Use of metabolic enhanced immune cells for the preparation of a medicament for the treatment of autoimmune diseases

Abstract

The present application relates to a kind of metabolic enhancement type immune cell preparation in the application of the drug for treating autoimmune diseases, the protein expressed by the metabolic enhancement type immune cell includes the CAR of antigen recognition domain and interleukin-10.In the present application, the immune cell expression includes the CAR of antigen recognition domain and interleukin-10, can significantly improve the immune regulation of this immune cell, alleviate immune cell exhaustion, improve the activity of immune cell and the killing effect to target cell.The immune cell can also express the antibody of anti-IFN gamma or anti-IFNAR1 simultaneously, reduce the side effect of treatment, improve the safety and curative effect of autoimmune disease treatment.

Description

Technical Field

[0001] This invention relates to the field of biological cell technology, and in particular to the application of a metabolically enhanced immune cell in the preparation of a drug for treating autoimmune diseases. Background Technology

[0002] Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in the treatment of hematologic malignancies. Based on the immunomodulatory capabilities of CAR-T cells, researchers have begun to explore their potential in treating autoimmune diseases.

[0003] Conventional treatments for autoimmune diseases include glucocorticoids, immunosuppressants, and monoclonal antibodies (mAbs). Glucocorticoids and immunosuppressants broadly suppress the immune system, leading to increased susceptibility to infections. Monoclonal antibodies offer targeted interventions, but a portion of patients respond poorly, the need for repeated dosing can affect adherence, and inherent pharmacokinetic limitations prevent the maintenance of therapeutic levels.

[0004] To date, drug-free remission strategies for autoimmune diseases remain exploratory, making CAR-T therapy an attractive avenue. Compared to existing treatments, CAR-T cells offer promise for precise therapy due to their targeted therapeutic strategy and their ability to persist in the body due to their capacity for self-expansion. The effectiveness of CAR-T therapy depends on its ability to recognize target antigens and perform cytotoxic effects. Theoretically, the deep depletion of CD19+ B cells and plasmablasts in tissues could trigger an immune reset in autoimmune diseases.

[0005] The durability of CAR-T therapy depends on the persistence of CAR-T cells in the body. Often, CAR-T cells will be exhausted and die due to their recognition of target cell antigens and repeated execution of cytotoxicity, making it difficult for them to persist in the body. This may lead to disease relapse.

[0006] Interleukin-10 (IL-10) is generally considered immunosuppressive, reducing tissue damage caused by uncontrolled inflammatory responses. Biotest AG reported that its humanized IL-10 monoclonal antibody BT-063 met its primary endpoint in a phase 2 clinical trial for the treatment of systemic lupus erythematosus, and BT-063 shows great promise in the treatment of autoimmune diseases.

[0007] Therefore, obtaining effective and safe drugs for autoimmune diseases has become an urgent problem to be solved. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an application of metabolically enhanced immune cells in the preparation of drugs for treating autoimmune diseases, wherein the immune cells can significantly enhance the killing effect on target cells.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides an application of metabolically enhanced immune cells in the preparation of drugs for treating autoimmune diseases, wherein the proteins expressed by the metabolically enhanced immune cells include CAR containing an antigen recognition domain and interleukin-10.

[0011] In this invention, the proteins expressed by the immune cells include CAR containing an antigen recognition domain and interleukin-10, which can significantly enhance the immunomodulatory effect of the immune cells, alleviate immune cell depletion, improve the activity of immune cells, and enhance the killing effect on target cells.

[0012] Preferably, the expression of CAR and interleukin-10 containing antigen recognition domains in the immune cells includes expression linked to CAR molecules and / or expression alone.

[0013] Preferably, the CAR molecule comprises a CAR containing an antigen recognition domain, a linker sequence, and interleukin-10.

[0014] Preferably, the linker sequence comprises a self-splitting peptide or an IRES sequence.

[0015] In this invention, the structural sequence of the CAR molecule is that the CAR containing the antigen recognition domain is linked to interleukin-10 via a cleavage peptide or IRES sequence. When the cleavage peptide in this CAR molecule is cleaved, interleukin-10 is secreted by immune cells or binds to the membrane and exerts its function.

[0016] Preferably, the amino acid sequence of the self-splitting peptide includes the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0017] SEQ ID NO. 1: GSGATNFSLLKQAGDVEENPGP.

[0018] SEQ ID NO. 2: GSGEGRGSLLTCGDVEENPGP.

[0019] SEQ ID NO. 3: GSGQCTNYALLKLAGDVESNPGP.

[0020] SEQ ID NO.4: GSGVKQTLNFDLLKLAGDVESNPGP.

[0021] The nucleotide sequence of the IRES sequence includes the sequence shown in SEQ ID NO.5.

[0022] SEQ ID NO.5:

[0023] .

[0024] Preferably, the amino acid sequence of interleukin-10 includes any one or a combination of at least two of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14.

[0025] SEQ ID NO.6:

[0026] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0027] SEQ ID NO.7:

[0028] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0029] SEQ ID NO.8:

[0030] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRWRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0031] SEQ ID NO.9:

[0032] SPGQGTQSENSCTHFPGWLPMMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0033] SEQ ID NO.10:

[0034] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVMSLGENLFTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0035] SEQ ID NO.11:

[0036] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLMTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0037] SEQ ID NO.12:

[0038] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0039] SEQ ID NO.13:

[0040] SPGQGTQSENSCTFFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0041] SEQ ID NO.14:

[0042] SPGQGTQSENSCWHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVLSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0043] Preferably, the N-terminus of interleukin-10 also includes a signal peptide.

[0044] Preferably, the amino acid sequence of the signal peptide includes any one of the sequences shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21.

[0045] SEQ ID NO. 15: MHSSALLCCLVLLTGVRA.

[0046] SEQ ID NO. 16: MALPVTALLLPLALLLHAARP.

[0047] SEQ ID NO. 17: MYRMQLLSCIALSLALVTNS.

[0048] SEQ ID NO. 18: ATMYRMQLLSCIALSLALVTNSISA.

[0049] SEQ ID NO. 19: MGAARSPSAVPGPLLGLLLLLLGVLAPGGAS.

[0050] SEQ ID NO. 20: MLCCMRRTKQVEKNDEDQKI.

[0051] SEQ ID NO. 21: MEFGLSWLFLVAILKGVQC.

[0052] Preferably, the CAR containing an antigen recognition domain includes an scFv that recognizes the antigen, a hinge and transmembrane region, a co-stimulatory domain, and an intracellular signal transduction domain.

[0053] Preferably, the antigen of the scFv that recognizes the antigen includes any one or a combination of at least two of CD19, CD20, CD22, BCMA, or CD70.

[0054] Preferably, the antigen of the scFv that recognizes the antigen includes CD19.

[0055] Preferably, the amino acid sequence of the scFv recognizing the antigen includes any one of the sequences shown in SEQ ID NO.22, SEQ ID NO.23 or SEQ ID NO.24.

[0056] SEQ ID NO.22:

[0057] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0058] SEQ ID NO.23:

[0059] GSDIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTLTVSS.

[0060] SEQ ID NO.24:

[0061] QAVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLGSRGGGGSGGGGSGGG GSLEEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSS.

[0062] Preferably, the hinge and transmembrane region include CD27, CD28, CD29, OX-40, 4-1BB / CD137, CD2, CD7, CD30, CD40, PD-1, ICOS, LFA-1, CD1-1α / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276, LIGHT, TNFSF14, NKG2C, NKG2D, CD79a, DAP-10, Fcγ receptor, and MHC-1 receptors. TNF receptor protein, immunoglobulin, cytokine receptor, integrin, lymphocyte-activating molecules involved in signal transduction, NK cell-activating receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, GITR, BAFFR, HVEM, KIRDS2, NKp80, NKp44, NKp30, NKp46, CD18, CD19, CD4, CD5, CD8, CD8α, CD8β, CD9, CD16, CD2 2. CD33, CD37, CD45, CD64, CD69, IL-2Rβ, IL-2Rγ, IL-7Rα, VLA-1, CD49a, ITGA4, ITGA6, CD49D, CD49f, VL A-6, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, ITGB2, ITGB7, TNFR 2. Any one or a combination of at least two of the following: TRANCE / RANKL, DNAM1, SLAMF4, SLAMF7, CD80, CD84, CD86, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / CBP, CD134, and CD154;

[0063] Preferably, the amino acid sequence of the hinge and transmembrane region includes the sequence shown in SEQ ID NO.25.

[0064] SEQ ID NO.25:

[0065] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSR.

[0066] Preferably, the co-stimulatory domain includes any one or a combination of at least two of 4-1BB, CD28, CD137, OX40, or ICOS.

[0067] Preferably, the amino acid sequence of the 4-1BB includes the sequence shown in SEQ ID NO.26.

[0068] SEQ ID NO.26:

[0069] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0070] Preferably, the amino acid sequence of CD28 includes the sequence shown in SEQ ID NO.27.

[0071] SEQ ID NO.27:

[0072] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV.

[0073] Preferably, the intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, BCR, NKp30, NKp44, NKp46, FcαR, FcRγ, CD16, or CD32.

[0074] Preferably, the intracellular signal transduction domain includes CD3ζ.

[0075] Preferably, the amino acid sequence of the intracellular signal transduction domain includes that shown in SEQ ID NO.28.

[0076] SEQ ID NO.28:

[0077] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0078] Preferably, the CAR molecule further includes Anti-IFNγscFv.

[0079] Preferably, the amino acid sequence of the Anti-IFNγscFv includes the sequence shown in SEQ ID NO.29.

[0080] SEQ ID NO.29:

[0081] MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGQ GTLVTVSSGGGGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSNRWMFGGGTKLTVL.

[0082] Preferably, the nucleic acid sequence of the Anti-IFNγscFv includes the sequence shown in SEQ ID NO.30.

[0083] SEQ ID NO.30:

[0084] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTAGCAGTGGCTGGTACGTACCACACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTAATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACTCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAACAGCGCCCGGGCAGTTCCCCCACCACTGTCATCTATGAGGATAACCAGAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAATTCTGCCTCCCTCACCATCTCTGGGCTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCGTTGGATGTTCGGCGGAGGGACCAAGCTGACCGTCCTA。

[0085] Preferably, the CAR molecule further comprises Anti-IFNAR1scFv.

[0086] Preferably, the amino acid sequence of the Anti-IFNAR1scFv comprises the sequence shown in SEQ ID NO. 31.

[0087] SEQ ID NO31:

[0088] EVQLVQSGAEVKKPGESLKISCKGSGYIFTNYWIAWVRQMPGKGLESMGIIYPGDSDIRYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHDIEGFDYWGRGTLVTVSSGGG GSGGGGSGGGGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFAWYQQKPGQAPRLLIYGASSRATGIPDRLSGSGSGTDFTLTITRLEPEDFFAVYYCQQYDSSAITFGQGTRLEIK.

[0089] Preferably, the nucleotide sequence of the Anti-IFNAR1scFv includes the sequence shown in SEQ ID NO.32.

[0090] SEQ ID NO.32:

[0091] .

[0092] Preferably, the immune cells are autologous, allogeneic, or heterologous.

[0093] Preferably, the immune cells include any one or a combination of at least two of T cells, CAR-T cells, NK cells, CAR-NK cells or iPS-derived CAR-NK cells.

[0094] Preferably, the immune cells also include a carrier or nucleic acid required for preparing metabolically enhanced immune cells.

[0095] Preferably, the carrier or nucleic acid is delivered to cells in vivo or in vitro to prepare the immune cells.

[0096] The present invention also provides a nucleic acid sequence encoding the CAR molecule described herein.

[0097] The present invention also provides a vector comprising a nucleic acid sequence encoding the CAR molecule described herein.

[0098] Preferably, the vector comprises a viral vector.

[0099] Preferably, the vector includes any one or a combination of at least two of the following: retroviral vector (such as pMSGV), DNA vector, mouse leukemia virus vector, SFG vector, RNA vector, adenovirus vector, pasteurellosis virus vector, vaccinia virus vector, herpes simplex virus vector or adenovirus-associated vector (AAV), and lentiviral vector (such as pGAR).

[0100] Preferably, the autoimmune disease includes B-cell-related autoimmune diseases.

[0101] Preferably, the autoimmune disease includes any one or a combination of at least two of the following: systemic lupus erythematosus, idiopathic inflammatory myositis, lupus nephritis, rheumatoid arthritis, pemphigus vulgaris, type 1 diabetes mellitus, inflammatory bowel disease, Sjögren's syndrome, antisynthetic enzyme syndrome, multifocal motor neuropathy, necrotizing crescentic glomerulonephritis, experimental autoimmune encephalitis, myasthenia gravis, neuromyelitis optica spectrum disorder, or multiple sclerosis.

[0102] In a second aspect, the present invention provides a combination drug composition for treating autoimmune diseases, the combination drug composition comprising the drug for treating autoimmune diseases prepared by metabolically enhanced immune cells as described in the first aspect and the therapeutic drug.

[0103] Preferably, the therapeutic agent includes any one or a combination of at least two of the following: antimalarial drugs, glucocorticoids, or immunosuppressants.

[0104] Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0105] Preferably, the formulation is any pharmaceutically acceptable dosage form.

[0106] Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0107] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0108] Compared with the prior art, the present invention has at least the following beneficial effects:

[0109] 1. In this invention, the immune cells express CAR containing an antigen recognition domain and interleukin-10, which can significantly improve the immunomodulatory effect of the immune cells, alleviate immune cell depletion and weakening, improve the activity of immune cells and the killing effect on target cells.

[0110] 2. In this invention, the structural sequence of the CAR molecule is that the CAR containing the antigen recognition domain is linked to interleukin-10 via a self-splitting peptide. When the self-splitting peptide in this CAR molecule is cleaved, interleukin-10 is secreted by immune cells or binds to the membrane and exerts its function. Attached Figure Description

[0111] Figure 1 A schematic diagram of the nucleic acid molecule used as a CAR construct.

[0112] Figure 2 Flow cytometry graph showing the positivity rate of CAR-T cells derived from peripheral blood in SLE patients.

[0113] Figure 3 Flow cytometry graph showing the positivity rate of CAR-T cells derived from peripheral blood in RA patients.

[0114] Figure 4 The images show a comparison of CAR-T cytotoxic B cells derived from SLE and RA patients. Figure A shows the cytotoxicity of CAR-T cells from SLE patients, and Figure B shows the cytotoxicity of CAR-T cells from RA patients.

[0115] Figure 5 The images show a comparison of IFN-γ and IL-10 secretion levels between CD19CAR-T cells that secrete IL-10 and traditional CAR-T cells. Figure A shows a comparison of IFN-γ secretion, and Figure B shows a comparison of IL-10 secretion.

[0116] Figure 6 The images show a comparison of B cell killing and IFN-γ secretion between IL10CD19CAR-T and IL10anti-IFNγCD19CAR-T. Figure A shows the killing effect, and Figure B shows the IFN-γ secretion effect.

[0117] Figure 7 This is a comparison of the B cell killing effects of IL10CD19CAR-T and IL10anti-IFNAR1CD19CAR-T.

[0118] Figure 8 This is a comparison of the effects of IL10CD19CAR-T and IL10anti-IFNAR1CD19CAR-T on the expression of MHC Class I and CD123 in DC cells. Detailed Implementation

[0119] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0120] Example 1

[0121] This embodiment focuses on the design of CAR molecular structures.

[0122] The specific structure is as follows: Figure 1 As shown (where 2A is a schistolytic peptide and SP is a signal peptide), a total of five CAR molecules were constructed, namely 1a, 1b, 1d, and 1e. First, a second-generation anti-CD19 CAR molecule was constructed, named 1a, which includes the CD8α signal peptide, CD19scFv, CD8 hinge region and transmembrane region, 4-1BB co-stimulatory domain, and CD3ζ. Based on 1a, the CD8α signal peptide and IL10 were linked using the schistolytic peptide T2A to obtain an anti-CD19 CAR molecule that secretes IL10, named 1b.

[0123] Adding anti-IFNγscFv to vector 1b, named 1d. Adding anti-IFNγscFv to vector 1b, named 1d. Adding anti-IFNAR1scFv to vector 1b, named 1e. Expressing IL-10 in another vector, named 1c. Simultaneous transduction of T cells with both vectors 1a and 1c can also yield CD19CARs that secrete IL-10.

[0124] Among them, the self-splitting peptide before IL10 is T2A, with the sequence SEQ ID NO.2; the sequence of IL10 is SEQ ID NO.6; the sequence of the signal peptide before the CAR structure and before IL10 is SEQ ID NO.16; the nucleic acid sequence of anti-IFNγscFv is SEQ ID NO.30; the nucleic acid sequence of anti-IFNAR1scFv is SEQ ID NO.32; the sequence of the signal peptide before anti-IFNγscFv and anti-IFNAR1scFv is SEQ ID NO.21; and the self-splitting peptide before anti-IFNγscFv and anti-IFNAR1scFv is P2A, with the sequence SEQ ID NO.1.

[0125] Example 2

[0126] This embodiment prepares lentiviruses and CAR-T cells.

[0127] CAR structural fragments containing 1a–1e of this application were artificially synthesized and constructed into the lentiviral vector pCDH-EF1α (manufacturer: Addgene) to obtain CAR expression vectors. Subsequently, the CAR expression vectors and three packaging plasmids were transfected into 293F cells. Cell supernatants were collected after 48 hours and concentrated to obtain functional lentiviral vectors. The three packaging plasmids were pMD2.G, pMDLg-pRRE, and pRSV-Rev (manufacturer: Porton Biotech).

[0128] Results: The titers of all lentiviruses were greater than 1×10⁻⁶. 8 The titer was TU / mL, and the specific titer results are shown in Table 1.

[0129] Table 1

[0130] Serial Number Structure of expression plasmids Viral titer (TU / mL) 1 1a <![CDATA[2.93×10 9 ]]> 2 1b <![CDATA[3.42×10 9 ]]> 3 1c <![CDATA[5.2×10 9 ]]> 4 1d <![CDATA[1.26×10 9 ]]> 5 1e <![CDATA[1.0×10 9 ]]>

[0131] Peripheral blood (PBMCs) were collected from SLE and rheumatoid arthritis (RA) patients (30 mL each) and obtained by density gradient centrifugation using Ficoll. T cells were activated and expanded using anti-CD3 / anti-CD28 magnetic beads (manufacturer: Tongli Haiyuan). After 24 h of magnetic bead stimulation, lentivirus (MOI = 10) was added and gently mixed by pipetting. After 72 h of magnetic bead stimulation, the beads were removed using a magnetic rack. CAR-T cells were passaged and expanded every other day using X-VIVO15 medium containing 2% serum substitute (SR), 2 mM L-glutamine, 5 ng / mL human IL-7, and 5 ng / mL human IL-15. All cells were cultured in a 37°C, 5% CO2 incubator. After 7 days of cell culture, CAR-T cells were harvested and the CAR positivity rate was detected by flow cytometry using PE anti-G4S antibody (manufacturer: GST). The specific results are shown in Table 2.

[0132] Table 2

[0133]

[0134] Specific results are as follows Figure 2 and Figure 3 As shown, Figure 2 Flow cytometry plot of CAR-T cell positivity rate from peripheral blood in SLE patients. Figure 3 Flow cytometry plot showing the positivity rate of CAR-T cells derived from peripheral blood in RA patients. The results indicate that CAR-T cells derived from peripheral blood of SLE and RA patients were successfully prepared.

[0135] Example 3

[0136] This embodiment conducts an in vitro killing experiment using human IL10CD19CAR-T cells.

[0137] PBMCs from SLE and RA patients were activated by CD3 / CD28 magnetic bead positive selection and cultured in vitro to prepare IL10CD19CAR-T cells or CD19CAR-T cells as effector cells. IL10CD19CAR-T cells were prepared using either a virus with structure 1b or a mixture of viruses with structures 1a and 1c, and were named (1b) and (1a+1c) respectively for distinction. The remaining positively selected cells were then isolated using a CD19 cell isolation kit to obtain CD19 cells. + Cells were used as target cells and co-cultured at an E:T ratio of 1:1 for 16 hours. The killing effect was then detected by flow cytometry.

[0138] The in vitro killing procedure was as follows: target cells were resuspended in killing medium (X-VIVO 15 + 5% FBS + Glutmax (1×)) and seeded into 12-well plates, 2×10⁶ cells per well. 5 Cells, 500 μL / well. Add 500 μL of CAR-T cells at an E:T ratio of 1:1, and incubate the cells at 37°C in a 5% CO2 incubator for 48 h. Gently pipette the cells from the wells, centrifuge at 400g for 5 min, collect the cell culture supernatant, and use the CBA method to detect the secretion levels of IFN-γ and IL10 (manufacturer: BD). Use Zombie Aqua... TM For staining to determine cell viability and deadness using the Fixable Viability Kit, refer to the reagent's instruction manual for detailed operating procedures. Stain with APC / Cyanine7 anti-human CD3 and APC anti-human CD19 antibodies (manufacturer: BioLegend) at 4°C for 25 min. Wash cells twice with 1×DPBS, then resuspend in 500 μL of 1×DPBS and count 200 μL.

[0139] The FSC-SSC gate circles all cells except for the fragments, and then, based on Zombie Aqua... TM Circle the viable cell population. Finally, circle the hCD3-positive and hCD19-positive cells within the viable cell population.

[0140] Kill efficiency (%) = (Number of live hCD19 positive cells in the target cell group - Number of live hCD19 positive cells in the experimental cell group) / Number of live hCD19 positive cells in the target cell group.

[0141] The results are as follows Figure 4 and Figure 5As shown, CD19CAR-T cells that secrete IL10 (1b or 1a+1c) exhibited higher cytotoxicity against target cells than conventional CD19CAR-T cells. They secreted large amounts of IL10 and IFN-γ into the cell culture supernatant. Furthermore, during cytotoxicity, the IFN-γ secretion level of IL10CD19CAR-T cells was lower compared to conventional CAR-T cells. This indicates that the IL10CD19CAR-T cells constructed in this invention can significantly enhance T cell cytotoxicity against CD19+ cells in patients with autoimmune diseases. + Reduces the killing ability of B cells and decreases IFN-γ secretion.

[0142] Example 4

[0143] This embodiment verifies that adding anti-IFNγ elements can improve the safety of treatment for autoimmune diseases.

[0144] CAR-T cells with two structures, 1b and 1d, were prepared using T cells derived from SLE patients, along with CD19 cells derived from SLE patients. + Cells were used as target cells and co-cultured at an E:T ratio of 1:1 for 24 hours. Flow cytometry was used to detect cell killing. Cell culture supernatant was collected, and IFN-γ secretion levels (manufacturer: BD) were detected using the CBA method.

[0145] The results are as follows Figure 6 As shown, IL10CD19CAR-T cells secreting anti-IFN-γ single-chain antibodies (1d, CAR19-IL10-antiIFNγ) exhibited comparable cytotoxic activity to CD19CAR-T cells secreting IL-10 (1b), indicating that IFNγ antibody secretion does not affect the in vitro cytotoxic ability of CAR-T cells. After co-culturing CAR19-IL10-antiIFNγ cells with CD19+ target cells, less IFN-γ was detected in the supernatant, suggesting that the anti-IFN-γ antibody secreted by CAR-T cells can effectively neutralize IFN-γ, thereby effectively reducing the occurrence of CRS.

[0146] Example 5

[0147] This embodiment verifies the effect of adding anti-IFNAR1 elements on enhancing the therapeutic effect of SLE.

[0148] 30 mL of peripheral blood from an SLE patient was collected, centrifuged at 3000 rpm for 10 min, and the supernatant was collected to obtain SLE patient serum. Two types of CAR-T cells, 1b and 1e, were prepared using T cells derived from SLE patients, along with CD19 cells derived from SLE patients. +Cells were used as target cells and co-cultured at an E:T ratio of 1:1 for 24 hours. Flow cytometry was used to detect cell killing, and the supernatant of the cytotoxic cells was collected. 10 mL of blood from a single healthy individual was diluted 1:2 with DPBS and gently added to a Ficoll layer. The cells were centrifuged at 500 g for 30 min at room temperature (increase speed 2, decrease speed 0). The white blood cell layer was collected, washed once with DPBS, and PBMCs were obtained. PBMCs were resuspended in RPMI-1640 + 10% FBS at 4 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of 1 / mL into T25 cells and incubated at 37°C with 5% CO2 for 2 hours. Non-adherent cells were removed to obtain monocytes. Monocytes were cultured using either RPMI-1640 + 20% SLE patient serum + 10% 1b cytotoxic cell culture supernatant or RPMI-1640 + 20% SLE patient serum + 10% 1e cytotoxic cell culture supernatant. After 7 days of incubation at 37°C with 5% CO2, cells were harvested, and the expression levels of cell surface molecules MHC Class I and CD123 (manufacturer: BioLegend) were detected by flow cytometry.

[0149] The results are as follows Figure 7 As shown, IL10CD19CAR-T cells that secrete anti-IFNAR1 single-chain antibodies (CAR19-IL10-antiIFNAR1) have comparable killing ability to CD19CAR-T cells that secrete IL-10 (CAR19-IL10), indicating that secreting IFNAR1 antibodies does not affect the in vitro killing ability of CAR-T cells.

[0150] The results are as follows Figure 8 As shown, after 7 days of culture, flow cytometry analysis revealed a significant decrease in the expression levels of MHC Class I and CD123 on the surface of dendritic cells (DCs) cultured in the supernatant of IL10CD19CAR-T cells that secreted anti-IFNAR1 single-chain antibodies, indicating inhibition of DC development. Therefore, IL10CD19CAR-T cells that secrete anti-IFNAR1 single-chain antibodies can not only directly kill B cells secreting large amounts of autoantibodies, but also act on DCs by secreting anti-IFNAR1 antibodies, reducing autoimmune responses and further improving the therapeutic effect on autoimmune diseases.

[0151] In summary, the CAR molecules expressed by immune cells in this invention include CAR molecules with an antigen recognition domain and interleukin-10, which can significantly enhance the immunomodulatory function of the immune cells, alleviate immune cell exhaustion, improve immune cell activity, and increase the killing effect on target cells. Furthermore, the immune cells can simultaneously express antibodies against IFNγ or IFNAR1, reducing treatment side effects and improving the safety and efficacy of treatment for autoimmune diseases.

[0152] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of a metabolically enhanced immune cell in the preparation of a drug for treating autoimmune diseases, characterized in that, The proteins expressed by the metabolically enhanced immune cells include CAR containing an antigen recognition domain and interleukin-10.

2. The application of the metabolically enhanced immune cells described in claim 1 in the preparation of a drug for treating autoimmune diseases, characterized in that, The expression forms of CARs and interleukin-10 containing antigen recognition domains in the immune cells include expression linked to CAR molecules and / or expression alone; Preferably, the CAR molecule comprises a CAR containing an antigen recognition domain, a linker sequence, and interleukin-10; Preferably, the linker sequence comprises a self-splitting peptide or an IRES sequence.

3. The application of the metabolically enhanced immune cells described in claim 1 or 2 in the preparation of a medicament for treating autoimmune diseases, characterized in that, The amino acid sequence of the self-splitting peptide includes the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; Preferably, the nucleotide sequence of the IRES sequence includes the sequence shown in SEQ ID NO.5; Preferably, the amino acid sequence of interleukin-10 includes any one or a combination of at least two of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14; Preferably, the N-terminus of interleukin-10 further includes a signal peptide; Preferably, the amino acid sequence of the signal peptide includes any one of the sequences shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.

21.

4. The use of metabolically enhanced immune cells prepared according to any one of claims 1-3 in the preparation of a medicament for treating autoimmune diseases, characterized in that, The CAR containing the antigen recognition domain includes an scFv that recognizes the antigen, a hinge and transmembrane region, a co-stimulatory domain, and an intracellular signal transduction domain.

5. The application of the metabolically enhanced immune cells described in claim 4 in the preparation of a drug for treating autoimmune diseases, characterized in that, The antigens of the scFv that recognize the antigens include any one or a combination of at least two of CD19, CD20, CD22, BCMA, or CD70. Preferably, the antigen of the scFv that recognizes the antigen includes CD19; Preferably, the amino acid sequence of the scFv that recognizes the antigen includes any one of the sequences shown in SEQ ID NO.22, SEQ ID NO.23, or SEQ ID NO.24; Preferably, the hinge and transmembrane region include CD27, CD28, CD29, OX-40, 4-1BB / CD137, CD2, CD7, CD30, CD40, PD-1, ICOS, LFA-1, CD1-1α / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276, LIGHT, TNFSF14, NKG2C, NKG2D, CD79a, DAP-10, Fcγ receptor, and MHC-1 receptors. TNF receptor protein, immunoglobulin, cytokine receptor, integrin, lymphocyte-activating molecules involved in signal transduction, NK cell-activating receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, GITR, BAFFR, HVEM, KIRDS2, NKp80, NKp44, NKp30, NKp46, CD18, CD19, CD4, CD5, CD8, CD8α, CD8β, CD9, CD16, CD2 2. CD33, CD37, CD45, CD64, CD69, IL-2Rβ, IL-2Rγ, IL-7Rα, VLA-1, CD49a, ITGA4, ITGA6, CD49D, CD49f, VL A-6, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, ITGB2, ITGB7, TNFR 2. Any one or a combination of at least two of the following: TRANCE / RANKL, DNAM1, SLAMF4, SLAMF7, CD80, CD84, CD86, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / CBP, CD134, and CD154; Preferably, the amino acid sequence of the hinge and transmembrane region includes the sequence shown in SEQ ID NO.

25.

6. The use of the metabolically enhanced immune cells described in claim 4 or 5 in the preparation of a medicament for treating autoimmune diseases, characterized in that, The costimulatory domains include any one or a combination of at least two of 4-1BB, CD28, CD137, OX-40, or ICOS; Preferably, the amino acid sequence of the 4-1BB comprises the sequence shown in SEQ ID NO.26; Preferably, the amino acid sequence of CD28 includes the sequence shown in SEQ ID NO.27; Preferably, the intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, BCR, NKp30, NKp44, NKp46, FcαR, FcRγ, CD16, or CD32; Preferably, the intracellular signal transduction domain includes CD3ζ; Preferably, the amino acid sequence of CD3ζ includes the sequence shown in SEQ ID NO.28; Preferably, the CAR molecule further includes Anti-IFNγscFv; Preferably, the amino acid sequence of the Anti-IFNγscFv includes the sequence shown in SEQ ID NO.29; Preferably, the nucleotide sequence of the Anti-IFNγscFv includes the sequence shown in SEQ ID NO.30; Preferably, the CAR molecule further includes Anti-IFNAR1 scFv; Preferably, the amino acid sequence of the Anti-IFNAR1 scFv includes the sequence shown in SEQ ID NO.31; Preferably, the nucleotide sequence of the Anti-IFNAR1 scFv includes the sequence shown in SEQ ID NO.32; Preferably, the immune cells further include a carrier or nucleic acid required for preparing metabolically enhanced immune cells; Preferably, the carrier or nucleic acid is delivered to cells in vivo or in vitro to prepare the immune cells.

7. The use of metabolically enhanced immune cells prepared according to any one of claims 1-6 in the preparation of a medicament for treating autoimmune diseases, characterized in that, The immune cells are autologous, allogeneic, or xenogeneic. Preferably, the immune cells include any one or a combination of at least two of T cells, CAR-T cells, NK cells, CAR-NK cells or iPS-derived CAR-NK cells.

8. The use of metabolically enhanced immune cells prepared according to any one of claims 1-7 in the preparation of a medicament for treating autoimmune diseases, characterized in that, The autoimmune diseases mentioned include B-cell-related autoimmune diseases; Preferably, the autoimmune disease includes any one or a combination of at least two of the following: systemic lupus erythematosus, idiopathic inflammatory myositis, lupus nephritis, rheumatoid arthritis, pemphigus vulgaris, type 1 diabetes mellitus, inflammatory bowel disease, Sjögren's syndrome, antisynthetic enzyme syndrome, multifocal motor neuropathy, necrotizing crescentic glomerulonephritis, experimental autoimmune encephalitis, myasthenia gravis, neuromyelitis optica spectrum disorder, or multiple sclerosis.

9. A combination drug composition for treating autoimmune diseases, characterized in that, The combined pharmaceutical composition comprises a drug for treating autoimmune diseases prepared from metabolically enhanced immune cells according to any one of claims 1-8, and a therapeutic drug.

10. The combination drug composition according to claim 9, characterized in that, The therapeutic agents include any one or a combination of at least two of antimalarial drugs, glucocorticoids, or immunosuppressants; Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations; Preferably, the formulation is any pharmaceutically acceptable dosage form; Preferably, the combined pharmaceutical composition further contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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