Regulatory T cell targeting IL12RB1 and application thereof

Abstract

The invention relates to the field of cell therapy, in particular to a regulatory T cell targeting IL12RB1 and application of the regulatory T cell. The regulatory T cell provided by the invention comprises a P40 subunit or a mutant thereof. According to the invention, a natural protein sequence is used, so that the risk that the antigen has immunogenicity caused by scFv can be avoided; the Treg cells are homed to the inflammation part, and the activated lymphocytes are inhibited.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to regulatory T cells targeting IL12RB1 and uses thereof. Background Art

[0002] In the treatment of autoimmune diseases, biologics, such as monoclonal antibodies against TNF-α, IL-17, and IL-23, can rapidly suppress inflammation (NEJM 2021, 385:231-240). However, long-term use is associated with secondary failure in 60% of patients within 5 years and an 8- to 10-fold increased risk of tuberculosis reactivation (Lancet 2022, 399:1564-1575). Newer therapies, such as dual antibodies against PD-1 and CTLA-4, despite attempting multi-target intervention, result in 37% of grade 3 or higher irAEs due to unintended immune activation and are unable to penetrate fibrotic tissue (Nat Med 2023, 29:1128). Drug development targeting the IL-12 signaling pathway has garnered significant attention in recent years. IL12RB1, a shared subunit of the IL-12 and IL-23 receptors, plays a central role in Th1 / Th17-mediated pathologies (Immunity 2019, 50:907). Clinical data show that the proportion of IL12RB1+ T cells in the intestinal mucosa of IBD patients is 3-5 times higher than that in healthy controls (Cell 2020, 182: 1460), and the intensity of IL12RB1 expression in psoriatic skin lesions is positively correlated with disease severity (NEJM 2021, 384: 1889). Existing drugs targeting this pathway, such as the anti-p40 monoclonal antibody ustekinumab, have been approved, but they carry the risk of CMV reactivation due to simultaneous blockade of both IL-12 and IL-23 (Gut 2020, 69: 1895). Preclinical studies have shown that selective inhibition of IL12RB1 can inhibit pathogenic T cells by blocking STAT4 phosphorylation (Nat Immunol 2022, 23: 1364), but traditional antibodies have difficulty penetrating inflamed tissues due to molecular weight limitations. Summary of the Invention

[0003] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a regulatory T cell targeting IL12RB1 and a use thereof, so as to solve the problems in the prior art.

[0004] To achieve the above objectives and other related objectives, the present invention provides use of an IL12RB1 activating factor in preparing a regulatory T cell product, wherein the amino acid sequence of IL12RB1 is shown in SEQ ID No. 1.

[0005] Preferably, the IL12RB1 activating factor comprises a P40 subunit or a mutant thereof, wherein the amino acid sequence of the P40 subunit is shown in SEQ ID No. 2; and / or the P40 subunit mutant has one or more of the following mutations: D109A, K80M, E67Y, N125H, D40L, K217R or T150Y.

[0006] The present invention also provides a P40 subunit mutant, wherein the P40 subunit mutant has one or more of the following mutations relative to the wild-type P40 subunit: D109A, K80M, E67Y, N125H, D40L, K217R, or T150Y, wherein the amino acid sequence of the wild-type P40 subunit is shown in SEQ ID No. 2. The amino acid sequences of the mutant P40 subunits are shown in SEQ ID Nos. 3-9.

[0007] The present invention also provides a chimeric antigen receptor, which comprises the aforementioned P40 subunit mutant.

[0008] The present invention also provides a polynucleotide encoding the aforementioned P40 subunit mutant or the aforementioned chimeric antigen receptor.

[0009] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide.

[0010] The present invention also provides a viral vector comprising the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0011] The present invention also provides a regulatory T cell targeting IL12RB1, wherein the regulatory T cell comprises a P40 subunit or a mutant thereof.

[0012] The present invention also provides the use of the aforementioned polynucleotides, the aforementioned nucleic acid constructs or the aforementioned regulatory T cells in the preparation of drugs for treating autoimmune diseases, transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome or drugs for treating diseases involving uncontrolled inflammatory reactions mediated by one or more inflammatory-related factors or caused by these uncontrolled inflammatory reactions.

[0013] As described above, the regulatory T cells targeting IL12RB1 and their uses of the present invention have the following beneficial effects:

[0014] The present invention uses natural protein sequences to avoid the immunogenicity risk of antigens caused by scFv; it enables Treg cells to home to the inflammatory site and inhibit activated lymphocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Shown is a schematic diagram of the CAR structure of the present invention.

[0016] Figure 2 Shown is a schematic diagram of affinity detection of different mutations of the p40 site of the present invention.

[0017] Figure 3 Shown is a schematic diagram of the construction and detection of CAR-Treg cells of the present invention.

[0018] Figure 4 Shown is a schematic diagram of the CAR-Treg cell positivity rate detection of the present invention.

[0019] Figure 5 Shown is a schematic diagram of the CAR-Treg cell-specific activation detection of the present invention.

[0020] Figure 6 Shown is a schematic diagram of the in vitro inhibitory function detection of CAR-Treg cells of the present invention. DETAILED DESCRIPTION

[0021] The present invention provides use of an IL12RB1 activating factor in preparing a regulatory T cell product, wherein the amino acid sequence of IL12RB1 is shown as SEQ ID No. 1.

[0022] In some specific embodiments, the IL12RB1 activator comprises a P40 subunit or a mutant thereof, wherein the amino acid sequence of the P40 subunit is shown in SEQ ID No. 2.

[0023] In some embodiments, the P40 subunit mutant has one or more of the following mutations: D109A, K80M, E67Y, N125H, D40L, K217R or T150Y, and its amino acid sequence is shown in any one of SEQ ID No. 3 to 9. In some embodiments, the IL12RB1 activator is a chimeric antigen receptor (CAR).

[0024] In some embodiments, the chimeric antigen receptor comprises one or more of a guide peptide, an antigen binding domain, a hinge domain, a transmembrane domain, or an intracellular signaling domain.

[0025] Furthermore, the P40 subunit or a mutant thereof is the antigen binding domain of the chimeric antigen receptor.

[0026] Furthermore, the guide peptide is selected from CD8 guide peptide or GM-CSF guide peptide, wherein the amino acid sequence of CD8 guide peptide is shown as SEQ ID No.10; or, the amino acid sequence of GM-CSF guide peptide is shown as SEQ ID No.11.

[0027] Furthermore, the hinge domain is selected from any one of a CD8α hinge domain, a CD28 hinge domain, a CD4 hinge domain, an IgG hinge domain, and an IgD hinge domain, wherein the amino acid sequence of the CD8α hinge domain is shown in SEQ ID No. 12; or, the amino acid sequence of the CD28 hinge domain is shown in SEQ ID No. 13; or, the amino acid sequence of the CD4 hinge domain is shown in SEQ ID No. 14; or, the amino acid sequence of the IgG hinge domain is shown in SEQ ID No. 15; or, the amino acid sequence of the IgD hinge domain is shown in SEQ ID No. 16. Preferably, the hinge domain is a CD28 hinge domain.

[0028] Further, the transmembrane domain is selected from the transmembrane domain of any of the following proteins: CD28, CD28T, OX-40, 4-1BB, CD137, CD2, CD7, CD8, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT, TNFSF14, NKG2C, Igα (CD79a), DAP10, Fcγ receptor, MHC class 1 molecule or TNF receptor protein, wherein the amino acid sequence of the CD28 transmembrane domain is shown in SEQ ID No. 17; or, the amino acid sequence of the 4-1BB transmembrane domain is shown in SEQ ID No. 18; or, the amino acid sequence of the OX-40 transmembrane domain is shown in SEQ ID or, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID No. 20. Preferably, the transmembrane domain is the CD28 transmembrane domain.

[0029] Furthermore, the intracellular signaling domain is selected from the intracellular signaling domain of any of the following proteins: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b or CD66d, wherein the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID No. 21. Preferably, the intracellular signaling domain is the CD3ζ intracellular signaling domain.

[0030] In some embodiments, the chimeric antigen receptor further comprises a cleavage peptide, and in particular, the cleavage peptide is selected from one or more of P2A, E2A, F2A, or T2A.

[0031] In some specific embodiments, the domains in the chimeric antigen receptor are connected in the following manner: guide peptide-P40 subunit or a mutant thereof-hinge domain-transmembrane domain-intracellular signaling domain.

[0032] In some embodiments, the regulatory T cell product is a chimeric antigen receptor-regulatory T cell (CAR-Treg). Specifically, the regulatory T cell product is an ex vivo chimeric antigen receptor-regulatory T cell.

[0033] The present invention also provides a P40 subunit mutant, wherein the P40 subunit mutant has one or more of the following mutations relative to the wild-type P40 subunit: D109A, K80M, E67Y, N125H, D40L, K217R or T150Y, wherein the amino acid sequence of the wild-type P40 subunit is shown in SEQ ID No. 2.

[0034] The present invention also provides a chimeric antigen receptor, which comprises the aforementioned P40 subunit mutant.

[0035] In some specific embodiments, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No. 22.

[0036] The present invention also provides a polynucleotide encoding the aforementioned P40 subunit mutant or the aforementioned chimeric antigen receptor.

[0037] In some specific embodiments, the nucleotide sequence of the polynucleotide is shown as SEQ ID No. 23.

[0038] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide.

[0039] In some embodiments, the nucleic acid construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector can be transformed, transduced, or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. The construct is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist in its entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0040] In some specific embodiments, the backbone plasmid of the expression vector can be selected from pLVX expression vector, PCDNA3.1, PIRES, PCDNA3.4, pET expression vector, pCW expression vector, pUC expression vector, pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPICZA, pPICZ B, pPICZ C, pGAPZ A, pGAPZ B or pGAPZ C.

[0041] The present invention also provides a viral vector comprising the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0042] In some embodiments, the viral vector is selected from one or more of lentivirus, adenovirus, or adeno-associated virus.

[0043] The present invention also provides a regulatory T cell targeting IL12RB1, wherein the regulatory T cell comprises a P40 subunit or a mutant thereof.

[0044] In some embodiments, the regulatory T cells contain the aforementioned chimeric antigen receptor.

[0045] Furthermore, the chimeric antigen receptor comprises one or more of a guide peptide, an antigen binding domain, a hinge domain, a transmembrane domain or an intracellular signaling domain.

[0046] Furthermore, the antigen binding domain is the P40 subunit or a mutant thereof, wherein the amino acid sequence of the P40 subunit is shown in SEQ ID No.2.

[0047] The present invention also provides the use of the aforementioned polynucleotides, the aforementioned nucleic acid constructs or the aforementioned regulatory T cells in the preparation of drugs for treating autoimmune diseases, transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome or drugs for treating diseases involving uncontrolled inflammatory reactions mediated by one or more inflammatory-related factors or caused by these uncontrolled inflammatory reactions.

[0048] In some embodiments, the autoimmune disease is selected from any one or more of rheumatoid arthritis, psoriatic arthritis, psoriasis, lupus, juvenile rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, or Crohn's disease.

[0049] In some embodiments, the transplant rejection is selected from any one or more of organ transplant rejection, stem cell transplant rejection, or bone marrow transplant rejection.

[0050] The present invention also provides a method for treating any one or more of the following diseases: autoimmune diseases, transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome, or any disease / disorder involving an uncontrolled inflammatory response mediated by one or more inflammation-related factors or caused by these uncontrolled inflammatory responses, wherein the method comprises administering a therapeutically effective number of the aforementioned regulatory T cells, thereby treating the autoimmune disease, transplant rejection, or graft-versus-host disease (GVHD).

[0051] The present invention also provides a method for inducing immune tolerance in a subject in need thereof, wherein the method comprises administering a therapeutically effective number of the aforementioned regulatory T cells, thereby inducing immune tolerance in the subject in need thereof.

[0052] The present invention also provides a method for locally or systemically downregulating inflammation in a subject in need thereof, wherein the method comprises administering a therapeutically effective number of the aforementioned regulatory T cells, thereby downregulating inflammation locally or systemically in the subject in need thereof.

[0053] In the present invention, the term "regulatory T cells" also refers to a T cell subset of suppressor T cells, which has at least one of the following characteristics: expression of CD4; expression of FOXP3; expression of CD25, CD4+, FOXP3+ and CD25+ T cells; downregulation of the ability to induce and proliferate effector T cells, CD4+FOXP3+CD25 (high) T cells; or greater T cell receptor (TCR) diversity than effector T cells.

[0054] In the present invention, the terms "polynucleotide" and "nucleic acid" (or singular form) are interchangeable. A polynucleotide or nucleic acid can be DNA, RNA, or a combination of DNA and RNA. Those skilled in the art will readily understand that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides."

[0055] In the present invention, the term "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (or entire polynucleotide), such as a gene, cDNA (including transgenic cDNA), or mRNA, to serve as a template for other polymers and macromolecules in synthetic biological processes. These polymers and macromolecules have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (which serves as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of the gene or cDNA. If, in its native state or when manipulated by methods well known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce an mRNA and / or polypeptide or fragment thereof, then the polynucleotide can be said to "encode" the polypeptide. The antisense strand is the complementary strand of this nucleic acid, and the coding sequence can be derived therefrom.

[0056] As used herein, the term "plasmid backbone" generally refers to a circular or linear DNA molecule that can autonomously replicate and express an inserted gene of interest within a cell. The backbone plasmid may contain regulatory sequences, such as promoters, replicons, and transcriptional and translational start and stop codons. The backbone plasmid is typically linked to the gene of interest to form a complete expression vector capable of expressing a specific output in cells.

[0057] As used herein, the term "autoimmune disease" refers to a disease or disorder that is the result of or leads to a host's reaction against itself. Thus, autoimmune diseases are the result of an inappropriate and excessive reaction to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, systemic rejection of transplanted organs, etc., graft-versus-host disease, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, inflammation, systemic lupus erythematosus (lupus), multiple sclerosis, inflammatory bowel disease, myasthenia gravis, pemphigus vulgaris, psoriatic arthritis, psoriasis, rheumatism, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, and the like. syndrome), spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia and ulcerative colitis, etc.

[0058] In the present invention, the term "subject" refers to any animal that can suffer from the following: autoimmune disease; transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome or any disease / disorder involving an uncontrolled inflammatory response mediated by one or more inflammation-related factors or caused by these uncontrolled inflammatory responses. Particularly interesting subjects are humans, and scientifically relevant species such as mice, rats, ferrets, guinea pigs, hamsters, non-human primates, dogs, pigs and sheep, or economically relevant animals such as horses, dogs, cats and cattle. In a preferred embodiment, the subject is a human.

[0059] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0061] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0062] The specific amino acid and nucleotide sequence information used in this application is as follows:

[0063] SEQ ID No.1

[0064] MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLLCYRISSD

[0065] RYECSWQYEGPTAGVSHFLRCC

[0066] LSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSV

[0067] KYEPPLGDIKVSKLAGQLRMEWE

[0068] TPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGS

[0069] QGSSWSKWSSPVCVPPENPPQPQ

[0070] VRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPKAKA

[0071] TRTLHLGKMPYLSGAAYNVAVIS

[0072] SNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQ

[0073] DGGLATCSLTAPQDPDPAGMATYS

[0074] WSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNH

[0075] SLDSVSVDWAPSLLSTCPGVLKE

[0076] YVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQR

[0077] FSIEVQVSDWLIFFASLGSFLSIL

[0078] LVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALV

[0079] VEMSWDKGERTEPLEKTELPEG

[0080] APELALDTELSLEDGDRCKAKM

[0081] SEQ ID No.2

[0082] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0083] GITWTLDQSSEVLGSGKTLTIQVK

[0084] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0085] GRFTCWWLTTISTDLTFSVKSSR

[0086] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0087] LKYENYTSSFFIRDIIKPDPPKN

[0088] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0089] VICRKNASISVRAQDRYYSSSWS

[0090] EWASVPCSAS

[0091] SEQ ID No.3

[0092] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0093] GITWTLDQSSEVLGSGKTLTIQVM

[0094] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0095] GRFTCWWLTTISTDLTFSVKSSR

[0096] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0097] LKYENYTSSFFIRDIIKPDPPKN

[0098] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0099] VICRKNASISVRAQDRYYSSSWS

[0100] EWASVPCSAS

[0101] SEQ ID No.4

[0102] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0103] GITWTLDQSSYVLGSGKTLTIQVK

[0104] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0105] GRFTCWWLTTISTDLTFSVKSSR

[0106] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0107] LKYENYTSSFFIRDIIKPDPPKN

[0108] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0109] VICRKNASISVRAQDRYYSSSWS

[0110] EWASVPCSAS

[0111] SEQ ID No.5

[0112] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0113] GITWTLDQSSEVLGSGKTLTIQVK

[0114] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKHKTFLRCEAKNYS

[0115] GRFTCWWLTTISTDLTFSVKSSR

[0116] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0117] LKYENYTSSFFIRDIIKPDPPKN

[0118] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0119] VICRKNASISVRAQDRYYSSSWS

[0120] EWASVPCSAS

[0121] SEQ ID No.6

[0122] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPLAPGEMVVLTCDTPEEDG

[0123] ITWTLDQSSEVLGSGKTLTIQVK

[0124] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0125] GRFTCWWLTTISTDLTFSVKSSR

[0126] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0127] LKYENYTSSFFIRDIIKPDPPKN

[0128] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0129] VICRKNASISVRAQDRYYSSSWS

[0130] EWASVPCSAS

[0131] SEQ ID No.7

[0132] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0133] GITWTLDQSSEVLGSGKTLTIQVK

[0134] QFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0135] GRFTCWWLTTISTDLTFSVKSSR

[0136] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0137] LKYENYTSSFFIRDIIKPDPPKN

[0138] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0139] VICRKNASISVRAQDRYYSSSWS

[0140] EWASVPCSAS

[0141] SEQ ID No.8

[0142] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0143] GITWTLDQSSEVLGSGKTLTIQVK

[0144] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0145] GRFTCWWLTTISTDLTFSVKSSR

[0146] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHR

[0147] LKYENYTSSFFIRDIIKPDPPKN

[0148] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0149] VICRKNASISVRAQDRYYSSSWS

[0150] EWASVPCSAS

[0151] SEQ ID No.9

[0152] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEED

[0153] GITWTLDQSSEVLGSGKTLTIQVK

[0154] EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS

[0155] GRFTCWWLTTISYDLTFSVKSSR

[0156] GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

[0157] LKYENYTSSFFIRDIIKPDPPKN

[0158] LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSAT

[0159] VICRKNASISVRAQDRYYSSSWS

[0160] EWASVPCSAS

[0161] SEQ ID No.10

[0162] MALPVTALLLPLALLLHAARP

[0163] SEQ ID No.11

[0164] METDTLLLWVLLLWVPGSTG

[0165] SEQ ID No.12

[0166] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0167] SEQ ID No.13

[0168] DIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0169] SEQ ID No.14

[0170] VWVNAGMWCSDSGVSNKVTWSTV

[0171] SEQ ID No.15

[0172] EPKSCDKTHTCPPCP

[0173] SEQ ID No.16

[0174] VPRDCGCSPGCPPAAPSVAAPAPPSPTPTPTPTPTPTPTPTPTPTPTPTPTPTPT

[0175] SEQ ID No.17

[0176] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0177] SEQ ID No.18

[0178] ALCVLGLVAGVLVGLLLPLGILG

[0179] SEQ ID No.19

[0180] LILLGTSLVCLVFLSLGILA

[0181] SEQ ID No.20

[0182] IYIYLVVLLLNSAVYLIHR

[0183] SEQ ID No.21

[0184] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQG

[0185] QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0186] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0187] SEQ ID No.22

[0188] MALPVTALLLPLALLLHAARPASMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVE

[0189] LDWYPDAPGEMVVLTCDTPEEDG

[0190] ITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEAGIWSTD

[0191] ILKDQKEPKNKTFLRCEAKNYS

[0192] GRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQE

[0193] DSACPAAEESLPIEVMVDAVHK

[0194] LKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQ

[0195] VQGKSKREKKDRVFTDKTSAT

[0196] VICRKNASISVRAQDRYYSSSWSEWASVPCSASDIYFCKIEVMYPPPYLDNEKSNGTII

[0197] HVKGKHLCPSPLFPGPSKPFW

[0198] VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP

[0199] PRDFAAYRSRVKFSRSADAPAYQ

[0200] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

[0201] AYSEIGMKGERRRGKGHDGLYQGLS

[0202] TATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIP

[0203] RKVCNGIGIGEFKDSLSINATN

[0204] IKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTD

[0205] LHAFENLEIIRGRTKQHGQ

[0206] FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRG

[0207] ENSCKATGQVCHALCSPEGC

[0208] WGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTG

[0209] RGPDNCIQCAHYIDGPHCVKTC

[0210] PAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0211] SEQ ID No.23

[0212]

[0213] Example 1 Affinity detection of mutation sites

[0214] The mutant P40 gene sequence (shown in SEQ ID No. 2-9) was submitted to a gene synthesis company (Shanghai Sangon Biotech) for gene synthesis. The synthesized gene was inserted into the pLVX-EF1 plasmid via the Xba1 and Not1 restriction sites. The functional composition of the inserted sequence is shown in Figure 1 First, construct the pLVX-EF1 vector to express wild-type P40 and different mutants carrying EGFR tags. Figure 1 293T cells grown to 80% confluence (seeding density 2×10 5 cells / well, DMEM+10% FBS medium) were transfected with 1 μg of plasmid and 3 μL of PEI (1 mg / mL), and cultured at 37°C, 5% CO2 for 48 hours until protein expression was high; the cells were digested and collected, washed with PBS (centrifuged at 300g for 5 min), and then distributed into flow cytometry tubes (5×10 5 Cells were isolated and plated at 4°C (cells / tube). Fc receptor blocking (anti-human CD16 / 32 antibody) was performed for 15 minutes, followed by incubation with detection antibody (recombinant IL12RB1-Fc fusion protein, 2 μg / mL) for 30 minutes at 4°C in the dark. The cells were then stained with APC-conjugated secondary antibody (anti-human Fc) and anti-EGFR-PE antibody for 30 minutes. After washing with PBS + 2% BSA, the cells were analyzed on a BD FACSCanto II. Data were analyzed using FlowJo software to calculate the MFI ratio of each group. Untransfected cells were used as background subtraction, and EGFR was used as the transfection efficiency to eliminate the influence of protein expression differences caused by varying transfection efficiency on protein binding. Binding specificity was verified by calculating relative binding using the formula: [(mutant IL12RB1 MFI – untransfected IL12RB1 MFI)] / [(mutant EGFR MFI – untransfected EGFR MFI] The resulting value for the mutant group was compared with the wild-type group and multiplied by 100 to obtain the final data.

[0215] The above results are as follows Figure 2 As shown, the results showed that the affinity of different p40 mutants to IL12RB1 was significantly different.

[0216] Example 2 Detection of CD69 Activation by CAR-Treg Cells

[0217] Preparation of CAR-Treg cells: PBMCs were obtained from healthy donors’ peripheral blood using Ficoll-Paque PLUS (Cytiva, 17-1440-03). CD8+ cells were removed using the Miltenyi Biotec Human CD8 MicroBeads negative selection kit (Miltenyi Biotec, 130-045-201). CD4+CD25+ Treg populations were then isolated from CD8- cells using CD25 positive selection magnetic beads (Human CD25 MicroBeads, Miltenyi Biotec, 130-092-983). The initial yield was approximately 2×10^6 cells / 100 mL whole blood (purity ≥92%). The sorted Treg cells were plated at a density of 1×10^6 cells / mL in TexMACS medium (Miltenyi Biotec, 130-092-983). After 48 h of activation with 300 IU / mL recombinant human IL-2 (PeproTech, 200-02) and CD3 / CD28 magnetic beads (Gibco, 11161D) at a bead-to-cell ratio of 4:1, cells were transduced with a third-generation lentiviral vector carrying an EGFR-tag (expressing a p40 subunit mutant + EGFR-tag fusion protein with the amino acid sequence shown in SEQ ID No. 22) (LV-CAR, titer 5 × 10^7 TU / mL) (MOI = 5, Polybrene 8 μg / mL, Sigma, TR-1003-G) and cultured for 72 h. On day 11 after transduction, cells were washed three times with 0.5 mM EDTA / PBS to remove residual viral particles and then stained with anti-EGFR APC antibody (BioLegend, 352906, 1:100 dilution) at 4°C for 30 min. Anti-APC microbeads (Miltenyi Biotechnology, Inc., MI) were then added. Biotec, 130-090-855) were used for positive sorting to obtain a cell population with a CAR-Treg positive rate of ≥85%. The sorted CAR-Tregs were cultured at a density of 5×10^5 cells / mL in X-VIVO 15 medium (Lonza, BE02-060F) containing 300 IU / mL IL-2 and activated with CD3 / CD28 magnetic beads (Gibco, 11161D) at a bead-to-cell ratio of 4:1. The medium was replaced by half every 48 hours. After a total of 16 days of culture, the cells could be harvested at a D of 1.2-1.8×10^8 cells were finally confirmed by flow cytometry (BD FACSAria III) to have a CAR expression rate ≥78% and a cell viability >95% (7-AAD negative rate). The cells were then cryopreserved in CryoStor CS10 (BioLife Solutions, 210102) containing 5% DMSO (STEMCELL, 07930) for subsequent experiments.

[0218] The above process is as follows Figure 3 As shown, the results of flow cytometry detection of CAR-Treg cells are as follows Figure 4 The results showed that the positive rate of G83FCAR-Treg cells was about 40%.

[0219] The CAR-Treg cell CD69 activation assay was performed as follows: frozen and revived CAR-Treg cells (5 × 10^5 cells / group) were divided into three treatment groups: ① CD3 / CD28 activation group was stimulated using Human T-Activator CD3 / CD28 magnetic beads (Gibco11161D) at a 1:1 bead-to-cell ratio; ② target activation group was stimulated using a complex of IL12RB1-His recombinant protein (Sino Biological10084-H08H) coupled to Tosyl magnetic beads (Dynabeads M-280 Tosylactivated, Invitrogen 14203), with 20 μg protein coupled to each 1 mg of magnetic beads (rotated at room temperature for 16 hours and then blocked with PBS + 0.1% BSA), at a 1:1 bead-to-cell ratio; ③ unactivated control group was stimulated using only TexMACS basal medium (Miltenyi 170-076-307). All three groups of cells were seeded in 48-well plates at a density of 2×10^5 cells / mL and supplemented with 300 IU / mL IL-2 (PeproTech 200-02). After incubation in a 37°C incubator for 24 hours, the cells were collected and washed three times with 0.5 mM EDTA / PBS. The cells were then labeled with FixableViability Dye eFluor 506 (eBioscience 65-0866-18) at room temperature in the dark for 15 minutes, and then stained with anti-human CD69-PE antibody (BioLegend310906, diluted 1:50) and EGFR-APC antibody (transduction marker, BioLegend352906) at 4°C in the dark for 30 minutes. Finally, the cells were resuspended in PBS + 2% FBS, detected by Beckman CytoFLEX LX flow cytometer, and analyzed using FlowJo v10.8 software: the gating strategy first excluded debris (FSC-A / SSC-A) and dead cells (FVD506+), and CAR-Treg were identified by EGFR-FITC.

[0220] The above results are as follows Figure 5 As shown, this indicates that D109A CAR-Treg cells can be activated specifically by the target. After activating D109A CAR Treg cells with CD212 protein-coated magnetic beads, CD69 protein in CAR-positive cells was significantly upregulated, comparable to the level after activation with CD3 / CD28 magnetic beads. However, CD69 in CAR-negative cells was not upregulated, and the level was comparable to that of the resting group, indicating that D109A CAR-Treg can be activated specifically by the target.

[0221] Example 3 In vitro verification of CAR-Treg inhibitory function

[0222] The specific operation of the validation experiment is as follows: first, mononuclear cells were isolated from the blood of healthy donors using human PBMC separation solution (Ficoll-Paque PLUS, Cytiva17-1440-03). CD4+CD25- Teff cells were obtained using the Miltenyi CD4+ T cell isolation kit (Miltenyi Biotec 130-096-533) combined with CD25 microbead negative selection (Miltenyi 130-092-983). Then, they were labeled with 5μM CFSE (Thermo Fisher C34554) at 37°C for 15 minutes, centrifuged (300g×5min) and washed three times; then, Teff cells were co-incubated with anti-CD3 / CD28 Dynabeads (Gibco 11161D, bead-to-cell ratio 3:1) at a density of 1×10^5 cells / well in a U-bottom 96-well plate (Corning 3799) for 24 hours to achieve pre-activation. Meanwhile, CAR-Treg cells were treated with three different pathways: ① Activation Group A: stimulation with Gibco CD3 / CD28 magnetic beads (bead-to-cell ratio 3:1); ② Target Activation Group B: incubation with IL12RB1-Fc fusion protein (R&D Systems 6579-RB)-coated streptavidin magnetic beads (M-280 Streptavidin Beads, Invitrogen 11205D) (20 μg protein / mg beads, bead-to-cell ratio 2:1); and ③ Unactivated Group C: no stimulation. Treated CAR-Treg cells were washed with PBS (300g × 5 min × 3 times) and then co-cultured with preactivated Teff cells at a target-to-target ratio of 1:1 (2 × 10^5 cells / well) in TexMACS medium (Miltenyi 170-076-307) supplemented with 300 IU / mL IL-2 (PeproTech 200-02) for 72 hours. After the cells were collected, CFSE signal attenuation was detected by Beckman CytoFLEX flow cytometer, and the inhibition rate was calculated as [(PI of Teff alone group - PI of co-culture group) / PI of Teff alone group]×100%.

[0223] The above results are as follows Figure 6 As shown, the inhibitory function of D109A CARTreg cells is higher than that of the Treg cell group that does not express CAR. Secondly, the inhibitory function of CARTreg is proportional to the amount of addition, indicating that the inhibitory function has an obvious dose effect. The more cells added, the higher the inhibitory function.

[0224] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of an IL12RB1 activating factor in the preparation of a regulatory T cell product, wherein the amino acid sequence of IL12RB1 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The IL12RB1 activating factor comprises a P40 subunit or a mutant thereof, wherein the amino acid sequence of the P40 subunit is shown in SEQ ID No. 2; and / or the P40 subunit mutant has one or more of the following mutations: D109A, K80M, E67Y, N125H, D40L, K217R or T150Y.

3. The use according to claim 1, characterized in that The IL12RB1 activating factor is a chimeric antigen receptor.

4. The use according to claim 3, characterized in that The chimeric antigen receptor has one or more of the following characteristics: 1) The P40 subunit or its mutant is the antigen binding domain of the chimeric antigen receptor; 2) The guide peptide is selected from CD8 guide peptide or GM-CSF guide peptide; 3) The hinge domain is selected from any one of a CD8α hinge domain, a CD28 hinge domain, a CD4 hinge domain, an IgG hinge domain, and an IgD hinge domain; 4) The transmembrane domain is selected from the transmembrane domain of any of the following proteins: CD28, CD28T, OX-40, 4-1BB, CD137, CD2, CD7, CD8, CD27, CD30, CD40, programmed death-1, inducible T cell co-stimulatory factor, lymphocyte function-associated antigen-1, CD3γ, CD3δ, CD3ε, CD247, CD276, LIGHT, TNFSF14, NKG2C, Igα, DAP10, Fcγ receptor, MHC class 1 molecule or TNF receptor protein; 5) The intracellular signaling domain is selected from the intracellular signaling domain of any of the following proteins: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b or CD66d; 6) The chimeric antigen receptor further comprises a cleavage peptide; preferably, the cleavage peptide is selected from one or more of P2A, E2A, F2A or T2A; 7) The domains in the chimeric antigen receptor are connected in the following manner: guide peptide-P40 subunit or its mutant-hinge domain-transmembrane domain-intracellular signaling domain.

5. A P40 subunit mutant, characterized in that: The P40 subunit mutant has one or more of the following mutations relative to the wild-type P40 subunit: D109A, K80M, E67Y, N125H, D40L, K217R or T150Y, wherein the amino acid sequence of the wild-type P40 subunit is shown in SEQ ID No.

2.

6. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the P40 subunit mutant according to claim 5.

7. A polynucleotide, characterized in that The polynucleotide encodes the P40 subunit mutant according to claim 5 or the chimeric antigen receptor according to claim 6.

8. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide of claim 7.

9. The nucleic acid construct according to claim 8, characterized in that The nucleic acid construct is constructed by inserting the isolated polynucleotide according to claim 7 into the multiple cloning site of an expression vector.

10. A viral vector, characterized in that The viral vector contains the polynucleotide according to claim 7 or the nucleic acid construct according to claim 8 or 9.

11. A regulatory T cell targeting IL12RB1, characterized in that The regulatory T cells contain the P40 subunit or a mutant thereof.

12. The regulatory T cell according to claim 11, characterized in that The regulatory T cells contain the chimeric antigen receptor according to claim 6.

13. Use of the polynucleotide of claim 7, the nucleic acid construct of claim 8 or 9, or the regulatory T cells of claim 11 or 12 in the preparation of drugs for treating autoimmune diseases, transplant rejection, graft-versus-host disease, cytokine release syndrome, or diseases involving uncontrolled inflammatory responses mediated by one or more inflammatory-related factors or caused by these uncontrolled inflammatory responses.

14. The use according to claim 13, characterized in that The autoimmune disease is selected from any one or more of rheumatoid arthritis, psoriatic arthritis, psoriasis, lupus, juvenile rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease or Crohn's disease; and / or the transplant rejection is selected from any one or more of organ transplant rejection, stem cell transplant rejection or bone marrow transplant rejection.

Images