statistical graph showing the plasma cell killing efficiency in the bone marrow of NHP patients in different drug administration groups in Example 24. The figure represents the average value of two biological replicates.

[0275] In each graph (if any), * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. Detailed Implementation

[0276] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0277] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0278] Example 1

[0279] Encapsulating CAR-mRNA to target CD8+ Preparation of T-cell lipid nanoparticles

[0280] This embodiment provides a method for encapsulating CAR-mRNA to target CD8. + T-cell lipid nanoparticles and their preparation method are detailed below:

[0281] 1. Preparation of LNPs containing coupling sites (TLNP precursors)

[0282] Table 1 CD8 + Preparation table of T cell-targeted LNP (CD8-TLNP)

[0283] Five lipid molecules, namely ionizable amino lipids (cationic lipids, E12LA6B6O3 / DLin-MC3), DSPC, CHOL (cholesterol), PEG (DMG-PEG2000, DSPE-PEG2000, DPPE-PEG2000), and PEG-Mal (DSPE-PEG2000-Mal / DSPE-Mal / CHO-Mal), were dissolved in ethanol according to the corresponding molar ratios (see Table 1). CAR-mRNA (SEQ ID NO:66) was dissolved in an acidic acetic acid buffer solution (25 mM, pH 4), with a molar ratio (N / P) of ionizable amino lipids to mRNA bases of 6.5:1.

[0284] The ethanol and aqueous phases were mixed using a Myanna microfluidic device, with the flow rate controlled at a volume ratio of 1:3 between the ethanol and aqueous phases. The total flow rate of the two phases was 12 mL / min, and the flow rate ratio of the ethanol and aqueous phases was 1:3, to obtain crude LNP. Then, 10 volumes of 25 mM Tris buffer were added to terminate the process.

[0285] The LNPs with stop solution were ultrafiltered once at 4°C and 1200 rcf; then, 25 mM Tris buffer was added and ultrafiltered again at 4°C and 1200 rcf. The prepared CAR-LNPs (TLNP precursors) were collected, sucrose was added to adjust the concentration to 8%, and the mixture was aseptically filtered in a laminar flow hood using a 0.22 μm filter. The sample was then diluted to the required concentration.

[0286] The preparation of ordinary four-component LNP (cLNP) is similar to the above procedure, except that no Mal lipid is added to the ethanol phase.

[0287] 2. Preparation of CD8 nanobody-modified cell-targeting LNPs (CD8-TLNP)

[0288] The TLNP precursor was mixed with the nanobody (SEQ ID NO:44) and incubated overnight at 4°C. 25 mM Tris-8% sucrose solution was added to the overnight incubated CD8-TLNP, and ultrafiltration purification was performed. After concentration and collection, the sample was aseptically filtered through a 0.22 μm filter in a clean bench. 30 μL of the sample was used to detect the particle size, dispersion index, and encapsulation efficiency (results are shown in Table 2). After the encapsulation efficiency was detected, the sample was diluted to the required concentration to obtain the final CD8-TLNP.

[0289] The cryo-electron microscopy images of cLNP and CD8-TLNP1 are shown in Figure 1.

[0290] Table 2 Quality control information for LNP

[0291] Experimental results show that the cell-targeting lipid nanoparticles (CD8-TLNPs) prepared by conjugating CD8 nanobodies all exhibited good characterization data, falling within the generally accepted numerical range in the LNP field. This indicates that the preparation process of CD8 cell-targeting LNPs in this invention is feasible and stable. From the above data, it can be seen that CD8-TLNP7 and CD8-TLNP15 showed the best performance.

[0292] Following the above method, the present invention also prepares CD8-TLNPs conjugated with different CD8 nanobodies, and the conjugated CD8 nanobodies sequences are shown in any one of SEQ ID NO:1-20, SEQ ID NO:44-45, SEQ ID NO:85-107 or SEQ ID NO:331-355.

[0293] 3. Preparation of mRNA encoding chimeric antigen receptor molecules

[0294] Most of the mRNA sequences in this invention employ a 120-A Poly A strategy. The 120-A Poly A sequence (mRNA sequence, 5′→3′) is specifically shown in SEQ ID NO:113.

[0295] To reduce the side effects of target genes (especially cytokines that trigger immune responses), introducing a complete or partial matching sequence of an organ-specific highly expressed miRNA into the 3'UTR can promote the degradation of the mRNA drug in these organs. The mRNA sequence described in this invention may also contain zero or at least one sequence from the target sequence of the tissue-specific highly expressed miRNA. The target sequence (mRNA, 5′→3′) of the tissue-specific highly expressed miRNA is specifically shown in SEQ ID NO:108.

[0296] The designed sequence containing 5'UTR (as shown in SEQ ID NO:109), 3'UTR (as shown in any one of SEQ ID NO:110-112), open reading frame sequence (hereinafter referred to as the target ORF sequence, as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419 or SEQ ID NO:457-461), miRNA (as shown in SEQ ID NO:108), and Poly A sequence (as shown in SEQ ID NO:113) was synthesized and constructed into a cloning plasmid vector by cloning.

[0297] The obtained plasmid was transformed into *E. coli* DH5α strain to obtain a recombinant strain. The next day, three single colonies were selected from each growth plate. Colonies with correctly sequenced sequences and a Poly A ≥ 110A were transferred to 200 mL of LB medium and incubated overnight at 37°C and 150 rpm in a shaker. The bacterial cells were harvested the following day, and plasmids were extracted using a plasmid extraction kit.

[0298] The plasmids obtained above were digested with restriction endonucleases, and the digested plasmids were purified and recovered using magnetic beads.

[0299] The obtained linearized plasmid was used as a template for in vitro transcription of mRNA. In vitro transcription was performed using an in vitro mRNA transcription kit to obtain mRNA. After in vitro transcription, DNase I was added to the reaction system at a final concentration of 0.15 U / μl, and the reaction was carried out at 37°C for 30 min to digest the DNA template in the reaction system. The mRNA obtained from in vitro transcription was then purified and concentrated using magnetic beads to obtain the final mRNA sample.

[0300] The full-length mRNA sequences obtained according to the above method include any one of SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, and SEQ ID NO:462-471, containing at least one sequence in the 5'UTR sequence listing, containing at least one sequence in the 3'UTR sequence listing, containing 1-3 target sequences of tissue-specific highly expressed miRNAs, and containing a range of 120 Ploy A sequences.

[0301] Example 2

[0302] Encapsulating CAR-mRNA to target CD5 + Preparation of T-cell lipid nanoparticles

[0303] The CD5 cell-targeting LNP preparation procedure encapsulating CD19-CAR-mRNA (SEQ ID NO:66) was performed in accordance with the description of CD8-TLNP7 preparation in Example 1, except that the antibody conjugated to the TLNP was replaced with the corresponding CD5 nanobody (as shown in SEQ ID NO:46). The quality control information for the prepared samples is shown in Table 3.

[0304] Table 3 Quality control information for LNP

[0305] Experimental results show that, similar to CD8 cell-targeting lipid nanoparticles (CD8-TLNP), CD5-TLNP also exhibits excellent physicochemical data, falling within the generally accepted numerical range in the LNP field. This indicates that the preparation process of cell-targeting LNPs in this invention has universal applicability.

[0306] Following the above method, the present invention also prepares CD5-TLNPs conjugated with different CD5 nanobodies, and the conjugated CD5 nanobodies sequences are shown in any one of SEQ ID NO:21-43 or SEQ ID NO:46.

[0307] Example 3

[0308] The efficiency of EGFP mRNA delivery in in vitro hPBMCs prepared with different CD8 antibodies was investigated.

[0309] 1. The CD8-TLNPs encapsulated with different EGFPs used in this embodiment were prepared according to the method described in Example 1.

[0310] 2. Peripheral blood was collected from healthy adults, and hPBMCs were obtained by separating them using lymphocyte separation medium. The collected hPBMCs were counted and analyzed at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 Cells were resuspended at a concentration of 1 / mL in complete RPMI-1640 medium (anti-CD3 and anti-CD28 activation, 300 U / mL IL2), and then CD8-TLNPs of 0.05 μg / mL, 0.1 μg / mL, and 0.5 μg / mL were added respectively (corresponding groups are shown in Table 4). hPBMC cells were then transfected at 37℃ and 5% CO2 for 24 h, and the expression rate of eGFP-mRNA in CD8 T cells was detected by flow cytometry. A statistical graph of in vitro EGFP expression data for different CD8-TLNPs is shown in Figure 2.

[0311] Table 4 Experimental Groups

[0312] Experimental results showed that the in vitro EGFP expression of different CD8-TLNPs was greater than 80%, among which the CD8-TLNP prepared using SEQ ID NO:99 had the highest efficiency in delivering EGFP mRNA.

[0313] Example 4

[0314] The efficiency of CD8-TLNP in delivering CAR mRNA in in vitro hPBMCs was investigated.

[0315] 1. The CD8-TLNP7 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP7 of Example 1 was replaced with the sequences shown in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:456, SEQ ID NO:454 and SEQ ID NO:455, respectively. The CD8 nanobody sequence used is SEQ ID NO:332.

[0316] 2. Collect peripheral blood from healthy adults, separate PBMCs using lymphocyte separation medium, count the collected PBMCs, and record the cells at a concentration of 0.5 × 10⁻⁶. 6Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (anti-CD3 and anti-CD28 activation, 300 U / mL IL2), and 1 μg / mL LNP was added (specific groupings are shown in Table 5; the CAR groups were CD8-TLNP7 groups encapsulated with different CAR mRNAs, specifically CAR1 encapsulated with CAR-BCMA (SEQ ID NO:71), CAR2 encapsulated with CAR-BCMA (SEQ ID NO:72), CAR3 encapsulated with CAR-BCMA (SEQ ID NO:456), CAR4 encapsulated with CAR-BCMA-CD19 (SEQ ID NO:454), and CAR5 encapsulated with CAR-CD19-BCMA (SEQ ID NO:455)). The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. The statistical graph of the expression ratio of mRNA corresponding molecules in PBMCs of different drug-treated groups in Table 5 is shown in Figure 3.

[0317] Table 5 Experimental Groups

[0318] Experimental results show that different molecules delivered by CD8-TLNP in vitro all have an expression rate of over 80%.

[0319] Example 5

[0320] This embodiment uses CD5. + The relationship between CAR molecule expression and drug dosage in mice was investigated using CAR-mRNA delivered by T cell-targeted lipid nanoparticles, as detailed below:

[0321] 1. The CD5-TLNP used in this embodiment was prepared according to the method described in Example 2. The only difference is that the CD5 antibody sequence in the CD5-TLNP of Example 2 was replaced with the sequence shown in SEQ ID NO:40.

[0322] 2. The relationship between the expression rate of CAR-mRNA (SEQ ID NO:66) in CD5 humanized mice and the drug dosage is studied as follows:

[0323] Twelve 6-8 week old humanized female hCD5 mice (from Biocytogen) that passed quarantine and met SPF (specific pathogen-free) standards were procured and divided into four groups. Mice were injected intravenously with different doses of CD5-TLNP. The doses of mRNA-LNP administered were 0, 0.25, 0.5, and 1.0 mpk (mg / kg), with an injection volume of 200 μL for each dose. Specific grouping details are shown in Table 6. Peripheral blood samples were collected at Day 0 (before administration), Day 1, Day 3, and Day 5 for flow cytometry analysis of CAR-mRNA expression rates. The statistical graph of changes in CAR molecule expression rates in vivo under different doses of CD5-TLNP is shown in Figure 4. The Vector 1 group refers to the group injected with unencapsulated mRNA empty vector LNP (0 mpk).

[0324] Table 6. In vivo experimental grouping of CAR-CD5-TLNP

[0325] Experimental results showed that CAR-mRNA expression peaked at Day 1, and with prolonged drug administration, CAR molecules were continuously consumed and degraded, leading to a gradual decrease in CAR molecule levels in peripheral blood, which were virtually undetectable by Day 5. Furthermore, CD5+ levels in mouse peripheral blood... + The T cell population exhibited the best CAR molecule expression rate at a drug dose of 1 mpk.

[0326] Example 6

[0327] This embodiment uses CD8 + The effect of T cell-targeted lipid nanoparticle-delivered CAR-mRNA on clearing B cells in mice was investigated, as detailed below:

[0328] 1. The CD8-TLNP8 used in this embodiment was prepared according to the method described in Example 1. The only difference is that the mRNA in CD8-TLNP7 of Example 1 was replaced with the sequence shown in SEQ ID NO:66, and the CD8 antibody sequence was replaced with the sequence shown in SEQ ID NO:331.

[0329] 2. The relationship between the clearance effect of B cells in hCD8 humanized mice and the drug dosage is studied as follows:

[0330] Nine 6-8 week old humanized female hCD8 mice (from Biocytogen) that passed quarantine and met SPF (specific pathogen-free) standards were procured and divided into three groups. Mice were injected via tail vein with CD8-TLNP8 containing different mRNAs. The Vector 1 group received an empty vector LNP without mRNA encapsulation, the eGFP group received an injection of mRNA encapsulated with enhanced green fluorescent protein, and the CAR group (SEQ ID NO: 66) received CD8-TLNP8 prepared according to the method described in Example 1 of this invention. Specific groupings are shown in Table 7. Mice were administered 1.0 mpk of mRNA-LNP at a dose of 200 μL. Peripheral blood samples were collected on day 0 (before administration) and on days 1, 3, 4, 7, 14, and 25 after administration to detect the proportion and number of B cells. Figure 5 shows the statistical data on the changes in the proportion of B cells in mice in different CD8-TLNP8 groups, and Figure 6 shows the statistical data on the changes in the total number of B cells in mice in different CD8-TLNP8 groups.

[0331] Table 7. In vivo experimental grouping of CD8-TLNP8

[0332] Experimental results showed that the proportion and number of B cells in mouse peripheral blood decreased transiently one day after CD8-TLNP8 administration, while the proportion and number of B cells decreased significantly after CAR-mRNA-CD8 TLNP8 administration, and then slowly increased after one week. CAR-mRNA-CD8 TLNP8 showed a significant scavenging effect on B cells.

[0333] Example 7

[0334] This embodiment uses CD5. + The relationship between the effect of T cell-targeted lipid nanoparticle-delivered CAR-mRNA on clearing B cells in mice and the dosage was investigated, as follows:

[0335] 1. The CD5-TLNP used in this embodiment was prepared according to the method described in Example 2. The only difference is that the CD5 antibody sequence in the CD5-TLNP of Example 2 was replaced with the sequence shown in SEQ ID NO:35.

[0336] 2. The relationship between the clearance effect of CD5 humanized mice and the drug dosage is studied as follows:

[0337] Twelve 6-8 week old humanized hCD5 female mice that passed quarantine and met SPF standards were purchased and divided into four groups. Different doses of CD5-TLNP were injected into the tail vein of the mice. The doses of mRNA-LNP administered were 0, 0.25, 0.5, and 1.0 mpk, with an injection volume of 200 μL for each group. Specific grouping details are shown in Table 8. Peripheral blood samples were collected on day 0 (before administration), day 3, day 5, and day 8 after administration to detect the proportion and number of B cells. The statistical data on the changes in B cell count in mice under different CD5-TLNP doses are shown in Figure 7. The Vector 1 group received an injection of empty vector LNP (0 mpk) without mRNA encapsulation, while the other groups received CD5-TLNP encapsulated with CAR-mRNA (SEQ ID NO: 66).

[0338] Table 8. CD5-TLNP in vivo experimental grouping

[0339] Experimental results showed that the number of B cells in the peripheral blood of mice decreased significantly after drug administration, and this decrease was correlated with the drug dose. Within a certain dosage range (0-1.0 mpk), the higher the CAR-mRNA-LNP dosage, the higher the expression level of CAR molecules in the peripheral blood, thus resulting in better B cell clearance. Current analysis indicates that a dosage of 1 mpk showed the best B cell clearance effect.

[0340] Example 8

[0341] This embodiment uses CD5. + The pharmacodynamics of CAR-mRNA delivered via T-cell-targeted lipid nanoparticles for the treatment of SLE mice was investigated, as detailed below:

[0342] (I) Construction of an induced mouse SLE model

[0343] 1) Preparation of DNA derived from activated lymphocytes (ALD-DNA):

[0344] BALB / c mice were euthanized by CO2 asphyxiation. The spleens were aseptically removed and placed in PBS on ice until all mice were prepared. At 60 mm... 2 Spleen cells were aseptically ground in a petri dish to prepare a single-cell suspension. The suspension was passed through a sieve and collected in a 50 mL centrifuge tube. The tube was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. 3 mL of erythrocyte lysis buffer (ACK) was added, and the mixture was incubated at room temperature for 3 minutes, then centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The spleen cells were resuspended in 10 mL of complete 1640 medium and seeded onto 100 mm thick plates. 2Add ConA to a final concentration of 5 μg / mL in culture dishes and incubate at 37°C in a 5% CO2 cell culture incubator. After 6 days, collect surviving spleen cells (mainly lymphocytes) for DNA extraction, and collect the cells in 1.5 mL EP tubes. Add 200 μL / 5 × 10⁻⁶ cells to the cells. 5 Cell lysis buffer (containing proteinase K) was used for digestion. The mixture was incubated at 70°C for 5 minutes, vortexed for 15 seconds until no obvious clumps were observed. 200 μl of phenol solution was added to a centrifuge tube, vortexed for 15 seconds, and centrifuged at 14000 rpm for 5 minutes. The supernatant (white layer) was pipetted into a new EP tube, an equal volume of chloroform was added, vortexed for 15 seconds, and centrifuged at 14000 rpm for 5 minutes. The supernatant was pipetted again into a new tube, 10% (v / v) of 3 mol sodium acetate was added, followed by two volumes of anhydrous ethanol, and the tube was gently inverted. The flocculated material was picked up with a sterile paperclip, washed twice in 70% ethanol, and then transferred to a new EP tube. The EP tube cap was opened and the tube was allowed to air dry at room temperature. The DNA was dissolved in sterile water, and the DNA concentration was measured using Nanodrop. The ALD-DNA was stored at 4°C for later use.

[0345] 2) Obtaining and validating bone marrow-derived dendritic cells (BMDCs):

[0346] C57BL / 6 mice were euthanized by CO2 asphyxiation and disinfected by immersion in 75% alcohol for 5 minutes. The thigh and tibiae of the mice were dissected, and the ligaments and Achilles tendons connecting the joints were removed. The muscles were removed to expose the bone, and the joints at both ends were cut off. A 1mL sterile syringe needle was used to pipette the red bone marrow cells into PBS, gently agitating until no visible cell clumps remained, with a total volume less than 10mL. The solution was transferred to a 50mL centrifuge tube equipped with a 40μm cell filter and filtered. The filtrate was transferred to a 15mL centrifuge tube and centrifuged at 500rcf for 5 minutes at 4°C. The supernatant was discarded, and 2mL of ACK erythrocyte lysis buffer was added. The cells were gently mixed by pipetting and lysed gently for 5 minutes. Lysis was terminated by adding 6mL of PBS solution. The cells were centrifuged at 500rcf for 5 minutes at 4°C. The supernatant was discarded, and 3-6mL of the culture medium was resuspended by pipetting. Transfer 10 μL of cell suspension to a 1.5 mL EP tube, add 10 μL of trypan blue for staining, mix well, and then transfer 10 μL to a cell counting chamber. Count the cells and calculate the dilution volume. Dilute the cell suspension to 1.5 × 10⁻⁶ cells / mL with culture medium. 6Cells / mL were seeded into 6-well plates. 2 mL of diluted cell suspension was added to each well. Culture conditions were as follows: BMDC induction medium (1640) containing 10% fetal bovine serum, 1% penicillin and streptomycin, 50 ng / mL recombinant cytokine GM-CSF, and 2.5 ng / mL recombinant cytokine IL-4. Incubation was performed at 37°C, 5% CO2, and 95% humidity in a cell culture incubator. The medium was changed halfway every 2 days until day 6, at which point tightly adhered cells were collected as BMDCs.

[0347] BMDCs were validated through morphological observation and cell phenotype analysis: most BMDCs grew in colonies, with multiple dendritic protrusions, which were more pronounced in mature BMDCs; flow cytometry was used to detect CD11c defining molecules on the surface of BMDC cells, and CD11c-positive cells were the target cells.

[0348] Experimental results show that after determining the location of the main population using forward and side-scattered light, and then outlining single cells and removing dead cells, the purity of induced DCs was finally determined to be 75% or higher using CD11c. The analytical results of determining the purity of BMDCs using CD11c are shown in Figure 8.

[0349] 3) BMDC coated with ALD-DNA was then transfused into CD5 humanized mice to induce a mouse SLE model:

[0350] The collected BMDCs were counted and analyzed at 5 × 10⁻⁶ cells. 6 Cells were resuspended at a concentration of 10 μg / mL in complete RPMI-1640 medium, and 10 μg / mL of activated lymphocyte-derived DNA was added. The cells were incubated at 37°C in a CO2 incubator for 12 hours. Cells were collected, washed twice with PBS, centrifuged at 1500 rpm for 5 minutes at 4°C, and then split at 5 × 10⁻⁶ cells / mL. 6 The cells were suspended in PBS at a concentration of / mL. The cells were then transfused into CD5 humanized mice, with 200 μl of cells injected via the tail vein into each mouse.

[0351] (II) CAR-mRNA drug treatment in model mice

[0352] 1) Detect the level of anti-double-stranded DNA antibody in mouse serum. An elevated level of double-stranded DNA antibody indicates that the mouse has developed systemic lupus erythematosus (symptoms usually appear about a week after modeling), and the mouse can be treated.

[0353] 2) Mice were randomly divided into groups of 5 mice each. SLE mice receiving unencapsulated empty vector LNPs (Vector 1) served as the model treatment control group. The other two groups received CD5-TLNPs containing CAR mRNA via tail vein injection. Mice were administered the drugs on day 6 after model establishment, with a dose of 1.0 mpk mRNA-LNPs and an injection volume of 200 μL. Specific groupings are shown in Table 9. The Vector 1 group received unencapsulated empty vector LNPs, the CAR1 group (SEQ ID NO: 66) received CD5-TLNPs encapsulated with CAR CD19 mRNA, and the CAR2 group (SEQ ID NO: 321) received CD5-TLNPs encapsulated with CAR CD19-BCMA mRNA. CAR1 and CAR2 groups received CD5-TLNPs prepared according to the method described in Example 2 of this invention.

[0354] Table 9. Grouping of CAR-CD5-TLNP Treatment Trials

[0355] 3) Regularly collect blood from the orbital rims of mice, separate serum, and detect the level of SLE symptoms in the mice.

[0356] 4) Regularly test the proteinuria level of mice.

[0357] 5) When the mice reached the experimental endpoint, they were euthanized by carbon dioxide anesthesia, dissected, and their kidneys were obtained. The kidneys were then fixed and sectioned for immunofluorescence staining to analyze lupus nephritis.

[0358] Figure 9 shows the statistical graph of changes in the proportion of B cells in SLE mice treated with different CD5-TLNPs. Figure 10 shows the statistical graph of changes in the number of B cells in SLE mice treated with different CD5-TLNPs. Figure 11 shows the statistical graph of changes in dsDNA IgG antibody levels in SLE mice treated with different CD5-TLNPs. Figure 12 shows the statistical graph of dsDNA IgG1 antibody levels in SLE mice treated with different CD5-TLNPs. Figure 13 shows the statistical graph of proteinuria levels in SLE mice treated with different CD5-TLNPs at 16 weeks. Figure 14 shows the immunofluorescence image of kidney tissue sections from SLE mice treated with different CD5-TLNPs at 20 weeks.

[0359] Experimental results showed that an elevated level of dsDNA antibody in the serum of CD5 humanized mice was detected on day 7 after modeling, indicating successful construction of the SLE mouse model. Compared with the control group without mRNA in the CD5-TLNP group, the CD5-TLNP treatment group with CAR-mRNA showed: a significant reduction in the proportion and number of B cells, i.e., significant B cell clearance; significantly reduced levels of dsDNA IgG and IgG1 antibodies in serum; and a significant reduction in proteinuria at week 16; and significantly reduced C3 complement and IgG antibodies in kidney tissue immunofluorescence at week 20. In summary, this indicates that SLE was alleviated, and CAR-mRNA CD5-TLNP can treat SLE. A schematic diagram of the mechanism of action of mRNA-LNP drugs for in vivo CAR T cell therapy in autoimmune diseases is shown in Figure 15.

[0360] Example 9

[0361] This embodiment uses CD8 + The pharmacodynamics of CAR-mRNA delivered via T-cell-targeted lipid nanoparticles for the treatment of myositis in mice was investigated, as detailed below:

[0362] (I) Construction of a mouse myositis model

[0363] A mouse myositis model was constructed using the classic myosin-induced experimental autoimmune myositis (EAM) model. Purified rabbit myosin was removed and thawed on ice. 0.3 μL of purified rabbit myosin (10 mg / mL) and 0.1 mL of complete Freund's adjuvant (CFA, containing 10 mg / mL of Mycobacterium tuberculosis) were added to each 0.2 mL of physiological saline. The mixture was stirred and vortexed until a white emulsion was formed. This emulsion was injected into the bilateral axillae, hind limbs, and tail base of the mice, with a total injection of 0.3 mL per mouse. Immunization was administered weekly for a total of four weeks. During the first two immunizations, each mouse was intraperitoneally injected with 200 μL (500 ng) of pertussis toxin.

[0364] (II) CAR-mRNA drug treatment in model mice

[0365] 1) Starting from day 0, monitor the mice's fur, body shape, food intake, activity level, and mental state throughout the process. Score the mice according to the Lennon LA criteria based on their weight, activity level, and respiratory capacity. The scoring criteria are shown in Table 10 (middle-range symptoms are scored as 0.5, 1.5, or 2.5).

[0366] Table 10 Scoring criteria for myositis mice

[0367] In addition, starting from day 0, the weight of the mice was measured and recorded on days 0, 7, 14, 21, and 28. The grip strength of the mice's limbs was measured: The mice were first placed next to the experimental apparatus to acclimatize. After half an hour, the mice gradually adapted. The apparatus was then turned on, set to N / kg unit mode, and zeroed. The mouse was slowly lifted by its tail with one hand, and its limbs were placed on a test grid connected to a force transmission detector. After the mouse gripped the grid tightly, it was slowly dragged horizontally to one side. The value on the display gradually increased. When the mouse's limbs left the grid and the value reached its highest point, this value was recorded. This value is the grip strength of the mouse's limbs. After a 30-second rest, the measurement was repeated. A total of five measurements were taken, and the average value was taken as the grip strength of the mouse's limbs.

[0368] 2) Mice exhibiting myasthenia gravis symptoms (score above 1) can be treated. Mice were randomly divided into groups of 3. Mice with myositis who did not receive mRNA-encapsulated CD8-TLNP3 served as the model control group (PBS), while the remaining groups were treated with mRNA-encapsulated CD8-TLNP3 via tail vein injection. The dosage of mRNA-LNP was 1.0 mpk, with an injection volume of 200 μL. The Vector 1 group was injected with unencapsulated empty vector LNP, the eGFP group was injected with mRNA encapsulated with enhanced green fluorescent protein, the CAR1 group (SEQ ID NO: 66) was injected with CD8-TLNP3 encapsulated with CAR CD19 mRNA, and the CAR2 group (SEQ ID NO: 322) was injected with CD8-TLNP3 encapsulated with CAR CD19-BCMA mRNA. CAR1 and CAR2 groups were injected with CD8-TLNP3 prepared according to the method described in Example 1 of this invention. The specific grouping is shown in Table 11.

[0369] Table 11 CAR-CD8-TLNP3 Treatment Trial Grouping

[0370] 3) Regularly collect blood from the orbital cavity of mice, separate the plasma, and detect the plasma creatine kinase level.

[0371] 4) Regularly assess the mice's general performance (see Table 10).

[0372] 5) Regularly measure the limb tension of mice.

[0373] 6) Perform electromyography (EMG) tests on mice regularly: Measure the strength of the tibialis anterior (TA) muscle: Fix the mouse's left hind limb in the corresponding position on the muscle testing system, and slowly insert the electrode into the subcutaneous tissue of the mouse's lower leg in a direction parallel to the muscle surface, so that the electrode is in close contact with the mouse's TA muscle. Then open the software and adjust the intensity of the stimulation current. When the optimal stimulation current intensity is found, record the maximum contractile force (twitch) of a single contraction. Then change the test program and stimulate the TA at intensities of 50Hz, 100Hz, and 150Hz respectively, and record the tetanic force (tetanic).

[0374] Figure 16 shows the general performance scores of different groups of myositis mice treated with CD8-TLNP3 for one week. Figure 17 shows the limb traction test statistics of different groups of myositis mice treated with CD8-TLNP3 for one week. Figure 18 shows the plasma creatine kinase statistics of different groups of myositis mice treated with CD8-TLNP3 for one week. Figure 19 shows the twitch statistics of mouse muscles under the strongest current stimulation when TA is stimulated in different groups of myositis mice treated with CD8-TLNP3 for one week. Figure 20 shows the tetanic statistics of mouse muscles under the strongest current stimulation when TA is stimulated in different groups of myositis mice treated with CD8-TLNP3 for one week.

[0375] The experimental results showed that, compared with the model control group without mRNA and the CD8-TLNP3 group given unrelated EGFP-mRNA, the general performance scores of myositis mice in the CD8-TLNP3 treatment group with CAR-mRNA were significantly reduced, the limb pulling ability of the mice was significantly improved after treatment, and the plasma creatine kinase level was also significantly reduced, indicating that myositis was alleviated and that CD8-TLNP3 with CAR-mRNA can treat myositis.

[0376] Example 10

[0377] This embodiment uses CD5. + The pharmacodynamics of CAR-mRNA delivered via T-cell-targeted lipid nanoparticles in treating a mouse model of neuromyelitis optica (NMOSD) was investigated, as detailed below:

[0378] (I) Construction of an induced mouse NMOSD model

[0379] 250 μg of myelin oligodendrocyte glycoprotein (MOG35-55) and 250 μg of aquaporin 4 (AQP4201-220) were mixed with CFA (containing 10 mg / ml of Mycobacterium tuberculosis) and stirred until a white emulsion was formed. This emulsion was subcutaneously injected into humanized mice for immunization, administered via bilateral axillae, bilateral hind limbs, and tail base, with a total injection of 0.1 mL per mouse. Additionally, each mouse was intraperitoneally injected with 300 ng of pertussis toxin. The mice's motor impairment was assessed daily. The scoring criteria are shown in Table 12.

[0380] Table 12 NMOSD Mouse Scoring Criteria

[0381] (II) CAR-mRNA drug treatment in model mice

[0382] 1) Mice exhibiting behavioral disturbances (score of 3 or higher) can be treated. Mice were randomly divided into groups of three. NMOSD mice without mRNA-encapsulated CD5-TLNP served as the model control group, while the remaining groups received CD5-TLNP mRNA via tail vein injection. The dosage of mRNA-LNP was 1.0 mpk, with an injection volume of 200 μL. The Vector 1 group received empty vector LNP without mRNA encapsulation, the eGFP group received mRNA encapsulated with enhanced green fluorescent protein, and the CAR group (SEQ ID NO: 66) received CD5-TLNP prepared according to the method described in Example 2 of this invention. Specific grouping details are shown in Table 13.

[0383] Table 13 CAR-CD5-TLNP Treatment Trial Grouping

[0384] 2) Regularly collect blood from the orbital rims of mice, separate serum, and detect anti-AQP4 antibody levels.

[0385] 3) Regularly assess the mice for behavioral impairment.

[0386] Figure 21 shows the statistical graph of behavioral impairment scores in NMOSD mice treated with different CD5-TLNPs for one week. Figure 22 shows the statistical graph of anti-AQP4 IgG antibodies in the serum of NMOSD mice treated with different CD5-TLNPs for one week.

[0387] The experimental results showed that, compared with the model control group without mRNA and the CD5-TLNP group given unrelated EGFP-mRNA, the NMOSD mice in the CD5-TLNP treatment group with CAR-mRNA had significantly lower behavioral impairment scores and significantly lower serum anti-AQP4 IgG antibody levels, indicating that NMOSD was alleviated and that CAR-mRNA CD5-TLNP can treat NMOSD.

[0388] Example 11

[0389] This embodiment uses CD8 + The pharmacodynamics of CAR-mRNA delivered via T-cell-targeted lipid nanoparticles in treating EAMG mice with myasthenia gravis was investigated, as detailed below:

[0390] (I) Construction of the induced EAMG model in mice

[0391] 250 μg of acetylcholine receptor (AChR97-116) was mixed with CFA (containing 10 mg / ml of Mycobacterium tuberculosis) and stirred until a white emulsion was formed. This emulsion was then subcutaneously injected into humanized mice for immunization. The injection sites were bilateral axillae, bilateral hind limbs, and the base of the tail, with a total injection of 0.1 mL per mouse. Immunization was performed three times, on days 0, 14, and 21. Additionally, after the first immunization, each mouse was intraperitoneally injected with 300 ng of pertussis toxin. The mice's motor impairment was assessed daily using the same scoring criteria as myositis mice (Table 10).

[0392] (II) CAR-mRNA drug treatment in model mice

[0393] 1) The EAMG model was considered successfully established when the mouse score was ≥1. Mice were randomly divided into groups of 3. EAMG mice that did not receive mRNA-encapsulated CD8-TLNP1 served as the model control group, while the remaining groups were treated with mRNA-encapsulated CD8-TLNP1 via tail vein injection. The dosage of mRNA-LNP was 1.0 mpk, and the injection volume was 200 μL. The Vector 1 group was injected with unencapsulated empty vector LNP, the eGFP group was injected with mRNA encapsulated with enhanced green fluorescent protein, the CAR1 group (SEQ ID NO: 66) was injected with CD8-TLNP1 encapsulated with CAR CD19 mRNA, the CAR2 group (SEQ ID NO: 323) was injected with CD8-TLNP1 encapsulated with CAR CD19-BCMA mRNA, and the CAR group was injected with CD8-TLNP1 prepared according to the method in Example 1 of this invention. The specific grouping is shown in Table 14.

[0394] Table 14 CAR-CD8-TLNP1 Treatment Trial Grouping

[0395] 2) Regularly collect blood from the orbital rims of mice, separate serum, and detect the level of anti-AChR IgG antibodies.

[0396] 3) Regularly assess the general performance of the mice (according to Table 10).

[0397] 4) Perform suspension time tests on mice regularly. Animals should acclimatize to the laboratory one hour before the test. Place the animal on a wire mesh so that it can grip the mesh with its limbs. Flip the mesh over, hang the animal upside down, and start the timer to measure the time the mouse can remain on the mesh.

[0398] 5) Perform electromyography tests on mice regularly (see Example 9 for the method).

[0399] Figure 23 shows the statistical graph of performance scores of EAMG mice treated with different CD8-TLNP1 for one week. Figure 24 shows the statistical graph of mouse suspension time. Figure 25 shows the statistical graph of anti-AChR IgG antibodies in the serum of EAMG mice treated with different CD8-TLNP1 for one week. Figure 26 shows the statistical graph of muscle twisting in EAMG mice treated with different CD8-TLNP1 groups for one week under the strongest current stimulation during TA stimulation.

[0400] The experimental results showed that, compared with the model control group without mRNA and the CD8-TLNP1 group given unrelated EGFP-mRNA, the EAMG mice in the CD8-TLNP1 treatment group with CAR-mRNA had significantly lower general performance scores, longer suspension time, significantly lower serum anti-AChR IgG antibody levels, and restored muscle twitch upon stimulation, indicating that EAMG disease was alleviated and that CD8-TLNP1 with CAR-mRNA can treat EAMG.

[0401] Example 12

[0402] This embodiment uses CD8 + The in vitro killing effect of NALM-6 cells by T cell-targeted lipid nanoparticle-delivered CAR-mRNA on NALM-6 cells was investigated, as detailed below:

[0403] 1. The CD8-TLNP1 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP1 of Example 1 was replaced with the sequences shown in SEQ ID NO:67 and SEQ ID NO:68, respectively. The CD8 nanobody sequence used is SEQ ID NO:44.

[0404] 2. The in vitro killing effect of PBMCs on NALM-6 cells in healthy adults is studied as follows:

[0405] Peripheral blood was collected from healthy adults, and peripheral blood cells (PBMCs) were obtained by separation using lymphocyte separation medium. The collected PBMCs were then counted and analyzed at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (anti-CD3 and anti-CD28 activation, 300 U / mL IL2), and 1 μg / mL of CD8-TLNP1 was added (specific groupings are shown in Table 15; Vector 2 group was the group containing mRNA encapsulated with enhanced green fluorescent protein, CAR1 group was the group containing CD19 CAR mRNA (SEQ ID NO: 68) encapsulated with the co-stimulatory sequence CD28, and CAR2 group was the group containing CD19 CAR mRNA (SEQ ID NO: 67) encapsulated with the co-stimulatory sequence 4-1BB). The cells were incubated at 37°C and 5% CO2 for 24 hours to reprogram them into CAR-T cells. Then, CAR-T cells were co-incubated with NALM-6 target cells for 8 hours as effector cells to kill the cells, with effector-target ratios of 1:1 and 1:2. The statistical chart of NALM-6 cell proportions in different drug administration groups is shown in Figure 27.

[0406] Table 15 Grouping of CD8-TLNP1 Drug-Induced Killing Experiment

[0407] Experimental results showed that after 8 hours of CAR1-mRNA and CAR2-mRNA administration, the proportion of surviving NALM-6 cells was significantly lower than that of the control group. The proportion of NALM-6 cells treated with CAR1-mRNA was even lower, indicating that CD8+... + T-cell-targeted lipid nanoparticle-delivered CAR CD19-mRNA exhibits significant killing effect on NALM-6 cells in vitro, with CD19 CAR mRNA (SEQ ID NO:68) having a more pronounced specific killing effect on NALM-6 cells.

[0408] Example 13

[0409] This embodiment uses CD8 + The in vitro study investigated the killing effect of T cell-targeted lipid nanoparticle-delivered CAR-mRNA on non-human primate (NHP) B cells, as detailed below:

[0410] 1. The CD8-TLNP7 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP7 of Example 1 was replaced with the sequences shown in SEQ ID NO:69 and SEQ ID NO:70, respectively. The CD8 nanobody sequence used is SEQ ID NO:331.

[0411] 2. The in vitro B-cell killing effect of NHP is studied as follows:

[0412] Peripheral blood was collected from NHP (cynomolgus monkey) animals that had passed quarantine and met the target weight criteria. Peripheral blood cells (PBMCs) were isolated using lymphocyte separation fluid. The collected PBMCs were then counted and analyzed at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (containing 0.5 μg / mL concanavalin A and 300 U / mL IL2), and 1 μg / mL of CD8-TLNP7 corresponding to the CAR8-TLNP7 mRNA was added (specific groupings are shown in Table 16; Vector 2 group was the group containing mRNA encapsulated with enhanced green fluorescent protein, and CAR1 and CAR2 groups were the groups containing mRNA encapsulated with different NHP CD20 CAR sequences, where the mRNA in CAR1 is SEQ ID NO:69 and the mRNA in CAR2 is SEQ ID NO:70). The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The statistical graph of B cell counts in peripheral blood PBMCs of NHP in each group in Table 16 is shown in Figure 28.

[0413] Table 16 CD8-TLNP7 in vitro experimental grouping

[0414] Experimental results showed that the number of B cells in peripheral blood PBMCs of NHP patients decreased significantly 24 hours after administration of CAR1-mRNA and CAR2-mRNA, indicating that CD8+... + CAR-CD20 mRNA delivered by T cell-targeting lipid nanoparticles exhibits significant killing effects on NHP B cells in vitro.

[0415] Example 14

[0416] This embodiment uses CD8 + The study investigated the in vitro killing effect of CAR-mRNA delivered by T cells via lipid nanoparticles on peripheral blood B cells from patients with systemic lupus erythematosus and healthy adults. Details are as follows:

[0417] 1. The CD8-TLNP7 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP7 of Example 1 was replaced with the sequences shown in SEQ ID NO:67 and SEQ ID NO:68, respectively.

[0418] 2. The in vitro B-cell killing effect was studied in SLE patients and healthy adults as follows:

[0419] Peripheral blood was collected from healthy adults and SLE patients. Peripheral blood cells (PBMCs) were separated using a lymphocyte separation medium. The collected PBMCs were then counted, and the cells were analyzed at a concentration of 0.5 × 10⁻⁶. 6 Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (containing 0.5 μg / mL concanavalin A and 300 U / mL IL2), and 1 μg / mL of CD8-TLNP7 was added (specific groupings are shown in Table 17; Vector 2 group added mRNA encapsulated with enhanced green fluorescent protein, CAR1 group added CD19 CAR mRNA (SEQ ID NO: 68) encapsulated with the co-stimulatory sequence CD28, and CAR2 group added CD19 CAR mRNA (SEQ ID NO: 67) encapsulated with the co-stimulatory sequence 4-1BB). The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. The statistical chart of B cell proportions in PBMCs from different drug-treated groups in Table 17 is shown in Figure 29.

[0420] Table 17 CD8-TLNP7 in vitro experimental grouping

[0421] Experimental results showed that 24 hours after administration of CAR1-mRNA and CAR2-mRNA, the proportion of B cells in peripheral blood PBMCs of healthy adults and SLE patients was significantly lower than that in the control group. This indicates that CD8+... + CAR-CD19 mRNA delivered by T cell-targeted lipid nanoparticles has a significant killing effect on B cells in healthy adults and SLE patients in vitro. It has also been demonstrated that CD19 CAR mRNA containing different co-stimulatory sequences has a significant killing effect on B cells in healthy adults and SLE patients in vitro.

[0422] Example 15

[0423] This embodiment uses CD8 + The study investigated the in vitro killing effect of different CAR-mRNAs delivered by T cells via lipid nanoparticles on peripheral blood B cells from healthy adults, as detailed below:

[0424] 1. The CD8-TLNP7 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP7 of Example 1 was replaced with the sequences shown in SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, respectively. The CD8 nanobody sequence used is SEQ ID NO:44.

[0425] The preparation process of the four-component conventional LNP (cLNP) for encapsulating CAR-mRNA is as follows:

[0426] Four lipid molecules—ionizable amino lipid E12LA6B6O3, phospholipid DSPC, cholesterol CHOL, and DSPE-PEG2000—were dissolved in ethanol at a molar ratio of 47.5:15:36:1.0. CAR-mRNA (SEQ ID NO:68) was dissolved in an acidic acetic acid buffer solution (25 mM, pH 4), with a molar ratio (N / P) of ionizable amino lipid to mRNA bases of 6.5:1.

[0427] The ethanol and aqueous phases were mixed using a Myanna microfluidic device, with the flow rate controlled at a volume ratio of 1:3 between the ethanol and aqueous phases. The total flow rate of the two phases was 12 mL / min, and the flow rate ratio of the ethanol and aqueous phases was 1:3, to obtain crude LNP. Then, 10 volumes of 25 mM Tris buffer were added to terminate the process.

[0428] The LNP with stop solution was ultrafiltered once at 4°C and 1200 rcf; then, 25 mM Tris buffer was added and ultrafiltered again at 4°C and 1200 rcf. The prepared LNP was collected, sucrose was added and the concentration was adjusted to 8%, and the mixture was aseptically filtered in a clean bench using a 0.22 μm filter to obtain ordinary four-component LNP (cLNP).

[0429] 2. The in vitro B-cell killing effect in healthy adults is studied as follows:

[0430] Peripheral blood was collected from healthy adults, and peripheral blood cells (PBMCs) were obtained by separation using lymphocyte separation medium. The collected PBMCs were then counted and analyzed at a concentration of 0.5 × 10⁻⁶ cells / mL. 6Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (containing 0.5 μg / mL concanavalin A and 300 U / mL IL2), and corresponding 1 μg / mL LNPs were added (specific groupings are shown in Table 18; the cLNP group added conventional LNPs (common four-component LNPs) encapsulated with CAR-CD19 (SEQ ID NO:68); CAR1 to CAR4 groups added CD8-TLNP7 groups encapsulated with different CAR mRNAs, where CAR1 encapsulated CAR-CD19 (SEQ ID NO:68), CAR2 encapsulated CAR-BCMA (SEQ ID NO:71), CAR3 encapsulated CAR-BCMA (SEQ ID NO:72), and CAR4 encapsulated NHP CAR-CD20 (SEQ ID NO:70)). The cells were incubated at 37°C and 5% CO2 for 24 hours. The statistical chart of B cell proportions in PBMCs of different drug-treated groups in Table 18 is shown in Figure 30.

[0431] Table 18 CD8-TLNP7 in vitro experimental grouping

[0432] Experimental results showed that 24 hours after CAR1-mRNA administration, the proportion of B cells in peripheral blood PBMCs of healthy adults was significantly lower than that in the control group and other CAR groups. Because the vast majority of B cells in healthy adult PBMCs express CD19 and almost none express BCMA, CAR1 (CAR-CD19) reprogrammed T cells in PBMCs killed corresponding CD19-expressing target cells (B cells). In contrast, other CAR-reprogrammed T cells did not have corresponding target cells in this system (NHP B cells; CAR-BCMA reprogrammed T cells, but almost none of the B cells expressed BCMA). This indicates that CD8... + CAR-CD19 mRNA delivered by T-cell-targeting lipid nanoparticles exhibits significant specific killing effects on B cells from healthy adults in vitro.

[0433] Example 16

[0434] This embodiment encapsulates CD20-CAR-mRNA targeting human CD8. + The study on the effect of T cell nucleic acid-nanoparticle drug (mRNA CD8-TLNP7) on B cell clearance in NHP is as follows:

[0435] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0436] The preparation method of the lipid nanoparticles used in this embodiment is as described in Example 1. The CD8 nanobody sequence used is SEQ ID NO:331.

[0437] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0438] Two cynomolgus monkeys (NHP7 and NHP12) that passed quarantine and met weight standards were selected and administered the prepared CD20-CAR-CD8-TLNP7 via intravenous bolus injection. The mRNA sequence used for this drug is SEQ ID NO:70, the drug concentration is 100 μg / mL, and the dosage is 0.2 mpk. Peripheral blood samples were collected before administration and on days 1, 2, 4, 7, 12, and 22 after administration for flow cytometry analysis of the proportion, number, and dynamic changes of B cells. The dynamic changes in the proportion of B cells in NHPs are shown in Figure 31, the changes in the number of B cells are shown in Figure 32, and the relative values ​​of the number of B cells are shown in Figure 33.

[0439] Experimental results showed that 24 hours after a single dose, B cells in the peripheral blood of NHP were almost completely eliminated, with a clearance rate exceeding 99%. Even on day 22, the number of B cells remained at approximately 30% of the pre-drug level. This indicates that the drug has a long-lasting effect in eliminating B cells and can achieve the goal of B cell reconstruction in vivo.

[0440] Example 17

[0441] This embodiment studies the effect of a single dose of a nucleic acid nanoparticle drug (mRNA CD8-TLNP15) encapsulating CD20-CAR-mRNA targeting human CD8+ T cells on B cell clearance in NHP at different doses, as detailed below:

[0442] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0443] The preparation method of the lipid nanoparticles used in this embodiment is as described in Example 1. The ratio of each component is: E12LA6B6O3:DSPC:CHOL:DSPE-PEG2000:DSPE-PEG2000-Mal = 47.5:15:36:1.0:0.1. The CD8 nanobody sequence used is SEQ ID NO:331.

[0444] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0445] Six cynomolgus monkeys that passed quarantine and met the target weight were divided into three groups of two each. The prepared CD20-CAR-CD8-TLNP15 was administered intravenously via bolus injection. The mRNA sequence used for this drug is SEQ ID NO:70, and the drug concentration was 100 μg / mL. The administered doses were 0.05 mpk, 0.5 mpk, and 1.0 mpk. Peripheral blood samples were collected before administration (D0) and on days 1, 2, 4, 7, 14, 21, and 30 after administration for flow cytometry analysis to determine the CAR mRNA expression level in CD8 T cells and the ratio of CAR mRNA expression in B cells.

[0446] Figure 34 shows the expression of CAR mRNA in CD8 T cells of NHP one day after administration, and Figure 35 shows the relative number of B cells at each time point. The results indicate that at a dose of 0.05 mpk, the expression level of CAR mRNA in CD8 T cells was slightly lower than in the other two dose groups. 24 hours after NHP administration, B cells in the peripheral blood of all three dose groups were significantly cleared. From day 4 after administration, the number of B cells in the peripheral blood of all groups increased, but the increase in the proportion of B cells in the peripheral blood of NHP in the high-dose groups (0.5 mpk and 1.0 mpk) was significantly slower than that in the 0.05 mpk dose group.

[0447] Example 18

[0448] This embodiment encapsulates CD20-CAR-mRNA targeting human CD8. + A study on the long-term effect of T-cell nucleic acid nanoparticle drug (mRNA CD8-TLNP15) on peripheral blood B-cell reconstitution after multiple administrations to NHP.

[0449] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0450] The preparation method of the lipid nanoparticles used in this embodiment is as described in Example 1. The ratio of each component is: E12LA6B6O3:DSPC:CHOL:DSPE-PEG2000:DSPE-PEG2000-Mal = 47.5:15:36:1.0:0.1. The CD8 nanobody sequence used is SEQ ID NO:331.

[0451] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0452] Six cynomolgus monkeys that passed quarantine and met weight requirements were selected and divided into three groups of two. Each group received the prepared CD20-CAR-CD8-TLNP15 via intravenous bolus injection. The mRNA sequence used for this drug is SEQ ID NO:70, the drug concentration is 100 μg / mL, and the dosage is 0.2 mpk. Dosing frequency was single, double, and triple administration. The multiple-dose group received the drug every two days (48 hours). The flow cytometry time points for peripheral blood NHP in the three experimental groups are shown in Table 19 below.

[0453] Table 19

[0454] Figure 36 shows the expression of CAR mRNA in peripheral blood 6 hours after NHP administration, and Figure 37 shows the relative number of B cells at each time point. The results indicate that CAR molecule expression in peripheral blood gradually accumulates 6 hours after multiple intravenous administrations of NHP. The number of B cells in the peripheral blood of the two- and three-dose groups was zero. A small increase in B cells began to appear on day 7 after the last administration.

[0455] Example 19

[0456] This embodiment encapsulates CD20-CAR-mRNA targeting human CD8. + Study on the effect of T cell nucleic acid nanoparticle drug (mRNA CD8-TLNP15) on B cell clearance in tissues after multiple administrations to NHP.

[0457] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0458] The preparation method of the lipid nanoparticles used in this embodiment is as described in Example 1.

[0459] The proportions of each component are: E12LA6B6O3:DSPC:CHOL:DSPE-PEG2000:DSPE-PEG2000-Mal = 47.5:15:36:1.0:0.1. The CD8 nanobody sequence used is SEQ ID NO:331.

[0460] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0461] Thirteen cynomolgus monkeys that passed quarantine and met weight standards were selected and divided into seven groups. One monkey in each of the untreated groups and two monkeys in each of the treated groups were administered the prepared CD20-CAR-CD8-TLNP15 via intravenous bolus injection. The mRNA sequence used for this drug is SEQ ID NO:70, the drug concentration is 100 μg / mL, and the dosage is 0.2 mpk and 0.5 mpk. Dosing frequency included single, double, and triple administrations. The multiple-dose groups received the drug every two days (48 hours). 72 hours after the last administration, flow cytometry was used to analyze the peripheral blood, spleen, bone marrow, and inguinal lymph nodes of the thirteen NHPs.

[0462] The clearance effect of B cells in peripheral blood from NHP is shown in Figure 38. The clearance effect of B cells in the spleen is shown in Figure 39. The clearance effect of B cells in the bone marrow is shown in Figure 40. The clearance effect of B cells in the inguinal lymph nodes is shown in Figure 41.

[0463] Experimental results showed that after NHP administration, the proportion of B cells in peripheral blood decreased to near zero in both the 0.2 mpk and 0.5 mpk groups. After a single NHP dose, both the 0.2 mpk and 0.5 mpk groups cleared 90% of B cells in the spleen; after two doses, all B cells in the spleen were cleared. After two to three doses of 0.2 mpk NHP, B cells in the bone marrow were essentially cleared. A single 0.5 mpk dose was sufficient to clear B cells from the bone marrow. After 0.2 mpk NHP administration, approximately two-thirds of B cells in the inguinal lymph nodes were cleared. The clearance effect of one or two 0.5 mpk doses was similar to that of 0.2 mpk, and three doses cleared the vast majority of B cells in the inguinal lymph nodes.

[0464] Example 20

[0465] This embodiment describes the encapsulation of different CD20-CAR-mRNAs targeting human CD8. + The study on the effect of T cell nucleic acid-nanoparticle drug (mRNA CD8-TLNP15) on B cell clearance in NHP is as follows:

[0466] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0467] The lipid nanoparticles used in this embodiment were prepared according to the description in Example 1, wherein the ratio of each component was: E12LA6B6O3:DSPC:CHOL:DSPE-PEG2000:DSPE-PEG2000-Mal = 47.5:15:36:1.0:0.1. The CD8 nanobody sequence used is SEQ ID NO:331.

[0468] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0469] Four healthy cynomolgus monkeys (NHPs) that passed quarantine and met weight standards were selected and divided into two groups of two. Each group was administered CD8-TLNP15 of the prepared CD20-CAR-mRNA, either modified or unmodified by miR-122, via intravenous bolus injection. The mRNA sequences used were CAR1 (SEQ ID NO:77) and CAR2 (SEQ ID NO:76), respectively, at a concentration of 100 μg / mL and a dose of 0.2 mpk. On day 1 after administration, liver and spleen samples were collected for absolute quantification of CAR CD20 content using qPCR. The CAR CD20 mRNA content in the liver and spleen of NHPs is shown in Figure 42.

[0470] The experimental results showed that 24 hours after administration, the miR-122 modified drug (CAR1 group) reduced the expression of drug molecules in NHP liver cells and increased the expression in the spleen.

[0471] Example 21

[0472] This embodiment describes a CD19-CAR-mRNA targeting human CD8 that encapsulates miR-122. + The study on the CAR expression of T cell nucleic acid-nanoparticle drug (mRNA CD8-TLNP15) and its effect on B cell clearance is as follows:

[0473] 1. The CD8-TLNP15 used in this embodiment was prepared according to the method described in Example 1. The only difference was that the mRNA in Example 1 was replaced with the sequences shown in SEQ ID NO:74 and SEQ ID NO:75, respectively, which are miR-122 modified CD19-CAR-mRNA. The CD8 nanobody sequence used is SEQ ID NO:331.

[0474] 2. In vitro CAR expression and B cell clearance effect of human PBMCs: Peripheral blood was collected from healthy adults, and PBMCs were isolated using lymphocyte separation medium. The collected PBMCs were counted and analyzed at a concentration of 0.5 × 10⁻⁶ cells / mL. 6Cells were resuspended at a concentration of 1 μg / mL in complete RPMI-1640 medium (anti-CD3 and anti-CD28 activation, 300 U / mL IL2). The control group was the PBS group. The CAR-treated group was given 1 μg / mL of CD8-TLNP15 encapsulated with CAR-CD19 (SEQ ID NO:74) and incubated at 37°C in a 5% CO2 incubator for 72 hours. The statistical graph of CAR expression ratio in PBMCs is shown in Figure 43. The statistical graph of B cell clearance effect (in vitro killing) is shown in Figure 44.

[0475] Experimental results showed that the miR-122-modified CD19-CAR-mRNA delivered in vitro by CD8-TLNP15 exhibited an expression rate of over 80% in PBMCs. The in vitro B cell killing efficiency reached over 90%.

[0476] 3. Elimination effect of B cells in hCD8 humanized mice: Six 6-8 week old hCD8 humanized female mice (from Biocytogen) that met quarantine standards and reached the SPF (specific pathogen-free) level were purchased and divided into two groups. PBS served as the control group. Mice in the CAR-administered group were injected intravenously with CD8-TLNP15 encapsulated with CAR-mRNA (SEQ ID NO: 75), prepared according to the method described in Example 1 of this invention. The dosage of mRNA-LNP was 1.0 mpk, and the injection volume was 200 μL. Peripheral blood samples were collected on day 0 (before administration) and on days 1, 2, 4, 8, 11, and 16 after administration to detect the number of B cells. The statistical data on changes in the number of B cells in mice are shown in Figure 45.

[0477] Experimental results showed that the number of B cells in the peripheral blood of mice decreased instantaneously one day after administration of CD8-TLNP15. After administration of miR-122 modified CD19-CAR-mRNA-CD8 TLNP15, the number of B cells decreased significantly and then slowly increased after one week, showing a significant scavenging effect on B cells.

[0478] Example 22

[0479] This embodiment uses CD8 + The study investigated the in vitro expression of different CAR-mRNA-related molecules delivered by T cells targeting lipid nanoparticles and their killing effect on healthy adult peripheral blood B cells, as well as the effect on CD8. + The study investigated the killing effect of different CAR-mRNAs delivered by lipid nanoparticles by T cells on target cells, as detailed below:

[0480] 1. The CD8-TLNP7 used in this embodiment was prepared according to the method described in Example 1, except that the mRNA in the CD8-TLNP7 of Example 1 was replaced with the sequences shown in SEQ ID NO:391, SEQ ID NO:400, SEQ ID NO:373 and SEQ ID NO:382, respectively. The CD8 nanobody sequence used is SEQ ID NO:348.

[0481] 2. The in vitro B-cell killing effect in healthy adults is studied as follows:

[0482] Peripheral blood was collected from healthy adults, and peripheral blood cells (PBMCs) were obtained by separation using lymphocyte separation fluid. The collected PBMCs were then counted, and the counts were recorded at a rate of 1 × 10⁻⁶ cells / mL. 6 Cells were resuspended at a concentration of 1 μg / mL in lymphocyte culture medium (containing 300 U / mL IL2), and 1 μg / mL LNP was added (specific groupings are shown in Table 20; CAR1 to CAR4 groups were CD8-TLNP7 groups encapsulated with different CAR mRNAs, where CAR1 group encapsulated CAR-CD19-BCMA (SEQ ID NO:391), CAR2 group encapsulated CAR-CD19-BCMA (SEQ ID NO:400), CAR3 group encapsulated CAR-BCMA-CD19 (SEQ ID NO:373), and CAR4 group encapsulated CAR-BCMA-CD19 (SEQ ID NO:382)). The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. The statistical graph of CAR CD19 (FMC63) expression in PBMCs of different treatment groups in Table 20 is shown in Figure 46. The statistical graph of CAR BCMA expression in PBMCs of different treatment groups is shown in Figure 47. Figure 48 shows the statistical graph of B cell killing efficiency in PBMCs of different drug administration groups.

[0483] Table 20 CD8-TLNP7 in vitro experimental grouping

[0484] The experimental results showed that, 72 hours after CAR administration, the expression of CAR CD19 and CAR BCMA in CD8 T cells and the killing rate of B cells in peripheral blood PBMCs of healthy adults were significantly increased compared with the control group (PBS group). This indicates that CD8... + The CD19 and BCMA dual-target CAR mRNA delivered by T cell-targeted lipid nanoparticles was significantly expressed in vitro and exhibited a significant killing effect on B cells of healthy adults.

[0485] 3. The in vitro killing effect of PBMCs on NCI-H929 target cells from healthy adults is studied as follows:

[0486] Peripheral blood was collected from healthy adults, and PBMCs were obtained by separating them using lymphocyte separation medium. CD8 T cells were then sorted and administered to CD8-TLNP7 groups encapsulated with different CAR mRNAs (as in step 2 of this example). These cells were incubated at 37°C and 5% CO2 for 48 hours to reprogram them into CAR-T cells. The CAR-T cells were then co-incubated with NCI-H929 target cells for 16 hours as effector cells, with effector-to-target ratios of 1:1 and 2:1. The statistical graph of NCI-H929 cell killing efficiency in different treatment groups is shown in Figure 49.

[0487] Experimental results showed that CAR-mRNA expression significantly increased the killing efficiency of target cells compared to the control group 16 hours after administration. This indicates that CD8 + T-cell-targeted CAR mRNA targeting CD19 and BCMA delivered by lipid nanoparticles exhibits significant killing effect on NCI-H929 cells in vitro.

[0488] Example 23

[0489] This embodiment uses CD8 + The effects of different CAR-mRNAs delivered by T cell-targeted lipid nanoparticles on the clearance of B cells in HSC mice were investigated, as detailed below:

[0490] 1. The CD8-TLNP8 used in this embodiment was prepared according to the method described in Example 1. The CD8 nanobody sequence used is SEQ ID NO:348.

[0491] 2. The effect of HSC on B cell clearance in humanized mice is studied as follows:

[0492] Eight HSC humanized female mice (from Biocytogen) that passed quarantine, met SPF (specific pathogen-free) standards, and had successfully undergone immune system remodeling were procured and divided into four groups. Mice were injected via tail vein with CD8-TLNP8 containing different mRNAs. The PBS group was injected with unencapsulated empty LNPs, and the CAR group was injected with CD8-TLNP8 prepared according to the method described in Example 1 of this invention. Specifically, CAR1 group encapsulated CAR-CD19-BCMA (SEQ ID NO:321), CAR2 group encapsulated CAR-CD19-BCMA (SEQ ID NO:322), and CAR3 group encapsulated CAR-CD19-BCMA (SEQ ID NO:323). Specific grouping details are shown in Table 21. Mice were administered 1.0 mpk of mRNA-LNP at a volume of 200 μL. Peripheral blood was collected on day 2 to assess B cell killing efficiency. The statistical data on B cell killing rates in mice from different CD8-TLNP8 groups are shown in Figure 50.

[0493] Table 21 In vivo experimental grouping of CD8-TLNP8

[0494] The experimental results showed that the killing rate of B cells in the peripheral blood of mice increased significantly 2 days after administration of CD8-TLNP8, indicating that CAR-mRNA-CD8 TLNP8 has a significant scavenging effect on B cells.

[0495] Example 24

[0496] This example describes the encapsulation of different CAR-mRNAs targeting human CD8. + The study on the effect of T cell nucleic acid-nanoparticle drug (mRNA CD8-TLNP7) on B cell clearance in NHP is as follows:

[0497] (I) Preparation of a target for human CD8 + T-cell nanoparticles

[0498] The preparation method of the lipid nanoparticles used in this embodiment is as described in Example 1. The CD8 nanobody sequence used is SEQ ID NO:348.

[0499] (II) Targeting human CD8 + Study on the effect of T cell nanoparticles on the elimination of B cells in NHP

[0500] Cynomolgus monkeys (NHPs) that passed quarantine and met weight standards were selected and administered CD8-TLNP7 cells encapsulated with different CAR mRNAs via intravenous bolus injection. The CAR1 group encapsulated CAR-CD20-BCMA (SEQ ID NO: 84), the CAR2 group encapsulated CAR-CD20-BCMA (SEQ ID NO: 329), the CAR3 group encapsulated CAR-BCMA-CD20 (SEQ ID NO: 64), and the CAR4 group encapsulated CAR-BCMA-CD20 (SEQ ID NO: 328). Specific groupings are shown in Table 22. The drug concentration was 100 μg / mL, and the dosage was 0.2 mpk. On the second day after administration, peripheral blood was collected for flow cytometry analysis of B cell killing. Figure 51 shows the statistical graph of peripheral blood B cell killing efficiency in NHPs. Figure 52 shows the statistical graph of bone marrow B cell killing efficiency. Figure 53 shows the statistical graph of bone marrow plasma cell killing efficiency.

[0501] Table 22 In vivo experimental grouping of CD8-TLNP7

[0502] Experimental results showed that 48 hours after a single administration, B cells in the peripheral blood of NHP patients and B cells and plasma cells in the bone marrow were almost completely eliminated, with a clearance rate exceeding 90%. This indicates that the drug has the effect of eliminating B cells and plasma cells.

[0503] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. Industrial applicability

[0504] This invention provides a drug, method, and application for treating diseases caused by excessive B cell proliferation and autoantibody production. The drug of this invention comprises: mRNA encoding a chimeric antigen receptor molecule and T cell-targeting lipid nanoparticles; the drug can generate chimeric antigen receptor T cells in vivo; the lipid nanoparticles comprise: cationic lipids, phospholipids, cholesterol, PEG lipids, and a targeting molecule, wherein the targeting molecule mediates T cell targeting. This invention uses lipid nanoparticles encapsulating chimeric antigen receptor molecule mRNA (mRNA-LNP) to deliver mRNA to T cells in vivo to generate chimeric antigen receptor T cells, thereby eliminating pathological autoreactive B cells and achieving the treatment of diseases caused by excessive B cell proliferation and autoantibody production. This method has good economic value and application prospects.

Claims

1. A composition, characterized in that, The composition comprises: mRNA encoding a chimeric antigen receptor molecule and T cell-targeting lipid nanoparticles; The composition can generate chimeric antigen receptor T cells in vivo and clear autologous B cells; The T-cell-targeting lipid nanoparticles comprise: cationic lipids, phospholipids, cholesterol, PEG lipids, and targeting molecules, wherein the targeting molecules mediate T-cell targeting.

2. The composition according to claim 1, characterized in that, The mRNA may encode CD19 CAR, CD20 CAR, BCMA CAR, CD19 and CD20 dual CAR, CD19 and BCMA dual CAR or CD20 and BCMA dual CAR, preferably the mRNA may encode CD19 CAR, CD20 CAR and / or BCMA CAR, more preferably the mRNA may encode CD19 CAR.

3. The composition according to claim 1 or 2, characterized in that, The surface antigens of T cells recognized by the T cell-targeting lipid nanoparticles include one or more of CD3, CD4, CD5, CD7, and CD8. Preferably, the surface antigen of the T cell recognized by the T cell-targeting lipid nanoparticles is one or more of CD3, CD8, CD7 or CD5, more preferably CD5 or CD8.

4. The composition according to any one of claims 1-3, characterized in that, The targeting molecule of the T cell-targeting lipid nanoparticles comprises: an antibody that recognizes a T cell surface antigen, a linker, and a hydrophobic tail; the T cell surface antigen includes one or more of CD8, CD4, CD5, CD7, or CD3; the linker comprises PEG or a PEG derivative, and the hydrophobic tail comprises one or more substituted or unsubstituted C-terminals. 12 -C 18 Alkyl group, wherein the antibody and the linker are connected by an amino acid containing a reaction site, wherein the amino acid includes, but is not limited to, cysteine ​​and lysine; Preferably, the antibody that recognizes T cell surface antigens includes nanobodies; More preferably, the nanobody shown includes an antigen-binding domain that is specific to the antigen, wherein the antigen is a membrane protein molecule; More preferably, the membrane protein molecule is an immune cell or immune-related cell membrane protein molecule.

5. The composition according to claim 4, characterized in that, The amino acid sequences of the antibodies that recognize T cell surface antigens, CDR1, CDR2, and CDR3, are shown in any of the following groups: (1) SEQ ID NO: 114; SEQ ID NO: 160; SEQ ID NO: 206; (2) SEQ ID NO: 115; SEQ ID NO: 161; SEQ ID NO: 207; (3) SEQ ID NO: 116; SEQ ID NO: 162; SEQ ID NO: 208; (4) SEQ ID NO: 117; SEQ ID NO: 163; SEQ ID NO: 209; (5) SEQ ID NO: 118; SEQ ID NO: 164; SEQ ID NO: 210; (6) SEQ ID NO: 119; SEQ ID NO: 165; SEQ ID NO: 211; (7) SEQ ID NO: 120; SEQ ID NO: 166; SEQ ID NO: 212; (8) SEQ ID NO: 121; SEQ ID NO: 167; SEQ ID NO: 213; (9) SEQ ID NO: 122; SEQ ID NO: 168; SEQ ID NO: 214; (10) SEQ ID NO: 123; SEQ ID NO: 169; SEQ ID NO: 215; (11)SEQ ID NO:124;SEQ ID NO:170;SEQ ID NO:216; (12)SEQ ID NO:125;SEQ ID NO:171;SEQ ID NO:217; (13)SEQ ID NO:126;SEQ ID NO:172;SEQ ID NO:218; (14)SEQ ID NO:127;SEQ ID NO:173;SEQ ID NO:219; (15)SEQ ID NO:128;SEQ ID NO:174;SEQ ID NO:220; (16)SEQ ID NO:129;SEQ ID NO:175;SEQ ID NO:221; (17)SEQ ID NO:130;SEQ ID NO:176;SEQ ID NO:222; (18)SEQ ID NO:131;SEQ ID NO:177;SEQ ID NO:223; (19)SEQ ID NO:132;SEQ ID NO:178;SEQ ID NO:224; (20)SEQ ID NO:133;SEQ ID NO:179;SEQ ID NO:225; (21)SEQ ID NO:134;SEQ ID NO:180;SEQ ID NO:226; (22)SEQ ID NO:135;SEQ ID NO:181;SEQ ID NO:227; (23)SEQ ID NO:136;SEQ ID NO:182;SEQ ID NO:228; (24)SEQ ID NO:137;SEQ ID NO:183;SEQ ID NO:229; (25)SEQ ID NO:138;SEQ ID NO:184;SEQ ID NO:230; (26)SEQ ID NO:139;SEQ ID NO:185;SEQ ID NO:231; (27)SEQ ID NO:140;SEQ ID NO:186;SEQ ID NO:232; (28)SEQ ID NO:141;SEQ ID NO:187;SEQ ID NO:233; (29)SEQ ID NO:142;SEQ ID NO:188;SEQ ID NO:234; (30)SEQ ID NO:143;SEQ ID NO:189;SEQ ID NO:235; (31)SEQ ID NO:144;SEQ ID NO:190;SEQ ID NO:236; (32)SEQ ID NO:145;SEQ ID NO:191;SEQ ID NO:237; (33)SEQ ID NO:146;SEQ ID NO:192;SEQ ID NO:238; (34)SEQ ID NO:147;SEQ ID NO:193;SEQ ID NO:239; (35)SEQ ID NO:148;SEQ ID NO:194;SEQ ID NO:240; (36)SEQ ID NO:149;SEQ ID NO:195;SEQ ID NO:241; (37)SEQ ID NO:150;SEQ ID NO:196;SEQ ID NO:242; (38)SEQ ID NO:151;SEQ ID NO:197;SEQ ID NO:243; (39)SEQ ID NO:152;SEQ ID NO:198;SEQ ID NO:244; (40)SEQ ID NO:153;SEQ ID NO:199;SEQ ID NO:245; (41)SEQ ID NO:154;SEQ ID NO:200;SEQ ID NO:246; (42)SEQ ID NO:155;SEQ ID NO:201;SEQ ID NO:247; (43)SEQ ID NO:156;SEQ ID NO:202;SEQ ID NO:248; (44)SEQ ID NO:157;SEQ ID NO:203;SEQ ID NO:249; (45)SEQ ID NO:158;SEQ ID NO:204;SEQ ID NO:250; (46)SEQ ID NO:159;SEQ ID NO:205;SEQ ID NO:251; (47)SEQ ID NO:252;SEQ ID NO:275;SEQ ID NO:298; (48)SEQ ID NO:253;SEQ ID NO:276;SEQ ID NO:299; (49)SEQ ID NO:254;SEQ ID NO:277;SEQ ID NO:300; (50)SEQ ID NO:255;SEQ ID NO:278;SEQ ID NO:301; (51)SEQ ID NO:256;SEQ ID NO:279;SEQ ID NO:302; (52)SEQ ID NO:257;SEQ ID NO:280;SEQ ID NO:303; (53) SEQ ID NO: 258; SEQ ID NO: 281; SEQ ID NO: 304; (54) SEQ ID NO: 259; SEQ ID NO: 282; SEQ ID NO: 305; (55) SEQ ID NO: 260; SEQ ID NO: 283; SEQ ID NO: 306; (56) SEQ ID NO: 261; SEQ ID NO: 284; SEQ ID NO: 307; (57) SEQ ID NO: 262; SEQ ID NO: 285; SEQ ID NO: 308; (58) SEQ ID NO: 263; SEQ ID NO: 286; SEQ ID NO: 309; (59) SEQ ID NO: 264; SEQ ID NO: 287; SEQ ID NO: 310; (60) SEQ ID NO: 265; SEQ ID NO: 288; SEQ ID NO: 311; (61) SEQ ID NO: 266; SEQ ID NO: 289; SEQ ID NO: 312; (62) SEQ ID NO: 267; SEQ ID NO: 290; SEQ ID NO: 313; (63) SEQ ID NO: 268; SEQ ID NO: 291; SEQ ID NO: 314; (64) SEQ ID NO: 269; SEQ ID NO: 292; SEQ ID NO: 315; (65) SEQ ID NO: 270; SEQ ID NO: 293; SEQ ID NO: 316; (66) SEQ ID NO: 271; SEQ ID NO: 294; SEQ ID NO: 317; (67) SEQ ID NO: 272; SEQ ID NO: 295; SEQ ID NO: 318; (68) SEQ ID NO: 273; SEQ ID NO: 296; SEQ ID NO: 319; (69) SEQ ID NO: 274; SEQ ID NO: 297; SEQ ID NO: 320; Preferably, the amino acid sequence of the antibody recognizing T cell surface antigen is as shown in any one of SEQ ID NO:1-46, 85-107, 331-355 or as shown in any one of SEQ ID NO:1-46, 85-107, 331-355, having ≥80% homology with the sequence shown in any one of SEQ ID NO:1-46, 85-107, 331-355; Preferably, the surface antigen of the T cells corresponding to SEQ ID NO:1-20, SEQ ID NO:44-45, SEQ ID NO:85-107 or SEQ ID NO:331-355 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:21-43 or SEQ ID NO:46 is CD5; More preferably, the surface antigen of the T cells corresponding to SEQ ID NO:98-105 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:46 is CD5.

6. The composition according to any one of claims 1-5, characterized in that, The molar ratio of the cationic lipid, phospholipid, cholesterol, PEG lipid, and target molecule is 30-70:5-30:0-65:0.2-5:0.005-1; preferably 40-60:10-20:30-60:0.5-2:0.01-0.5; more preferably 45-55:10-20:30-40:0.75-1.5:0.05-0.25; A further preferred ratio is 47-52:12-18:32-38:1-1.2:0.05-0.20; A further preferred ratio is 47-50:13-16:34-36:1.0-1.1:0.05-0.15; Further preferred ratios are 47.5:15:36:1.0:0.05 or 47.5:15:36:1.0:0.

1.

7. The composition according to any one of claims 1-6, characterized in that, The molar ratio of the cationic lipid to the mRNA is 4-8:1, preferably 5-7.5:1, more preferably 6-7.5:1, and even more preferably 6.5-7.5:

1.

8. The composition according to any one of claims 1-7, characterized in that, The mRNA contains a coding region encoding a polypeptide, a 5′ untranslated region, a 3′ untranslated region, and a polyadenylated tail at the end of the 3′ untranslated region; Preferably, the 3' untranslated region includes at least one microRNA binding site for expression in a specific cell type, such that mRNA expression can be reduced, wherein the mRNA is targeted for degradation or reduced translation in the presence of the microRNA; the at least one microRNA binding site includes miR-122; the sequence of miR-122 is shown in SEQ ID NO:108; more preferably, the sequence of the 3' untranslated region is shown in any one of SEQ ID NO:110-112; Preferably, the sequence of the 5' untranslated region is as shown in SEQ ID NO:109; Preferably, the polyadenylate tail sequence is as shown in SEQ ID NO:

113.

9. The composition according to claim 8, characterized in that, The coding region sequence of the polypeptide encoding the mRNA is as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461, or as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461, or as shown in any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, or SEQ ID NO:457-461. Sequences showing ≥80% homology to any one of the sequences shown in NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419 or SEQ ID NO:457-461; Preferably, the full-length sequence of the mRNA is as shown in any one of SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471, or as shown in any one of SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, or .... The sequence is a sequence with ≥80% homology to any one of the sequences shown in SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456 or SEQ ID NO:462-471; Preferably, the nucleic acid sequence encoding the CD19 antigenic domain as shown in any one of SEQ ID NO:47-49, SEQ ID NO:54-56, SEQ ID NO:356-357, or SEQ ID NO:461; the nucleic acid sequence encoding the BCMA antigenic domain as shown in any one of SEQ ID NO:52-53, SEQ ID NO:63, SEQ ID NO:406-407, SEQ ID NO:412-413, or SEQ ID NO:418-419; the nucleic acid sequence encoding the CD20 antigenic domain as shown in any one of SEQ ID NO:50-51 or SEQ ID NO:57-60; the nucleic acid sequence encoding the BCMA-CD20 antigenic domain as shown in any one of SEQ ID NO:64-65; and the nucleic acid sequences encoding the BCMA-CD20 antigenic domain as shown in any one of SEQ ID NO:61-62, SEQ ID NO:324-326, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:461, SEQ ID NO:64-65, SEQ ID NO:379-381, SEQ ID NO:64-65, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:64-65 ... The nucleic acid sequence shown in any one of SEQ ID NO:388-390, SEQ ID NO:397-399 or SEQ ID NO:457-460 encodes the antigenic domain of CD19-BCMA; Preferably, the amino acid sequences of the antigenic domain of CD19 encoded by nucleic acid sequences shown in SEQ ID NO:47-49, SEQ ID NO:54-56, SEQ ID NO:356-357, and SEQ ID NO:461 are as shown in SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444, and SEQ ID NO:476, respectively; the amino acid sequences of the antigenic domain of BCMA encoded by nucleic acid sequences shown in SEQ ID NO:52-53, SEQ ID NO:63, SEQ ID NO:406-407, SEQ ID NO:412-413, and SEQ ID NO:418-419 are as shown in SEQ ID NO:429-430, SEQ ID NO:440, and SEQ ID NO:451-453, respectively; and the amino acid sequences of the antigenic domain of CD20 encoded by nucleic acid sequences shown in SEQ ID NO:50-51 and SEQ ID NO:57-60 are as shown in SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444, and SEQ ID NO:476, respectively. The amino acid sequences of the antigenic domains of BCMA-CD20 encoded by the nucleic acid sequences shown in SEQ ID NO:427-428 and SEQ ID NO:434-437 are shown in SEQ ID NO:64-65, respectively; the amino acid sequences of the antigenic domains of CD19-BCMA encoded by the nucleic acid sequences shown in SEQ ID NO:61-62, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, and SEQ ID NO:457-460 are shown in SEQ ID NO:438-439, SEQ ID NO:445-450, and SEQ ID NO:472-475, respectively.

10. The composition according to any one of claims 8-9, characterized in that, The mRNA is a modified mRNA, and the modified mRNA contains a substitute uridine, which is selected from pseudouridine or 1-methylpseudouridine.

11. The use of the composition according to any one of claims 1-10 in the preparation of a product that reduces one or more of the following: autoreactive pathological B cells in a patient, improves the patient's autoantibody level, C-reactive protein level, proteinuria level, type I interferon level, vascular inflammation, kidney involvement, lung involvement, and heart involvement.

12. A method for treating a disease caused by excessive proliferation of B cells producing autoantibodies, the method comprising administering the composition according to any one of claims 1-10.

13. The method according to claim 12, characterized in that, Diseases caused by excessive B cell proliferation producing autoantibodies include one or more of the following: systemic lupus erythematosus, myositis, myasthenia gravis, multiple sclerosis, Sjögren's syndrome, neuromyelitis optica, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antisynthetic enzyme syndrome, antineutrophil-associated vasculitis, sensitized kidney transplantation, autoimmune encephalitis, immune-associated necrotizing myositis, refractory rheumatoid arthritis, systemic sclerosis, stiff-person syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, Lambert-Eton myasthenia gravis, and DAGLA encephalitis. Preferably, the disease caused by the excessive proliferation of B cells producing autoantibodies is myositis, systemic lupus erythematosus, systemic sclerosis, neuromyelitis optica, or myasthenia gravis; more preferably, it is systemic lupus erythematosus.

14. A method for targeted delivery of mRNA to a subject's T cells, characterized in that, The method includes the step of contacting the composition of any one of claims 1-10 with subject T cells; the mRNA can generate a chimeric antigen receptor within the T cells.

15. A method for expressing chimeric antigen receptor T cells in subject T cells, characterized in that, The method includes the step of contacting the composition of any one of claims 1-10 with subject T cells; the mRNA can generate a chimeric antigen receptor within the T cells.

16. A pharmaceutical composition, characterized in that, Includes the composition according to any one of claims 1-10 and a pharmaceutically acceptable carrier.

17. An antibody that recognizes a T cell surface antigen, characterized in that, The CDR1, CDR2, and CDR3 amino acid sequences are shown in any of the following groups: (1) SEQ ID NO: 114; SEQ ID NO: 160; SEQ ID NO: 206; (2) SEQ ID NO: 115; SEQ ID NO: 161; SEQ ID NO: 207; (3) SEQ ID NO: 116; SEQ ID NO: 162; SEQ ID NO: 208; (4) SEQ ID NO: 117; SEQ ID NO: 163; SEQ ID NO: 209; (5) SEQ ID NO: 118; SEQ ID NO: 164; SEQ ID NO: 210; (6) SEQ ID NO: 119; SEQ ID NO: 165; SEQ ID NO: 211; (7) SEQ ID NO: 120; SEQ ID NO: 166; SEQ ID NO: 212; (8) SEQ ID NO: 121; SEQ ID NO: 167; SEQ ID NO: 213; (9) SEQ ID NO: 122; SEQ ID NO: 168; SEQ ID NO: 214; (10) SEQ ID NO: 123; SEQ ID NO: 169; SEQ ID NO: 215; (11) SEQ ID NO: 124; SEQ ID NO: 170; SEQ ID NO: 216; (12) SEQ ID NO: 125; SEQ ID NO: 171; SEQ ID NO: 217; (13)SEQ ID NO:126;SEQ ID NO:172;SEQ ID NO:218; (14)SEQ ID NO:127;SEQ ID NO:173;SEQ ID NO:219; (15)SEQ ID NO:128;SEQ ID NO:174;SEQ ID NO:220; (16)SEQ ID NO:129;SEQ ID NO:175;SEQ ID NO:221; (17)SEQ ID NO:130;SEQ ID NO:176;SEQ ID NO:222; (18)SEQ ID NO:131;SEQ ID NO:177;SEQ ID NO:223; (19)SEQ ID NO:132;SEQ ID NO:178;SEQ ID NO:224; (20)SEQ ID NO:133;SEQ ID NO:179;SEQ ID NO:225; (21)SEQ ID NO:134;SEQ ID NO:180;SEQ ID NO:226; (22)SEQ ID NO:135;SEQ ID NO:181;SEQ ID NO:227; (23)SEQ ID NO:136;SEQ ID NO:182;SEQ ID NO:228; (24)SEQ ID NO:137;SEQ ID NO:183;SEQ ID NO:229; (25)SEQ ID NO:138;SEQ ID NO:184;SEQ ID NO:230; (26)SEQ ID NO:139;SEQ ID NO:185;SEQ ID NO:231; (27)SEQ ID NO:140;SEQ ID NO:186;SEQ ID NO:232; (28)SEQ ID NO:141;SEQ ID NO:187;SEQ ID NO:233; (29)SEQ ID NO:142;SEQ ID NO:188;SEQ ID NO:234; (30)SEQ ID NO:143;SEQ ID NO:189;SEQ ID NO:235; (31)SEQ ID NO:144;SEQ ID NO:190;SEQ ID NO:236; (32)SEQ ID NO:145;SEQ ID NO:191;SEQ ID NO:237; (33)SEQ ID NO:146;SEQ ID NO:192;SEQ ID NO:238; (34)SEQ ID NO:147;SEQ ID NO:193;SEQ ID NO:239; (35)SEQ ID NO:148;SEQ ID NO:194;SEQ ID NO:240; (36)SEQ ID NO:149;SEQ ID NO:195;SEQ ID NO:241; (37)SEQ ID NO:150;SEQ ID NO:196;SEQ ID NO:242; (38)SEQ ID NO:151;SEQ ID NO:197;SEQ ID NO:243; (39)SEQ ID NO:152;SEQ ID NO:198;SEQ ID NO:244; (40)SEQ ID NO:153;SEQ ID NO:199;SEQ ID NO:245; (41)SEQ ID NO:154;SEQ ID NO:200;SEQ ID NO:246; (42)SEQ ID NO:155;SEQ ID NO:201;SEQ ID NO:247; (43)SEQ ID NO:156;SEQ ID NO:202;SEQ ID NO:248; (44)SEQ ID NO:157;SEQ ID NO:203;SEQ ID NO:249; (45)SEQ ID NO:158;SEQ ID NO:204;SEQ ID NO:250; (46)SEQ ID NO:159;SEQ ID NO:205;SEQ ID NO:251; (47)SEQ ID NO:252;SEQ ID NO:275;SEQ ID NO:298; (48)SEQ ID NO:253;SEQ ID NO:276;SEQ ID NO:299; (49)SEQ ID NO:254;SEQ ID NO:277;SEQ ID NO:300; (50)SEQ ID NO:255;SEQ ID NO:278;SEQ ID NO:301; (51)SEQ ID NO:256;SEQ ID NO:279;SEQ ID NO:302; (52)SEQ ID NO:257;SEQ ID NO:280;SEQ ID NO:303; (53)SEQ ID NO:258;SEQ ID NO:281;SEQ ID NO:304; (54)SEQ ID NO:259;SEQ ID NO:282;SEQ ID NO:305; (55) SEQ ID NO: 260; SEQ ID NO: 283; SEQ ID NO: 306; (56) SEQ ID NO: 261; SEQ ID NO: 284; SEQ ID NO: 307; (57) SEQ ID NO: 262; SEQ ID NO: 285; SEQ ID NO: 308; (58) SEQ ID NO: 263; SEQ ID NO: 286; SEQ ID NO: 309; (59) SEQ ID NO: 264; SEQ ID NO: 287; SEQ ID NO: 310; (60) SEQ ID NO: 265; SEQ ID NO: 288; SEQ ID NO: 311; (61) SEQ ID NO: 266; SEQ ID NO: 289; SEQ ID NO: 312; (62) SEQ ID NO: 267; SEQ ID NO: 290; SEQ ID NO: 313; (63) SEQ ID NO: 268; SEQ ID NO: 291; SEQ ID NO: 314; (64) SEQ ID NO: 269; SEQ ID NO: 292; SEQ ID NO: 315; (65) SEQ ID NO: 270; SEQ ID NO: 293; SEQ ID NO: 316; (66) SEQ ID NO: 271; SEQ ID NO: 294; SEQ ID NO: 317; (67) SEQ ID NO: 272; SEQ ID NO: 295; SEQ ID NO: 318; (68) SEQ ID NO: 273; SEQ ID NO: 296; SEQ ID NO: 319; (69) SEQ ID NO: 274; SEQ ID NO: 297; SEQ ID NO: 320; Preferably, the amino acid sequence of the antibody recognizing T cell surface antigen is as shown in any one of SEQ ID NO:1-46, 85-107, 331-355 or as shown in any one of SEQ ID NO:1-46, 85-107, 331-355, having ≥80% homology with the sequence shown in any one of SEQ ID NO:1-46, 85-107, 331-355; Preferably, the surface antigen of the T cells corresponding to SEQ ID NO:1-20, SEQ ID NO:44-45, SEQ ID NO:85-107 or SEQ ID NO:331-355 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:21-43 or SEQ ID NO:46 is CD5; More preferably, the surface antigen of the T cells corresponding to SEQ ID NO:98-105 is CD8, and the surface antigen of the T cells corresponding to SEQ ID NO:46 is CD5.

18. A peptide chain, characterized in that, Obtained by the mRNA encoded by any one of SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419 or SEQ ID NO:457-461; Alternatively, it may be obtained by encoding an mRNA as shown by a sequence having ≥80% homology to any of the sequences shown in SEQ ID NO:47-65, SEQ ID NO:324-326, SEQ ID NO:356-357, SEQ ID NO:364-365, SEQ ID NO:370-372, SEQ ID NO:379-381, SEQ ID NO:388-390, SEQ ID NO:397-399, SEQ ID NO:406-407, SEQ ID NO:412-413, SEQ ID NO:418-419, or SEQ ID NO:457-461; Preferably, the mRNA is encoded by any one of the mRNAs shown in SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471; Alternatively, it may be obtained by encoding an mRNA as shown by a sequence having ≥80% homology to any of the sequences shown in SEQ ID NO:66-84, SEQ ID NO:321-323, SEQ ID NO:327-330, SEQ ID NO:358-363, SEQ ID NO:366-369, SEQ ID NO:373-378, SEQ ID NO:382-387, SEQ ID NO:391-396, SEQ ID NO:400-405, SEQ ID NO:408-411, SEQ ID NO:414-417, SEQ ID NO:420-423, SEQ ID NO:454-456, or SEQ ID NO:462-471; Preferably, as shown in any one of SEQ ID NO:424-426, SEQ ID NO:431-433, SEQ ID NO:443-444; or as shown in any one of SEQ ID NO:429-430, SEQ ID NO:440, SEQ ID NO:451-453; or as shown in any one of SEQ ID NO:427-428, SEQ ID NO:434-437; or as shown in any one of SEQ ID NO:441-442; or as shown in any one of SEQ ID NO:438-439, SEQ ID NO:445-450, SEQ ID NO:472-476.

Images