Novel gene engineering modified three-target chimeric autoantibody receptor and application thereof

Abstract

The invention belongs to the technical field of autoimmune skin disease immunotherapy, and provides a three-target novel chimeric autoantibody receptor structure which comprises three chimeric receptors through two connecting peptides, specifically, a first chimeric autoantibody receptor comprises a Dsg1 extracellular structural domain; the second chimeric autoantibody receptor comprises a Dsg3 extracellular structural domain; the third chimeric antigen receptor comprises an extracellular domain capable of targeting a B cell associated antigen; wherein the Dsg1 extracellular structural domain is an extracellular structural domain of desmosome core glycoprotein 1; the Dsg3 extracellular structural domain is the extracellular structural domain of desmosome core glycoprotein 3. In-vitro experiments and animal experiments verify that the novel three-target CAAR-T cells can significantly increase the release of cytokines, enhance the targeted killing activity of the cytokines, and show better multiplication capacity; a novel cellular immunotherapy technology is provided for treating autoimmune skin diseases, and the application of CAAR-T in the field of autoimmune disease treatment can be expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy for autoimmune skin diseases, and specifically relates to a novel genetically engineered three-target chimeric autoantibody receptor and a use thereof. Background Art

[0002] Pemphigus is an autoimmune skin disease that affects the skin and mucous membranes and is characterized by widespread blisters. The clinical manifestations are severe, recurrent loose blisters and bullae, accompanied by intractable, painful mucosal erosions and ulcers, and characteristic acantholysis in histopathology. The international standard first-line treatment for pemphigus is a combination of systemic corticosteroids and immunosuppressants (such as azathioprine or mycophenolate mofetil (MMF)), as well as short-term corticosteroids combined with anti-CD20 monoclonal antibodies (such as rituximab, veltuzumab, etc.). However, the current treatment methods only relieve symptoms and cannot effectively control disease progression and prolong patient survival. The disease is prone to recurrence and drug resistance, and patients are often accompanied by adverse reactions such as increased risk of infection, diabetes, hypertension, abnormal lipid metabolism, and osteonecrosis.

[0003] The pathogenesis of pemphigus is not yet fully understood. It is generally believed to be an autoimmune disease. Under the stimulation of viral infection, ultraviolet radiation, drugs and other factors, the adhesion proteins between the spinous cell layers become autoantigens, inducing autoimmune reactions and activating B lymphocytes to produce antibodies, usually IgG type antibodies against two major adhesion proteins, namely Desmoglein 3 (Dsg3) and Desmoglein 1 (Dsg1); autoantibodies bind to the adhesion antigens of epidermal cells to produce immune responses, leading to loosening of the spinous layer and separation of keratinocytes from each other, thus forming intraepidermal blisters. Among them, the persistent production of autoantibodies by activated B lymphocytes against Dsg1 and Dsg3 is the main cause of pemphigus. Therapy that targets CD20 monoclonal antibodies to deplete B cells can lead to short-term disease remission in 95% of pemphigus patients, but 81% of patients may have relapses and fatal infections. Relapsed patients showed the same specific anti-Dsg1 and Dsg3 B lymphocyte clones as those observed in the active phase of the disease, and disease remission was closely associated with the clearance of anti-Dsg3 B cells in the blood. Therefore, we speculate that targeted elimination of specific anti-Dsg1 and Dsg3 memory B cells may cure pemphigus without causing systemic immunosuppression and reducing the risk of infection.

[0004] In recent years, chimeric antigen receptor T cell (CAR-T) therapy, as an emerging precision targeted cell therapy technology, has achieved unprecedented breakthrough efficacy in relapsed / refractory B-cell malignancies, and has completely changed the tumor treatment model. A number of products targeting CD19 and BCMA have been approved for marketing at home and abroad. Among them, the safety and efficacy of CD19 CAR-T in patients with systemic lupus erythematosus (SLE) have been preliminarily verified, opening the door to the clinical application of CAR-T cell therapy in autoimmune diseases.

[0005] Therefore, a new chimeric autoantibody receptor was developed that can enhance the specific cell lysis function and proliferation activity of CAAR-T cells, and will not cause widespread immunosuppression and hypoimmunoglobulinemia in pemphigus patients due to the elimination of overall B cells, providing a new strategy for the treatment of pemphigus, which is of great significance. Summary of the invention

[0006] The production of autoantibodies by B lymphocytes against desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) is the main cause of pemphigus. CD20-targeted B cell elimination therapy is prone to disease recurrence and may bring risks of widespread immunosuppression and fatal infection. In response to the above difficulties in the clinical treatment of pemphigus, the present invention innovatively provides a genetically engineered three-target chimeric autoantibody receptor structure and its host cell.

[0007] The main purpose of the present invention is to provide a new three-target chimeric autoantibody receptor structure and its application that can completely cure pemphigus, in view of the fact that the existing treatment methods for pemphigus have a short disease remission period, are prone to relapse, and may produce side effects such as immunosuppression and infection.

[0008] A novel genetically engineered triple-target chimeric autoantibody receptor (CAAR), characterized in that it comprises three chimeric receptors connected by two connecting peptides; specifically,

[0009] The first chimeric autoantibody receptor comprises a Dsg1 extracellular domain; the second chimeric autoantibody receptor comprises a Dsg3 extracellular domain; and the third chimeric antigen receptor comprises an extracellular domain capable of targeting a B cell-associated antigen;

[0010] Among them, the Dsg1 extracellular domain is the extracellular domain of desmoglein 1; the Dsg3 extracellular domain is the extracellular domain of desmoglein 3.

[0011] Among them, in the above-mentioned novel chimeric autoantibody receptor, the chimeric autoantibody receptor 1 has the structural characteristics of comprising an extracellular signal peptide, a Dsg1 extracellular structural region, an intracellular co-stimulatory signal and an intracellular activation signal.

[0012] The chimeric autoantibody receptor 2 is structurally characterized by comprising an extracellular signal peptide, a Dsg3 extracellular structural region, an intracellular co-stimulatory signal and an intracellular activation signal.

[0013] Among them, the chimeric antigen receptor 3 is structurally characterized by comprising an extracellular signal peptide, an antibody structural fragment that can specifically target B cell-associated antigens, and an intracellular co-stimulatory signal.

[0014] The Dsg1 extracellular structural region refers to the extracellular structural domain of desmoglein 1, and the amino acid sequence is shown in SEQ ID NO: 1, or a polypeptide sequence having 80%-99% similarity to the amino acid sequence.

[0015] .

[0016] Furthermore, the nucleotide sequence encoding the extracellular domain of Dsg1 is shown in SEQ ID NO: 2:

[0017]

[0018] Furthermore, the Dsg1 extracellular domain is preferably the cadherin-like subunit 1, 2, 3, 4 (EC1, EC2, EC3, EC4) region, i.e., amino acids 50-497 (EC1-4), the amino acid sequence of which is shown in SEQ ID NO: 3, or a polypeptide sequence having 80%-99% similarity to its amino acids.

[0019] EWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMAT FAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSS YTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVF RPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPN.

[0020] Furthermore, the coding nucleotide sequence of the Dsg1 extracellular domain EC1-4 is shown in SEQ ID NO: 4:

[0021]

[0022] Furthermore, the Dsg1 extracellular domain is preferably the cadherin-like subunit 1, 2, and 3 region, namely amino acids 50-385 (EC1-3), the amino acid sequence of which is shown in SEQ ID NO: 5, or a polypeptide sequence having 80%-99% similarity to its amino acids.

[0023] EWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGE IRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVF.

[0024] Furthermore, the coding nucleotide sequence of the Dsg1 extracellular domain EC1-3 is shown in SEQ ID NO: 6:

[0025]

[0026] Furthermore, in the extracellular domain of Dsg1, the cadherin-like subunits 1 (EC1), 2 (EC2), 3 (EC3) and 4 (EC4) regions, i.e., amino acids 50-158, 159-270, 271-385, 386-497, have amino acid sequences as shown in SEQ ID NOs: 7, 8, 9, 10, respectively, or polypeptide sequences having 80%-99% similarity to their amino acids.

[0027] SEQ ID NO: 7

[0028] EWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTG EINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFS.

[0029] SEQ ID NO: 8

[0030] MATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEI RTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYME.

[0031] SEQ ID NO: 9

[0032] QSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGI LKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVF.

[0033] SEQ ID NO: 10

[0034] RPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTG KLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPN.

[0035] Furthermore, the coding nucleotide sequences of the extracellular domains EC1, 2, 3, and 4 of Dsg1 are shown in SEQ ID NOs: 11, 12, 13, and 14, respectively:

[0036] SEQ ID NO: 11

[0037] GAATGGATTAAGTTTGCCGCCGCGTGCAGAGAAGGCGAGGATAATTCGAAGAGGAACCCTATAGCTAAGATTCATAGTGACTGTGCTGCTAATCAACAGGTTACGTACCGTATTAGTGGCGTAGGCATAGATCAGCCCCCCTACGGCATCTTTGTTATCAATCAAAAAACGGGGGAGATAAACATCACTTCAATAGTCGATAGAGAGGTAACGCCTTTCTTTATTATCTACTGTAGAGCCCTAAACTCCATGGGACAGGACCTCGAGCGTCCTCTAGAGCTGCGCGTTAGAGTGTTAGATATCAACGATAACCCCCCGGTTTTTTCC。

[0038] SEQ ID NO:12

[0039] ATGGCCACTTTTGCGGGCCAAATAGAGGAGAATTCCAATGCGAATACATTAGTAATGATCCTTAACGCAACTGACGCCGACGAGCCCAATAACCTCAACTCGAAGATAGCGTTCAAGATCATACGACAAGAGCCCTCAGATAGTCCAATGTTTATCATAAATCGCAACACAGGTGAAATCAGGACTATGAACAACTTCCTCGATAGGGAGCAATATGGACAATATGCACTCGCTGTGCGCGGGTCAGACCGGGACGGAGGAGCAGATGGGATGAGTGCGGAGTGTGAATGTAACATTAAGATACTAGACGTTAACGACAATATACCTTACATGGAG。

[0040] SEQ ID NO:13

[0041] CAAAGCTCTTATACTATCGAAATCCAGGAAAATACTTTGAACTCAAATTTGTTAGAGATAAGGGTCATTGATTTGGATGAAGAGTTCAGCGCGAACTGGATGGCCGTCATATTTTTCATTTCAGGGAACGAGGGAAATTGGTTTGAAATTGAGATGAATGAACGAACAAACGT TGGTATCCTAAAGGTGGTTAAACCACTGGACTATGAGGCCATGCAGAGTCTTCAGCTGTCTATAGGGGTACGGAACAAGGCTGAATTCCACCATTCGATCATGTCACAATACAAGTTAAAGGCGAGTGCAATTTCCGTCACGGTTCTGAATGTTATCGAAGGTCCGGTTTTC.

[0042] SEQ ID NO: 14

[0043] AGACCCGGGAGTAAGACCTATGTCGTGACGGGGAACATGGGTTCAAATGATAAAGTTGGCGATTTCGTAGCGACCGATCTGGACACAGGGCGCCCGTCTACGACGGTCAGATACGTAATGGGCAACAATCCCGCCGATCTCTTAGCTGTAGATTCGAGGACCGGTAAG CTCACATAAAGAACAAAGTGACGAAAGAGCAATACAACATGTTAGGAGGAAAATATCAGGGGGACTATACTCTCGATAGATGACAACTTGCAGCGAACATGCACCGGAACTATAAATATTAACATACAGTCGTTTGGCAACGACGACCGAACTAATACTGAGCCCAAT.

[0044] Furthermore, the Dsg1 extracellular domain is preferably at least one of the EC1, EC2, EC3 and EC4 regions.

[0045] The Dsg3 extracellular structural region refers to the extracellular structural domain of desmoglein 3, and the amino acid sequence is shown in SEQ ID NO: 15, or a polypeptide sequence having 80%-99% similarity to the amino acid sequence thereof.

[0046] MMGLFPRTTGALAIFVVVILVHGELRIETKGQYDEEEMTMQQAKRRQKREWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEKDAVCSSSPSVVVSARTLNNRYTGPYTFALEDQPVKLPAVWSITTLNATSALLRAQEQIPPGVYHISLVLTDSQNNRCEMPRSLTLEVCQCDNRGICGTSYPTTSPGTRYGRPHSGR。

[0047] Furthermore, the coding nucleotide sequence of the extracellular domain of Dsg3 is shown as SEQ ID NO: 16:

[0048]

[0049] Furthermore, the Dsg3 extracellular domain is preferably the cadherin-like subunit 1, 2, 3, 4 (EC1, EC2, EC3, EC4) region, i.e., amino acids 50-499 (EC1-4), the amino acid sequence of which is shown in SEQ ID NO: 17, or a polypeptide sequence having 80%-99% similarity to its amino acids.

[0050] EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQI FMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSA RIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPAS KTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEK.

[0051] Furthermore, the coding nucleotide sequence of Dsg3 extracellular domain EC1-4 is shown in SEQ ID NO: 18:

[0052]

[0053] Furthermore, the Dsg3 extracellular domain is preferably the cadherin-like subunit 1, 2, and 3 region, namely amino acids 50-383 (EC1-3), the amino acid sequence of which is shown in SEQ ID NO: 19, or a polypeptide sequence having 80%-99% similarity to its amino acids.

[0054] EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTG EVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAF.

[0055] Furthermore, the coding nucleotide sequence of the Dsg3 extracellular domain EC1-3 is shown in SEQ ID NO: 20:

[0056]

[0057] Furthermore, in the extracellular domain of Dsg3, the cadherin-like subunits 1 (EC1), 2 (EC2), 3 (EC3) and 4 (EC4) regions, i.e., amino acids 50-158, 159-268, 269-383, 386-499, have amino acid sequences as shown in SEQ ID NOs: 21, 22, 23, 24, respectively, or polypeptide sequences having 80%-99% similarity thereto.

[0058] SEQ ID NO: 21

[0059] EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFS.

[0060] SEQ ID NO: 22

[0061] QQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGE VRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFR.

[0062] SEQ ID NO: 23

[0063] DSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNE GILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAF.

[0064] SEQ ID NO: 24

[0065] ASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKT AEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEK.

[0066] Furthermore, the coding nucleotide sequences of Dsg3 extracellular domains EC1, 2, 3, and 4 are shown in SEQ ID NOs: 25, 26, 27, and 28, respectively:

[0067] SEQ ID NO:25

[0068] GAATGGGTTAAGTTTGCCAAACCCTGCAGAGAAGGTGAAGACAACTCCAAGCGCAACCCAATAGCCAAAATAACGTCTGATTACCAAGCGACGCAGAAGATTACTTATCGTATTTCAGGAGTGGGTATCGACCAGCCTCCCTTCGGAATCTTCGTAGTTGATAAAAATACGGGCGATATTAATATCACTGCTATAGTCGACCGAGAAGAGACGCCTTCGTTCCTAATAACCTGCAGGGCGCTGAACGCCCAGGGTCTGGACGTGGAAAAACCCTTGATTCTGACAGTAAAGATACTAGATATCAATGATAACCCTCCCGTCTTTTCA。

[0069] SEQ ID NO:26

[0070] CAGCAAATTTTCATGGGGGAGATTGAAGAGAACTCAGCAAGCAATTCGCTCGTAATGATCCTAAACGCAACGGATGCGGATGAGCCCAATCACTTGAATTCCAAAATTGCGTTCAAAATAGTCTCACAGGAACCGGCCGGTACCCCTATGTTCCTCCTATCTCGAAACACAGGGGAAGTGCGAACACTGACAAATTCTTTAGATAGAGAACAAGCCTCGTCCTACCGGCTAGTAGTGTCAGGAGCAGATAAAGACGGTGAAGGCTTGTCGACTCAATGTGAATGTAATATTAAAGTTAAGGATGTAAATGACAATTTTCCGATGTTCCGG。

[0071] SEQ ID NO:27

[0072] GATTCTCAATATTCTGCGCGTATCGAGGAGAATATCCTCAGCTCGGAACTGTTACGGTTCCAAGTGACTGACTTGGACGAGGAATACACAGATAACTGGTTAGCGGTCTATTTCTTCACGTCAGGTAATGAAGGTAATTGGTTCGAGATACAAACGGATCCGCGAACCAACGA GGGGATTCTTAAGGTAGTTAAGGCGCTTGATTATGAGCAACTTCAGAGTGTAAAACTCTCAATTGCAGTCAAGAACAAAGCAGAGTTTCATCAATCTGTCATATCAAGATATCGCGTGCAATCGACTCCGGTGACGATACAGGTCATAAACGTTAGGGAAGGGATAGCATTT.

[0073] SEQ ID NO: 28

[0074] GCGAGTAAAACTTTTACGGTGCAGAAGGGTATTAGCAGCAAAAAGCTTGTCGACTACATCCTAGGGACGTACCAAGCCATCGACGAGGACACGAACAAAGCGGCCTCCAATGTTAAGTATGTAATGGGTCGCAATGACGGCGGGTACCTGATGATCGATTCAAAAACCGCC GAGATCAAATTCGTGAAGAATATGAACCGGGATAGTACTTTCATTGTAAACAAGACGATAACTGCCGAGGTCCTGGCGATAGATGAGTATACTGGCAAGACTTCTACCGGTACTGTATACGTCCGGGTACCAGACTTCAACGACAATTGCCCTACTGCAGTGTTGGAAAAA.

[0075] Furthermore, the Dsg3 extracellular domain is preferably at least one of EC1, EC2, EC3 and EC4 regions.

[0076] Among them, the chimeric antigen receptor 3 can specifically target B cell-associated antigens. Furthermore, the B cell-associated antigens can be at least one of CD19, CD20, CD22, CD10, CD24, CD30, CD38, CD56, CD70, CD79, CD123, CD138, BCMA, FMC7, TACI, CS1 or other B cell antigens.

[0077] The extracellular domain specifically targeting B cell-associated antigens refers to an antibody region fragment capable of binding to the above-mentioned B cell-associated antigens, and further the antibody region fragment is at least one of a monoclonal antibody, a single chain antibody, Fab, scFv, VHH or other antibody fragments, and the preferred antibody region fragment is scFv or VHH.

[0078] The B cell-associated antigen is preferably at least one of CD19, CD20 or BCMA. Further, the antibody region fragment that specifically binds to the antigen CD19 is VHH, and the amino acid sequence is shown in SEQ ID NO: 29; the extracellular domain that binds to the antigen CD20 is scFv, and the amino acid sequence is shown in SEQ ID NO: 31; the extracellular domain that binds to the antigen BCMA is VHH, and the amino acid sequence is shown in SEQ ID NO: 33.

[0079] SEQ ID NO: 29 is:

[0080] EVQLVESGGGLVQPGGSLRLSCAASGFLFSDHAMSWVRQAPGKGLEWVSDISNGGF TTRYRDSVKGRFTISRDNAKNTLYLQLNSLKTEDTAMYYCARTEDQKRGQGTQVTVSS.

[0081] Furthermore, the encoding nucleotide sequence of the antibody fragment binding to the antigen CD19 is shown in SEQ ID NO: 30:

[0082] GAAGTTCAACTTGTGGAATCGGGCGGGGTTGGTACAGCCTGGGGGCTCTTTGCGACTCTCCTGTGCGGCTAGTGGCTTTCTCTTCCGACCATGCTATGTCGTGGGTGCGTCAAGCTCCAGGCAAAGGACTCGAGTGGGTATCGGATATCTCAAACGGAGGATTCACG ACGCGCTACCGGGACTCCGTAAAGGGACGTTTTACTATCTCCCGCGACAACGCAAAGAACACATTGTACCTCCAATTGAATAGCTTGAAGACAGAGGACACAGCGATGTACTATTGCGCGAGGACGGAGGATCAGAAGAGAGGGCAGGGGACACAAGTCACCGTGTCGTCC.

[0083] SEQ ID NO:31 is:

[0084] EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS GGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR.

[0085] Furthermore, the nucleotide sequence encoding the antibody fragment that binds to the antigen CD20 is shown in SEQ ID NO: 32:

[0086] GAAGTTCAGCTCGTTGAATCAGGTGGGGGTCTCGTTCAACCCGGAGGTTCCCTGCGATTATCCTGCGCTGCTTCGGGTTACACATTCACGAGTTATAACATGCATTGGGTCCGACAGGCACCGGGTAAAGGGCTGGAGTGGGTAGGGGCAATCTATCCTGGAAACGGAGACACTAGCTACAACCAAAAATTCAAGGGTCGTTTCACGATTAGCGTTGACAAATCTAAAAATACTCTATATCTTCAGATGAACTCTCTCCGAGCCGAGGATACGGCAGTGTATTACTGTGCCAGAGTCGTATATTATTCCAATTCTTATTGGTACTTTGACGTATGGGGCCAAGGTACACTTGTGACGGTAAGCAGCGGGGGGGGTGGATCGGGTGGAGGTGGATCAGGCGGCGGCGGAAGTGATATACAAATGACACAATCCCCATCGTCATTGTCCGCCTCCGTCGGGGACCGAGTCACCATAACGTGTAGGGCCTCTTCGTCGGTATCATACATGCACTGGTACCAGCAGAAGCCTGGCAAAGCTCCAAAACCTCTTATCTACGCACCTTCAAACTTAGCGAGCGGTGTTCCAAGCCGGTTTTCGGGATCAGGTTCGGGAACAGACTTTACACTTACCATAAGCTCTCTTCAACCAGAAGACTTTGCGACTTACTACTGTCAGCAATGGTCATTCAACCCCCCAACCTTCGGCCAGGGTACTAAGGTTGAAATCAAGAGA。

[0087] SEQ ID NO: 33 is as follows:

[0088] QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREAVSCISSSGGSTVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADKLYGPTWCGSEYDYWGQGTQVIVSS。

[0089] Furthermore, the coding nucleotide sequence of the antibody fragment that binds to antigen BCMA is shown as SEQ ID NO: 34:

[0090] CAGGTTCAACTGGTGGAATCAGGTGGCGGGTTGGTGCAGCCTGGCGGTTCGCTGCGATTGAGTTGCGCGGCGTCAGGATTCACCCTGGACTATTACGCAATAGGGTGGTTCAGGCAAGCTCCGGGTAAGGAACGAGAAGCGGTTTCATGCATTTCGAGCTCTGGGGGAAGCACGGTATATGCGGAT TCCGTTAAAGGTCGGTTTACGATATCTCGCGACAATGCGAAGAATACAGTATATTTGCAAATGAACAGCCTGAAACCCGAAGACACAGCTGTCTATTATTGTGCAGCAGACAAGTTGTATGGCCCCACATGGTGTGGATCTGAATACGATTACTGGGGCCAGGGAACGCAAGTAATCGTCTCATCA.

[0091] Wherein, the second intracellular co-stimulatory signal is at least one of CD28, 4-1BB, ICOS, OX40, CD27, CD30, CD40L, CD2, DAP10, DAP12, HVEM, DR3, SLAM, GITR or TIM-1. Preferably, the intracellular co-stimulatory signal is preferably CD28 or 4-1BB.

[0092] The first intracellular activation signal comprises an immunoreceptor tyrosine-based activation motif selected from at least one of CD3ζ, FcεRI, TREM1, TREM1, NKG2D, NKp30, or NKp44. Further, the immunoreceptor tyrosine-based activation motif signal is preferably CD3ζ.

[0093] Among them, the extracellular signal peptide is selected from CD8α, CD4, IL-2, CSF-2, TNF-α or IFN-γ signal guiding polypeptide, and further, CD8α signal peptide is preferred.

[0094] Wherein, the connecting peptide refers to a Furin protein or a 2A polypeptide having a self-cleavage function, wherein the 2A polypeptide is selected from T2A, P2A, E2A, and F2A, and further, T2A and P2A are preferred.

[0095] The above-mentioned novel triple-target chimeric autoantibody receptor is composed of: CD8α extracellular signal peptide, Dsg1 extracellular domain EC1-4, CD28 co-stimulatory signal, intracellular CD3ζ activation domain, T2A self-cleavage peptide, CD8α extracellular signal peptide, Dsg3 extracellular domain EC1-4, 4-1BB co-stimulatory signal, intracellular CD3ζ activation domain, P2A self-cleavage peptide, VHH specifically targeting CD19 or scFv targeting CD20 or VHH targeting BCMA, and 4-1BB co-stimulatory signal.

[0096] Furthermore, the novel chimeric autoantibody receptor structure of the present invention includes the following:

[0097] CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-CD19 VHH—4-1BB (Dsg1-Dsg3-CD19 CAAR)

[0098] CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-CD20 scFv—4-1BB (Dsg1-Dsg3-CD20 CAAR)

[0099] CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-BCMAVHH—4-1BB (Dsg1-Dsg3-BCMACAAR)

[0100] The amino acid sequence of CD8α signal peptide is shown in SEQ ID NO: 35:

[0101] MALPVTALLLPLALLLHAARP

[0102] Furthermore, the coding nucleotide sequence of the CD8α signal peptide is shown in SEQ ID NO: 36:

[0103] ATGGCACTGCCCGTGACCGCTTTTGCTCCTCCCCCTAGCACTTCTGCTACACGCGG CAAGACCT.

[0104] The amino acid sequence of the CD28 co-stimulatory signal is shown in SEQ ID NO: 37:

[0105] KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV TVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

[0106] Furthermore, the coding nucleotide sequence of CD28 is shown in SEQ ID NO: 38:

[0107] ATCGAGGTCATGTATCCCCCTCCTTACCTTGACAACGAAAAAAGTAATGGTACGATTATACATGTCAAGGGGAAGCATCTCTGCCCCTCTCCTTTGTTTCCCGGCCCTTCGAAGCCGTTCTGGGTGCTTGTCGTGGTTGGCGGGGTCTTGGCATGCTACAG CCTCCTGGTAACAGTGGCCTTTATCATATTTTGGGTTAGGAGTAAGCGGTCCCGCCTGCTACATAGCGACTATATGAACATGACACCGCGACGTCCGGGACCCACTAGAAAACACTATCAGCCTTACGCACCACCGCGTGACTTTGCTGCTTATAGATCC.

[0108] The amino acid sequence of the 4-1BB co-stimulatory signal is shown in SEQ ID NO: 39:

[0109] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0110] Furthermore, the nucleotide sequence encoding 4-1BB is shown in SEQ ID NO: 40:

[0111] AAGCCGGTCGCAAGAAGCTTCCTATATTTTCAAACAGCCGTTCATGAGGCCGG TCCAAACTACGCAGGAGGAAGACGGTTGCAGTTGCCGCTTTCCTGAAGAAGAGGAAG GTGGTTGTGAACTC.

[0112] The amino acid sequence of the CD3ζ activation domain is shown in SEQ ID NO: 41:

[0113] APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0114] Furthermore, the coding nucleotide sequence of CD3ζ is shown in SEQ ID NO: 42:

[0115] CGAGTCAAATTTAGCCGATCAGCGGATGCCCCGGCCTACCAGCAAGGACAGAACCAATTATACAACGAACTTAATCTCGGGCGGCGAGAAGAATATGACGTTCTTGACAAGCGACGAGGGAGGGATCCAGAGATGGGTGGAAAGCCAAGGAGGAAGAACCCTCAAGAA GGACTCTATAACGAGCTGCAGAAAGACAAAATGGCGGAGGCATATTCAGAAATTGGTATGAAGGGGGAACGTCGACGAGGCAAAGGTCACGATGGCTTGTATCAAGGTCTGAGTACTGCCACGAAGGATACTTACGACGCCCTTCACATGCAAGCGTTACCGCCTAGA.

[0116] The amino acid sequence of T2A is shown in SEQ ID NO: 43:

[0117] GSGEGRGSLLTCGDVEENPGP.

[0118] Furthermore, the coding nucleotide sequence of T2A is shown in SEQ ID NO: 44:

[0119] GGAAGTGGTGAAGGAAGGGGGTCCCTCCTCACGTGTGGCGACGTTGAAGAGAAT CCTGGTCCC.

[0120] The amino acid sequence of P2A is shown in SEQ ID NO: 45:

[0121] GSGATNFSLLKQAGDVEENPGP.

[0122] Furthermore, the coding nucleotide sequence of P2A is shown in SEQ ID NO: 46:

[0123] GGTAGTGGCGCGACAAACTTTTCTCTGCTAAAACAAGCGGGTGATGTTGAGGAG AACCCGGGTCCA.

[0124] The present invention provides a recombinant gene expression vector, which can stably express the novel three-target chimeric autoantibody receptor. Further, the expression vector is a eukaryotic or prokaryotic expression vector, the eukaryotic expression vector is a plasmid; the prokaryotic expression vector is a viral vector, the viral vector includes a retrovirus, a recombinant lentivirus, and a recombinant adenovirus; further, the viral vector is preferably a lentiviral vector pWPXLd.

[0125] The present invention provides a host cell expressing the novel chimeric autoantibody receptor. The host cell is preferably an immune cell; more preferably, an immune cell differentiated from a T cell, a NK cell, a monocyte, a macrophage, a dendritic cell, a neutrophil, a Treg cell, a mesenchymal stem cell, a hematopoietic stem cell, or a pluripotent stem cell; more preferably, a T cell or a NK cell.

[0126] The present invention provides a technical method for preparing the above-mentioned novel autoantibody receptor-modified T cells, comprising the separation of peripheral blood mononuclear cells, the sorting and activation of T lymphocytes, lentiviral transduction, and CAAR-T cell function verification.

[0127] The present invention also provides a use of the novel three-target chimeric autoantibody receptor, a recombinant vector containing the chimeric autoantibody receptor, and immune cells expressing the recombinant vector in the preparation of a drug for treating autoimmune skin diseases.

[0128] In the above use, the autoimmune skin disease is preferably at least one of pemphigus, systemic lupus erythematosus, scleroderma, pemphigoid, psoriasis, epidermolysis bullosa, dermatitis herpetiformis, linear IgA bullous dermatosis and alopecia areata.

[0129] The three-target novel chimeric autoantibody receptor structure design and the immune cells containing the novel chimeric autoantibody receptor described in the present invention are related to the application in the preparation of anti-autoimmune skin disease drugs and cell immunotherapy.

[0130] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0131] The present invention provides a novel chimeric autoantibody receptor structure with three targets, including three chimeric receptors formed by two connecting peptides; specifically, the first chimeric autoantibody receptor includes the Dsg1 extracellular domain; the second chimeric autoantibody receptor includes the Dsg3 extracellular domain; the third chimeric antigen receptor includes an extracellular domain capable of targeting B cell-related antigens; wherein the Dsg1 extracellular domain is the extracellular domain of desmoglein 1; and the Dsg3 extracellular domain is the extracellular domain of desmoglein 3. In vitro experiments and animal experiments have verified that the novel three-target CAAR-T cells can significantly increase the release of cytokines, enhance their targeted killing activity, and show better proliferation ability. Taking antigens CD19 and CD20 as examples, compared with the existing Dsg3 single-target CAAR structure, the novel CAAR structure of the invention significantly enhances the specific cell lysis ability and proliferation activity of T lymphocytes. A novel cell immunotherapy technology is provided for the treatment of autoimmune skin diseases, which can expand the application of CAAR-T in the field of autoimmune disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 This is a diagram of the interaction between T cells modified with a novel triple-target chimeric autoantibody receptor and pathogenic B cells.

[0133] Figure 2 It is a schematic diagram of the CAAR structure involved in the present invention.

[0134] Figure 3 To detect the positive rate of CAAR molecule expression on the surface of T cells by flow cytometry.

[0135] Figure 4 To detect the expression of Anti-Dsg1 scFv and Anti-Dsg3 scFv molecules on the surface of Raji cells by flow cytometry.

[0136] Figure 5 Flow cytometry was used to detect the expression of CD19 and CD20 molecules on the surface of Raji cells.

[0137] Figure 6 To detect the specific killing ratio of CAAR-T cells against Raji.

[0138] Figure 7 To compare the release of IFN-γ when CAAR-T cells kill Raji cells in vitro.

[0139] Figure 8 To compare the release of IL-2 when CAAR-T cells kill Raji cells in vitro.

[0140] Fig. 9These are the results of triple-target CAAR-T therapy in the Raji cell subcutaneous implantation model in mice. DETAILED DESCRIPTION

[0141] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0142] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0143] The present invention will be described in detail below through specific embodiments in conjunction with the accompanying drawings. In the following examples, all the specific experimental conditions and methods not specified are based on conventional conditions well known to those skilled in the art, such as the conditions described in Sambrook J, Russell DW, 2001, Molecular Cloning: A laboratory manual (3rd ed), Spring Harbor Laboratory Press, or the conditions recommended by the manufacturer. The reagents and consumables used in the following examples were purchased from qualified biochemical reagent sales merchants.

[0144] Example 1

[0145] Construction of a novel triple-target chimeric autoantibody receptor recombinant lentiviral vector

[0146] The novel three-target chimeric autoantibody receptor provided by the present invention is composed of a signal peptide-Dsg1 extracellular domain-intracellular co-stimulatory signal-intracellular activation signal-self-cleavage peptide-signal peptide-Dsg3 extracellular domain-intracellular co-stimulatory signal-intracellular activation signal-self-cleavage peptide-signal peptide-extracellular B cell antigen binding domain-co-stimulatory signal in series. During the transcription and translation process, two self-cleavage peptides are broken to form three independent CAAR structures that are expressed in parallel on the surface of immune cells ( Figure 1 In this example, four chimeric antigen receptors (CARs) were designed and constructed by targeting B cell-associated antigens CD19, CD20, and BCMA, respectively. Figure 2 ), the expression framework 5 ends to 3 ends are:

[0147] Dsg3 CAAR: CD8α signal peptide—Dsg3 EC1-4—CD28—CD3ζ;

[0148] Dsg1-Dsg3-CD19 CAAR: CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-CD19 VHH—4-1BB;

[0149] Dsg1-Dsg3-CD20 CAAR: CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-CD20 scFv—4-1BB;

[0150] Dsg1-Dsg3-BCMACAAR: CD8α signal peptide—Dsg1 EC1-4—CD28—CD3ζ—T2A—CD8α signal peptide—Dsg3 EC1-4—4-1BB—CD3ζ—P2A—CD8α signal peptide—anti-BCMA VHH—4-1BB.

[0151] The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 35; the nucleotide sequence encoding the CD8α signal peptide is shown in SEQ ID NO: 36.

[0152] The amino acid sequence of Dsg1 EC1-4 is shown in SEQ ID NO:3; the nucleotide sequence encoding Dsg1 EC1-4 is shown in SEQ ID NO:4.

[0153] The amino acid sequence of Dsg3 EC1-4 is shown in SEQ ID NO: 17; the nucleotide sequence encoding Dsg3 EC1-4 is shown in SEQ ID NO: 18.

[0154] The amino acid sequence of the CD28 costimulatory signal is shown in SEQ ID NO: 37; the nucleotide sequence encoding CD28 is shown in SEQ ID NO: 38.

[0155] The amino acid sequence of the 4-1BB co-stimulatory signal is shown in SEQ ID NO: 39; the nucleotide sequence encoding 4-1BB is shown in SEQ ID NO: 40.

[0156] The amino acid sequence of the CD3ζ activation domain is shown in SEQ ID NO:41; the nucleotide sequence encoding CD3ζ is shown in SEQ ID NO:42.

[0157] The amino acid sequence of the anti-CD19 VHH is shown in SEQ ID NO: 29; the nucleotide sequence encoding the anti-CD19 VHH is shown in SEQ ID NO: 30.

[0158] The amino acid sequence of the anti-CD20 scFv is shown in SEQ ID NO: 31; the nucleotide sequence encoding the anti-CD20 scFv is shown in SEQ ID NO: 32.

[0159] The amino acid sequence of the anti-BCMA VHH is shown in SEQ ID NO: 33; the nucleotide sequence encoding the anti-BCMA VHH is shown in SEQ ID NO: 34.

[0160] The amino acid sequence of the self-cleaving peptide T2A is shown in SEQ ID NO: 43, and the nucleotide sequence encoding T2A is shown in SEQ ID NO: 44. The amino acid sequence of P2A is shown in SEQ ID NO: 45, and the nucleotide sequence encoding P2A is shown in SEQ ID NO: 46.

[0161] The nucleotide sequence of the chimeric autoantibody receptor was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and inserted into the lentiviral vector pWPXLd (Addgene) through the 5' end restriction site BamHI and the 3' end restriction site EcoRI to form a recombinant gene vector containing the CAAR sequence; the recombinant vector was then transformed into Escherichia coli competent cells (Stbl3), plated, and cultured at 37°C for 12-18h, and then a single clone colony was picked. After the gene sequencing was correct, the single clone colony continued to be cultured in a shaking incubator at 37°C for 12-18h, and the plasmid was extracted and purified using Qiagen's plasmid purification kit. The specific purification steps refer to the kit manual to obtain the recombinant expression vector plasmid.

[0162] Example 2

[0163] Transformation of T cells with recombinant expression vector

[0164] 1.293T cell recovery and culture:

[0165] The frozen 293T cells were taken out from liquid nitrogen, and quickly placed in a 37°C water bath to shake and thaw. The completely dissolved 293T frozen suspension was then added to 10 mL of high-glucose DMEM medium (containing 10% fetal bovine serum and 100 U / ml penicillin and streptomycin), centrifuged and the supernatant was discarded. The cell pellet was then resuspended in 10 mL of complete high-glucose DMEM medium, and cultured in an incubator at 37°C and 5% CO2. The status of the 293T cells was observed regularly. When the cell density grows to 80%-90%, subculture is carried out, the culture medium in the culture dish is discarded, 1mL of sterile PBS is added to wash the remaining culture medium, and then 1-2mL of trypsin is dripped into the culture dish. When the cells are digested, 6-8mL of fresh complete culture medium is added to the culture dish to neutralize the trypsin, centrifuge and discard the supernatant, and then use high-glucose DMEM culture medium (containing 10% fetal bovine serum and 100U / ml penicillin and streptomycin) to resuspend the cell pellet and divide it into other culture dishes (it is generally recommended to subculture 3-5 dishes in 1 dish), and finally put it into the incubator for continued culture, and observe the cell status and density regularly.

[0166] 2. Preparation of lentivirus:

[0167] (1) Production of lentiviral stock solution:

[0168] Day 1: Plate 293T cells in the logarithmic growth phase and culture for 24 hours until the cell density is about 70-80%.

[0169] Day 2: Plasmid transfection. The plasmid dosage per 10cm dish is 6μg of pSPAX2 (packaging plasmid), 3μg of pMD.2G (envelope plasmid), and 12μg of pWPXLd (lentivirus vector). Use Highgene transfection reagent to bring the plasmid mixture into 293T cells. For specific experimental operation steps, refer to the Highgene transfection reagent manual; 4-6h after transfection, half of the medium is replaced with fresh complete medium.

[0170] Day 4: 48 hours after transfection, collect the viral supernatant, filter it with a 0.45μm filter, and store it at 4°C (can be stored for 1 week). Add 10mL of complete culture medium to the culture dish.

[0171] Day 5: 72 hours after transfection, continue to collect the viral supernatant, filter it with a 0.45μm filter, and store it at 4°C (can be stored for 1 week).

[0172] (2) Preparation of lentiviral concentrate:

[0173] Place the filtered virus stock solution in a Beckman ultracentrifuge tube, ensuring that each centrifuge tube is strictly balanced with an error of no more than ±0.001g; centrifugation conditions: 4°C, 70,000g, 2h; after centrifugation, discard the supernatant, invert the centrifuge tube on sterile filter paper to absorb the remaining supernatant. Resuspend the virus precipitate in serum-free and antibiotic-free DMEM medium, aliquot the resuspended virus concentrate, and store in a -80°C refrigerator.

[0174] 3. Isolation of human peripheral blood mononuclear cells and T cell activation:

[0175] Draw 15-20mL of peripheral venous blood from healthy volunteers, and slowly drip the blood onto the surface of FICOLL lymphocyte separation fluid to ensure that the ratio of lymphocyte separation fluid to blood is 1:1. Then centrifuge at 1000g, 30min, 18℃, acceleration / deceleration of 1. After centrifugation, the mixture is divided into 3 layers, and the lymphocytes are in the middle white film layer. Slowly draw the middle white film layer with the tip of the gun, add it to 20mL of serum-free and antibiotic-free X-VIVO15 culture medium, centrifuge, pour out the supernatant, resuspend the cell pellet with 10mL of sterile lysis solution, lysis time 2-3min, then centrifuge and discard the supernatant. The mononuclear cell pellet was resuspended in 4 mL of X-VIVO15 medium containing 5% fetal bovine serum and IL-2 (100 IU / mL), and the cells were counted. CD3 / CD28 Dynabeads immune stimulatory magnetic beads (Invitrogen) were added to activate T cells according to the number of cells. The usage and dosage of the magnetic beads were carried out according to the instructions. Finally, the monocytes were cultured in an incubator for 2 days to fully activate the T cells.

[0176] 4. Detection of virus concentrate infection of T cells and surface CAAR molecule expression:

[0177] One day before virus infection of T cells, 12-well plates were coated with fibronectin (RetroNectin) diluent (diluted with sterile PBS, concentration of 50 μg / mL), 1 mL per well, and sealed overnight at 4°C. On the day of infection, the RetroNectin diluent was removed, and 2% BSA solution was used to block for 30 min, and BSA was removed and washed once with PBS. 1×10 6 The activated T cells were mixed with an appropriate amount of virus concentrate, slowly adhered to the 12-well plate, centrifuged at 32°C, 1000g for 2h, and then cultured in a 37°C, 5% CO2 cell culture incubator. Fresh culture medium was completely replaced after 24h. After 48h of continuous culture, the expression of CAAR molecules on the surface of T lymphocytes was detected by flow cytometry ( Figure 3 ). When lentivirus is used for infection, the MOI value is generally controlled between 4 and 30.

[0178] 5. Cultivation of CAAR-T cells:

[0179] After 24 h of virus infection, the T cells were replaced with fresh complete culture medium (X-VIVO15, containing 5% fetal bovine serum and 100 IU / mL IL-2). At this time, the density of CAAR-T cells was adjusted to 5×10 5 / mL, subculture for 48h, and keep the cell density at 1×10 6 / mL is conducive to the rapid growth of CAAR-T. T cell complete medium is best prepared and used immediately, and can be stored at 4°C for up to 1 week. The recommended centrifugation conditions for T cells are 300g / min at room temperature for 5 minutes.

[0180] Example 3

[0181] Construction of target cell Raji cell stable strain and antigen detection

[0182] As in Example 1, Anti-Dsg1 scFv and Anti-Dsg3 scFv lentivirus were packaged, wherein the scFv sequence was provided by Chengdu Shengshi Junlian Biotechnology Co., Ltd. Raji cells were revived and cultured according to conventional techniques. When the cells were in the logarithmic growth phase, 1×10 6 Raji cells were centrifuged to remove the culture medium, and the cells were resuspended with Anti-Dsg1 scFv and Anti-Dsg3 scFv lentiviral stock solutions, respectively, and placed in a 12-well plate. The cells were centrifuged at 32°C, 1000g for 2 hours, and then cultured in a 37°C, 5% CO2 cell culture incubator. After 24 hours, the RPMI 1640 culture medium was completely replaced with fresh medium. After continuing to culture for 48 hours, the expression of Anti-Dsg1 scFv, Anti-Dsg3 scFv, CD19 and CD20 molecules on the surface of Raji cells were detected by flow cytometry ( Figure 4 and Figure 5 ).

[0183] Example 4

[0184] Detection of the killing activity of CAAR-T cells in vitro

[0185] Cell Trace for flow cytometry validation TM CFSE Cell Proliferation Kit (Thermo) and CellTrace TM Far Red Cell Proliferation Kit (Thermo) was used to stain CAAR-T cells and Raji cells, respectively. The staining steps were carried out according to the instructions. CAAR-T cells and Raji cells were added to a 12-well plate at an effector-target ratio of 1:1 and 2:1. The number of Raji cells in each well was 1×10 6, add control wells with only CAAR-T cells or Raji cells. By flow cytometry, select FITC channel for CFSE and APC channel for Far Red to observe the death or proliferation of Raji cells to reflect the in vitro killing ability of CAAR-T ( Figure 6 ). Figure 6 The results showed that the new triple-target Dsg1-Dsg3-CD19 CAAR-T and Dsg1-Dsg3-CD20 CAAR-T cells had significantly better killing abilities against Raji cells than the single-target Dsg3 CAAR-T, demonstrating the advantages of the present invention.

[0186] Example 5

[0187] Determination of IFN-γ and IL-2 secretion when CAAR-T cells were co-cultured with target cells

[0188] CAAR-T cells and Raji cells were mixed in a 96-well plate at an effector-target ratio of 1:1 and 2:1, with 5000 Raji cells, and resuspended in X-VIVO15 medium without cytokines and co-cultured for 24 hours; the supernatant was centrifuged and the release of cytokines was detected by ELISA. In this example, Human IFN-γ / IL-2 ELISA Kit (Biolegend) was used, and the specific operation was carried out according to the instructions.

[0189] On the first day, remove the ELISA plate, add 100 μL of coating solution containing the coating antibody to each well, and place in a 4°C refrigerator overnight. On the second day, add 300 μL of cleaning solution / well and wash 4 times with shaking; add 200 μL of 1X ELISA / ELISPOT solution to each well and block at room temperature for 1 hour; wash 4 times under the above cleaning conditions; then add the sample to be tested and the standard, 100 μL / well, set up 3 replicate wells, and incubate in a 4°C refrigerator overnight. On the third day, the cells were washed 4 times under the above-mentioned washing conditions, and 100 μL / well of detection antibody was added, and the cells were incubated at room temperature for 1 hour. The cells were washed 4 more times, and 100 μL / well of Avidin-HRP secondary antibody was added, and the cells were incubated at room temperature for 30 minutes. The cells were washed 6 times, and 100 μL of TMB colorimetric solution was added to each well. After incubation at room temperature for 20 minutes, 50 μL of stop solution was added to each well. The cells were tested on the microplate reader at a wavelength of 450 nm as the detection absorbance value, and 570 nm as the reference wavelength. The absorbance value at 450 nm was subtracted from the absorbance value at 570 nm. The data were obtained for statistical analysis.

[0190] Result judgment: The result is valid only when the value of the duplicate well is within the difference range of ±20%; the OD value of each standard or sample should be subtracted from the OD value of the background correction well; draw the standard curve manually. Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis, and connect the coordinate points of each standard with a smooth line. The concentration of the sample to be tested can be calculated on the standard curve through the OD value of the sample; if the OD value of the sample is higher than the upper limit of the standard curve, it should be appropriately diluted and re-tested, and the concentration should be multiplied by the dilution factor ( Figure 7 and Figure 8 ). Figure 7 and Figure 8 The data showed that the novel triple-target CAAR-T cells provided by the present invention released higher levels of IFN-γ and IL-2 when co-incubated with Raji cells, indicating that the novel optimized CAAR-T cells have stronger in vitro killing ability.

[0191] Example 6

[0192] A novel triple-target CAAR-T cell therapy for subcutaneous implantation and metastasis of human Raji cells in mice

[0193] Female NOD.Cg-PrkdcscidIl2rgtmWjl / SzJ (NSG) mice aged 8-10 weeks were purchased from Biocytogen Biotech and injected subcutaneously with 1×10 7 Raji cells, 7 days later when the graft volume reaches 50mm 3 At 14:00, the cells were divided into groups and injected with 5×10 6 Different designs of CAAR-T cells. The changes in graft size were recorded regularly after CAAR-T injection ( Fig. 9 ) to observe the control of Raji grafts by different designs of CAAR-T in animals. Fig. 9 The results clearly show that the efficacy of the new triple-target Dsg1-Dsg3-CD19 CAAR-T and Dsg1-Dsg3-CD20 CAAR-T on subcutaneously implanted metastatic tumors in Raji mice is significantly better than that of the single-target Dsg3 CAAR-T cells.

[0194] From the results of the above embodiments, it can be seen that:

[0195] The novel three-target chimeric autoantibody receptor structure provided by the present invention can be stably expressed on the surface of T lymphocytes; and through this three-target CAAR-T cell, not only can the release of cytokines IFN-γ and IL-2 be increased, but also the activity of lysing target cells is significantly enhanced. The present invention constructs a novel chimeric autoantibody receptor structure containing three targets, and verifies its efficient killing effect in both in vitro and animal experiments. It has obvious advantages over the existing chimeric autoantibody receptor technology, and provides a novel cell immunotherapy technology for the treatment of autoimmune skin diseases.

[0196] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel genetically engineered three-target chimeric autoantibody receptor, characterized in that: It comprises three chimeric receptors connected by two connecting peptides; specifically, The first chimeric autoantibody receptor comprises a Dsg1 extracellular domain; the second chimeric autoantibody receptor comprises a Dsg3 extracellular domain; and the third chimeric antigen receptor comprises an extracellular domain capable of targeting a B cell-associated antigen; Among them, the Dsg1 extracellular domain is the extracellular domain of desmoglein 1; the Dsg3 extracellular domain is the extracellular domain of desmoglein 3.

2. The novel genetically engineered triple-target chimeric autoantibody receptor according to claim 1, characterized in that: The Dsg1 extracellular domain includes at least one of Dsg1 EC1, Dsg1 EC2, Dsg1 EC3 and Dsg1 EC4; and / or the Dsg3 extracellular domain includes at least one of Dsg3 EC1, Dsg3 EC2, Dsg3 EC3 and Dsg3 EC4.

3. The novel genetically engineered three-target chimeric autoantibody receptor according to claim 2, characterized in that: The amino acid sequence of Dsg1 EC1 is shown in SEQ ID NO: 7; the amino acid sequence of Dsg1 EC2 is shown in SEQ ID NO: 8; the amino acid sequence of Dsg1 EC3 is shown in SEQ ID NO: 9; the amino acid sequence of Dsg1 EC4 is shown in SEQ ID NO: 10; and / or, The nucleotide sequence encoding Dsg1 EC1 is shown in SEQ ID NO: 11; the nucleotide sequence encoding Dsg1 EC2 is shown in SEQ ID NO: 12; the nucleotide sequence encoding Dsg1 EC3 is shown in SEQ ID NO: 13; the nucleotide sequence encoding Dsg1 EC4 is shown in SEQ ID NO: 14; and / or, The amino acid sequence of Dsg3 EC1 is shown in SEQ ID NO: 21; The amino acid sequence of Dsg3 EC2 is shown in SEQ ID NO: 22; The amino acid sequence of Dsg3 EC3 is shown in SEQ ID NO: 23; The amino acid sequence of Dsg3 EC4 is shown in SEQ ID NO: 24; and / or, The nucleotide sequence encoding Dsg3 EC1 is shown in SEQ ID NO: 25; The nucleotide sequence encoding Dsg3 EC2 is shown in SEQ ID NO: 26; The nucleotide sequence encoding Dsg3 EC3 is shown in SEQ ID NO: 27; The nucleotide sequence encoding Dsg3 EC4 is shown in SEQ ID NO:

28.

4. The novel genetically engineered three-target chimeric autoantibody receptor according to claim 1, characterized in that: The extracellular domain targeting a B cell-associated antigen includes at least one of a single-chain antibody, Fab, scFv, and VHH that can bind to a B cell-associated surface antigen; and / or the B cell-associated surface antigen is at least one of CD19, CD20, CD22, CD10, CD24, CD30, CD38, CD56, CD70, CD79, CD123, CD138, BCMA, FMC7, TACI, and CS1.

5. The novel genetically engineered triple-target chimeric autoantibody receptor according to any one of claims 1 to 4, characterized in that: The first chimeric autoantibody receptor includes a first extracellular signal peptide, a Dsg1 extracellular domain, a first intracellular second co-stimulatory signal, and a first intracellular first activation signal connected in series; The second chimeric autoantibody receptor comprises: a second extracellular signal peptide, a Dsg3 extracellular domain, a second intracellular second co-stimulatory signal, and a second intracellular first activation signal; The third chimeric antigen receptor includes: a third extracellular signal peptide, an extracellular domain capable of targeting a B cell-associated antigen, and a second co-stimulatory signal within the third cell.

6. The novel genetically engineered three-target chimeric autoantibody receptor according to claim 5, characterized in that: The first extracellular signal peptide, the second extracellular signal peptide and the third extracellular signal peptide are CD8α signal peptides, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 35; the nucleotide sequence encoding the CD8α signal peptide is shown in SEQ ID NO: 36; and / or, The second co-stimulatory signal in the first cell is a CD28 co-stimulatory molecule, the amino acid sequence of the CD28 co-stimulatory molecule is shown in SEQ ID NO: 37; the nucleotide sequence encoding the CD28 co-stimulatory molecule is shown in SEQ ID NO: 38; The second co-stimulatory signal in the second cell and the second co-stimulatory signal in the third cell are 4-1BB co-stimulatory molecules, the amino acid sequence of the 4-1BB co-stimulatory molecule is shown in SEQ ID NO: 39; the nucleotide sequence encoding the 4-1BB co-stimulatory molecule is shown in SEQ ID NO: 40; and / or, The first intracellular first activation signal and the second intracellular first activation signal are CD3ζ, the amino acid sequence of CD3ζ is shown in SEQ ID NO:41; the nucleotide sequence encoding CD3ζ is shown in SEQ ID NO:

42.

7. A recombinant vector, characterized in that: The novel genetically engineered triple-target chimeric autoantibody receptor described in any one of claims 1 to 6 can be expressed.

8. An immune cell, characterized in that An immune cell modified by the novel genetically engineered triple-target chimeric autoantibody receptor as described in any one of claims 1-6, wherein the immune cell is at least one of T cells, NK cells, monocytes, macrophages, dendritic cells, neutrophils, Treg cells, mesenchymal stem cells, hematopoietic stem cells, and immune cells differentiated from pluripotent stem cells.

9. Use of the novel genetically engineered three-target chimeric autoantibody receptor according to any one of claims 1 to 6, the recombinant vector according to claim 7, and the immune cell according to claim 8 in the preparation of drugs for preventing and / or treating autoimmune skin diseases.

10. The use according to claim 9, characterized in that The autoimmune skin disease is at least one of pemphigus, systemic lupus erythematosus, scleroderma, pemphigoid, psoriasis, epidermolysis bullosa, dermatitis herpetiformis, linear IgA bullous dermatosis and alopecia areata.

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