A p40 subunit mutant and uses thereof

Abstract

The present application relates to the field of cell therapy, in particular to a p40 subunit mutant and use thereof. The p40 subunit mutant provided by the present application has a mutation at the 83rd site compared with a wild-type p40 subunit, and the amino acid sequence of the wild-type p40 subunit is shown as SEQ ID No. 1. The present application constructs a CAR-Treg with the following functions by specifically targeting a structure combining a double signal regulation module of IL12RB1 p40 subunit: accurately recognizing IL12RB1 high-expression cells at an inflammatory site; and activating Treg cells at the target site to enhance the inhibitory function.

Description

Technical Field

[0001] This invention relates to the field of cell therapy, and in particular to a p40 subunit mutant and its uses. Background Technology

[0002] In the field of immunotherapy, the IL-12 / IL-23 pathway and CAR-Treg therapy are two important research directions. IL-12 and IL-23 are cytokines with similar structures that play a crucial role in immune regulation, participating in the body's immune defense and being closely related to the occurrence and development of various autoimmune diseases. Traditional antibody drugs have significant limitations when targeting this pathway, as they cannot effectively distinguish between protective and pathogenic signals within the pathway. For example, while inhibiting the IL-12 / IL-23 pathway can alleviate inflammatory symptoms in the treatment of certain inflammatory diseases, it may also weaken the body's normal immune defense function, making patients more susceptible to infection and potentially disrupting the normal balance of the immune system, thereby triggering other potential adverse reactions.

[0003] CAR-Treg, as an emerging immunotherapy approach, aims to precisely regulate immune responses by genetically engineering regulatory T cells, showing great potential in treating autoimmune diseases and preventing transplant rejection. However, current CAR-Treg therapy still faces the problem of insufficient target specificity, making it prone to off-target effects. Off-target effects may cause CAR-Treg to attack non-target cells, damage normal tissues, and trigger a series of adverse reactions. For example, in the treatment of solid tumors, since tumor-associated antigens are also expressed in normal tissues, CAR-Treg may attack these normal cells, causing tissue damage and functional impairment. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a p40 subunit mutant and its uses to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides a p40 subunit mutant, wherein the p40 subunit mutant has a mutation at position 83 compared to the wild-type p40 subunit, and the amino acid sequence of the wild-type p40 subunit is shown in SEQ ID No. 1.

[0006] Preferably, the mutation is performed by changing glycine to any one of leucine, phenylalanine, isoleucine, glutamic acid, or aspartic acid.

[0007] The present invention also provides a chimeric antigen receptor containing the aforementioned p40 subunit mutant.

[0008] The present invention also provides an in vitro 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 polynucleotides.

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

[0011] The present invention also provides a CAR-Treg cell, wherein the CAR-Treg cell comprises the aforementioned P40 subunit mutant or the aforementioned chimeric antigen receptor.

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

[0013] As described above, the p40 subunit mutant of the present invention and its use have the following beneficial effects:

[0014] This invention constructs a CAR-Treg with the following functions by combining an IL12RB1 p40 subunit-specific targeting structure with a dual signal regulation module: accurately identifying IL12RB1-highly expressing cells at inflammatory sites; activating Treg cells at the target site and enhancing inhibitory function. Attached Figure Description

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

[0016] Figure 2 The diagram shows the affinity detection of different mutations at the p40 site according to the present invention.

[0017] Figure 3 The diagram shown illustrates the construction and detection of CAR-Treg cells according to the present invention.

[0018] Figure 4 The diagram shown illustrates the CAR-Treg cell positivity rate detection method of this invention.

[0019] Figure 5 The diagram shown illustrates the CAR-Treg cell-specific activation detection method of this invention.

[0020] Figure 6 The diagram shown illustrates the in vitro inhibitory function detection of CAR-Treg cells according to the present invention.

[0021] Figure 7 The diagram shows the changes in body weight in a mouse model of GVHD inhibition by CAR-Treg cells according to the present invention.

[0022] Figure 8 The diagram shows the changes in the in vivo GVHD suppression mouse score in the CAR-Treg cell model of the present invention. Detailed Implementation

[0023] The present invention provides a p40 subunit mutant, wherein the p40 subunit mutant has a mutation at position 83 compared with the wild-type p40 subunit, and the amino acid sequence of the wild-type p40 subunit is shown in SEQ ID No. 1.

[0024] In some specific embodiments, the mutation is that glycine is mutated into any one of alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0025] Further, the mutation is a mutation of glycine into any one of leucine, phenylalanine, isoleucine, glutamic acid, or aspartic acid. Preferably, the mutation is G83F.

[0026] In some specific embodiments, the amino acid sequence of the p40 subunit mutant is shown in SEQ ID No. 2.

[0027] In some specific embodiments, the p40 subunit mutant, compared to the wild-type p40 subunit, also exhibits one or more of the following mutations: G83E, G83L, G83N, G83I. Preferably, the amino acid sequence of the p40 subunit mutant is shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6.

[0028] The present invention also provides a chimeric antigen receptor containing the aforementioned p40 subunit mutant.

[0029] In some specific 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.

[0030] Furthermore, the P40 subunit or its mutant is the antigen-binding domain of the chimeric antigen receptor.

[0031] Further, 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 in SEQ ID No. 7; or, the amino acid sequence of GM-CSF guide peptide is shown in SEQ ID No. 8.

[0032] Further, the hinge domain is selected from any one of the CD8α hinge domain, CD28 hinge domain, CD4 hinge domain, IgG hinge domain, or IgD hinge domain, wherein the amino acid sequence of the CD8α hinge domain is as shown in SEQ ID No. 9; or, the amino acid sequence of the CD28 hinge domain is as shown in SEQ ID No. 10; or, the amino acid sequence of the CD4 hinge domain is as shown in SEQ ID No. 11; or, the amino acid sequence of the IgG hinge domain is as shown in SEQ ID No. 12; or, the amino acid sequence of the IgD hinge domain is as shown in SEQ ID No. 13. Preferably, the hinge domain is a CD28 hinge domain.

[0033] Further, the transmembrane domain is selected from the transmembrane domains of any of the following proteins: CD28, CD28T, OX-40, 4-1BB, CD137, CD2, CD7, CD8, CD27, CD30, CD40, programmed cell death-1 (PD-1), inducible T cell co-stimulatory factor (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 molecules, or TNF receptor protein, wherein the amino acid sequence of the CD28 transmembrane domain is as shown in SEQ ID No. 14; or, the amino acid sequence of the 4-1BB transmembrane domain is as shown in SEQ ID No. 15; or, the amino acid sequence of the OX-40 transmembrane domain is as shown in SEQ ID No. 14. As shown in No. 16; or, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID No. 17. Preferably, the transmembrane domain is the CD28 transmembrane domain.

[0034] Further, the intracellular signaling domain is selected from the intracellular signaling domains 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. 18. Preferably, the intracellular signaling domain is the CD3ζ intracellular signaling domain.

[0035] In some specific embodiments, the chimeric antigen receptor further comprises a cleavage peptide. Specifically, the cleavage peptide is selected from one or more of P2A, E2A, F2A, or T2A.

[0036] In some specific embodiments, the domains in the chimeric antigen receptor are connected in the following manner: guide peptide - P40 subunit mutant - hinge domain - transmembrane domain - intracellular signaling domain.

[0037] In some specific embodiments, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 19.

[0038] The invention also provides an in vitro polynucleotide that encodes the aforementioned P40 subunit mutant or the aforementioned chimeric antigen receptor.

[0039] In some specific embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID No. 20.

[0040] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotides.

[0041] In some specific embodiments, the nucleic acid construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. The expression vector can be transformed, transduced, or transfected into host cells, allowing its carried genetic material elements to be expressed within the host cells. The construct can be a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0042] In some specific embodiments, the backbone plasmid of the expression vector may 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.

[0043] The present invention also provides a viral vector containing the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0044] In some specific embodiments, the viral vector is selected from one or more of lentiviruses, adenoviruses, or adeno-associated viruses.

[0045] The present invention also provides a CAR-Treg cell, wherein the CAR-Treg cell comprises the aforementioned P40 subunit mutant or the aforementioned chimeric antigen receptor.

[0046] The present invention also provides the use of the aforementioned polynucleotides, the aforementioned nucleic acid constructs, the aforementioned viral vectors, or the aforementioned regulatory T cells in the preparation of therapeutic drugs for autoimmune diseases, transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome, or in therapeutic drugs relating to uncontrolled inflammatory responses mediated by one or more inflammatory factors or diseases caused by such uncontrolled inflammatory responses.

[0047] In some specific 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.

[0048] In some specific embodiments, the transplant rejection is selected from any one or more of organ transplant rejection, stem cell transplant rejection, or bone marrow transplant rejection.

[0049] 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 / condition involving or caused by an uncontrolled inflammatory response mediated by one or more inflammatory-related factors, wherein the method comprises administering a therapeutically effective number of the aforementioned CAR-Treg cells to treat the autoimmune disease, transplant rejection, or graft-versus-host disease (GVHD).

[0050] The present invention also provides a method for inducing immune tolerance in a subject in need, wherein the method comprises administering a therapeutically effective number of the aforementioned CAR-Treg cells, thereby inducing immune tolerance in the subject in need.

[0051] The present invention also provides a method for locally or systemically downregulating inflammation in a subject in need, wherein the method comprises administering a therapeutically effective number of the aforementioned CAR-Treg cells, thereby locally or systemically downregulating inflammation in the subject in need.

[0052] In this invention, the term "Treg cell" also refers to a subset of regulatory T cells or 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.

[0053] In this invention, the terms "polynucleotide" and "nucleic acid" (or the singular form) are used interchangeably. 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".

[0054] In this invention, the term "encoding" refers to the inherent characteristic of a specific nucleotide sequence in a polynucleotide (or the entire polynucleotide) such as a gene, cDNA (including transgenic cDNA), or mRNA, to serve as a template for other polymers and macromolecules in the process of synthesizing biology. These polymers and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes said 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 gene or cDNA transcription) can be referred to as encoding the protein or other product of that gene or cDNA. If, in its natural state or when manipulated by methods well known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce mRNA and / or fragments of a polypeptide or a polypeptide or a polypeptide or a fragment thereof, then the polynucleotide may be said to "encode" that polypeptide. The antisense strand is the complementary strand of such nucleic acid, from which the coding sequence can be deduced.

[0055] In this invention, the term "plasmid backbone" is generally a circular or linear DNA molecule that can autonomously replicate and express the inserted target gene within a cell. The backbone plasmid may contain regulatory sequences, such as promoters, replicons, and transcription and translation initiation and termination codons. The backbone plasmid is typically linked with the target gene to form a complete expression vector capable of expressing a specific output within the cell.

[0056] In this invention, the term "autoimmune disease" refers to a disease or disorder resulting from or causing a host's response to itself. Therefore, an autoimmune disease is the result of an inappropriate and excessive response to its own antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, type I diabetes, malnutrition-related epidermolysis bullosa, epididymitis, glomerulonephritis, systemic rejection of transplanted organs, 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, and Sjögren's syndrome. (syndrome), spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, etc.

[0057] In this invention, the term "subject" refers to any animal capable of suffering from: autoimmune diseases; transplant rejection, graft-versus-host disease (GVHD), cytokine release syndrome, or any disease / condition involving or caused by one or more inflammatory factors. Subjects of particular interest are humans, as well as science-related 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.

[0058] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0059] Before further describing 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 embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0060] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0061] SEQ ID No.1

[0062] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDP QGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0063] SEQ ID No.2

[0064] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFFDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0065] SEQ ID No.3

[0066] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFEDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0067] SEQ ID No.4

[0068] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFLDAQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0069] SEQ ID No.5MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFNDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0070] SEQ ID No. 6

[0071] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFIDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSAS

[0072] SEQ ID No.7

[0073] MALPVTALLLPLALLLHAARP

[0074] SEQ ID No.8

[0075] METDTLLLWVLLLWVPGSTG

[0076] SEQ ID No.9

[0077] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0078] SEQ ID No.10

[0079] DIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0080] SEQ ID No.11

[0081] VWVNAGMWCSDSGVSNKVTWSTV

[0082] SEQ ID No.12

[0083] EPKSCDKTHTCPPCP

[0084] SEQ ID No.13

[0085] VPRDCGCSPGCPPAAPSVAAPAPPSPTPTPTPTPTPTPTPTPTPTPTPTPTPTPT

[0086] SEQ ID No.14

[0087] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0088] SEQ ID No.15

[0089] ALCVLGLVAGVLVGLLLPLGILG

[0090] SEQ ID No.16

[0091] LILLGTSLVCLVFLSLGILA

[0092] SEQ ID No.17

[0093] IYIYLVVLLLNSAVYLIHR

[0094] SEQ ID No.18

[0095] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQG

[0096] QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

[0097] EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0098] SEQ ID No.19

[0099] MALPVTALLLPLALLLHAARPMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVE

[0100] LDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFFDAGQYTCHK

[0101] GGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTIST

[0102] DLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIE

[0103] VMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHS

[0104] YFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASV

[0105] PCSDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVL

[0106] ACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR

[0107] VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG

[0108] LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID No.20

[0109]

[0110] SEQ ID No.21

[0111] MALPVTALLLPLALLLHAARPMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFFDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0112] Example 1 Detection of Mutation Site Affinity

[0113] The mutated P40 amino acid sequence (SEQ ID No. 1-6) was given to a gene synthesis company (Shanghai Sangon Biotech) for gene synthesis. The synthesized gene was inserted into the pLVX-EF1 plasmid via Xba1 and Not1 restriction sites. The functional composition of the inserted sequence is shown in [reference needed]. Figure 1 First, the pLVX-EF1 vector was constructed to express wild-type P40 carrying the EGFR tag and different mutants, as shown in the following structure. Figure 1 ; 293T cells grown to 80% confluence (seeding density 2×10⁶) 5 Cells / well, DMEM + 10% FBS medium, were transfected with 1 μg plasmid and 3 μL PEI (1 mg / mL) and cultured at 37°C and 5% CO2 for 48 hours until high protein expression was achieved. Cells were then digested, washed with PBS (centrifuged at 300g × 5 min), and aliquoted into flow cytometry tubes (5 × 10⁻⁶ cells / well). 5 Cells / tubes were sequentially subjected to 15 minutes of Fc receptor blocking (anti-human CD16 / 32 antibody), 30 minutes of incubation at 4°C in the dark with detection antibody (recombinant IL12RB1-Fc fusion protein, 2 μg / mL), 30 minutes of APC-labeled secondary antibody (anti-human Fc), and staining with anti-EGFR-PE antibody. After washing with PBS + 2% BSA, the samples were analyzed using FlowJo software to calculate the MFI ratio for each group, with untransfected cells subtracted as background. EGFR was used as the transfection efficiency value to exclude the influence of protein expression differences caused by different transfection efficiencies on the protein binding detection results. To verify binding specificity, the relative binding number was calculated using the formula: [(mutant IL12RB1 MFI – untransfected IL12RB1 MFI)] / [(mutant EGFRMFI – untransfected EGFRMFI]. The calculated mutant group value was multiplied by 100% to obtain the final data.

[0114] The above results are as follows Figure 2 As shown, the results indicate that, compared with the wild-type p40 sequence, the mutant p40 protein has altered its affinity for IL12RB1. Specifically, the affinity of G83F is greatly enhanced, the affinity of G83E and G83L is not significantly different from that of the wild type, the affinity of G83I is reduced, and the affinity of G83N is greatly reduced.

[0115] Example 2: Detection of CD69 activation by CAR-Treg cells

[0116] Preparation of CAR-Treg cells: PBMCs were isolated from healthy donor peripheral blood using Ficoll-Paque PLUS (Cytiva, 17-1440-03). CD8+ cells were then knocked out using the Miltenyi Human CD8 MicroBeads negative sorting kit (Miltenyi Biotec, 130-045-201). Subsequently, CD4+CD25+ Treg populations were sorted from CD8- cells using CD25 positive magnetic beads (Human CD25 MicroBeads, Miltenyi Biotec, 130-092-983), with an initial yield of approximately 2×10^6 cells / 100mL whole blood (purity ≥92%). The sorted Treg cells were then seeded at a density of 1×10^6 cells / mL on TexMACS medium (Miltenyi Biotec). In Biotec (170-076-307), 300 IU / mL recombinant human IL-2 (PeproTech, 200-02) and CD3 / CD28 magnetic beads (Gibco, 11161D) were added at a bead-to-cell ratio for 48 hours. After activation, a third-generation lentiviral vector carrying an EGFR-tag (expressing the p40 subunit mutant + EGFR-tag fusion protein with the amino acid sequence shown in SEQ ID No. 21) (LV-CAR, titer 5 × 10^7 TU / mL) was added for transduction (MOI = 5, Polybrene 8 μg / mL, Sigma, TR-1003-G), and the cells were cultured for 72 hours. On day 11 after transduction, the cells were washed three times with 0.5 mM EDTA / PBS to remove residual viral particles. They were then stained with anti-EGFR APC antibody (BioLegend, 352906, 1:100 dilution) at 4°C for 30 minutes, and then treated with anti-APC microbeads (Miltenyi). Biotec (130-090-855) performed positive sorting to obtain a CAR-Treg positive rate ≥85% cell population; the sorted CAR-Tregs were placed at a density of 5×10^5 cells / mL in X-VIVO 15 medium (Lonza, BE02-060F) containing 300 IU / mL IL-2, and CD3 / CD28 magnetic beads (Gibco, 11161D) were used to activate dynamic expansion at a bead-to-cell ratio of 4:1. Half of the medium was changed every 48 hours. After a total of 16 days of culture, 1.2-1 cells were harvested.Eight × 10^8 cells were collected, and flow cytometry analysis (BD FACSAria III) confirmed CAR expression ≥78% and cell viability >95% (7-AAD negative rate). Cells were then cryopreserved in CryoStor CS10 (BioLife Solutions, 210102) containing 5% DMSO (STEMCELL, 07930) for subsequent experiments.

[0117] 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 As shown in the figure. The results showed that the positive rate of G83FCAR-Treg cells was around 40%.

[0118] The CD69 activation detection procedure for CAR-Treg cells was implemented as follows: Frozen and thawed CAR-Treg cells (5×10^5 cells / group) were divided into three groups for treatment: ① CD3 / CD28 activation group was stimulated with Human T-Activator CD3 / CD28 magnetic beads (Gibco11161D) at a 1:1 bead-to-cell ratio; ② Target activation group was stimulated with a complex of IL12RB1-His recombinant protein (Sino Biological10084-H08H) and Tosyl magnetic beads (Dynabeads M-280Tosylactivated, Invitrogen 14203), with 20μg of protein conjugated to each 1mg of magnetic beads (conjugated by rotation at room temperature for 16 hours and then blocked with PBS+0.1% BSA) at a 1:1 bead-to-cell ratio; ③ The non-activated control group was treated with only TexMACS basal medium (Miltenyi 170-076-307). All three groups of cells were seeded at a density of 2×10^5 cells / mL in 48-well plates, supplemented with 300 IU / mL IL-2 (PeproTech 200-02), and incubated at 37°C for 24 hours. After incubation, the cells were collected, washed three times with 0.5 mM EDTA / PBS, and labeled with FixableViability Dye eFluor 506 (eBioscience 65-0866-18) at room temperature in the dark for 15 minutes. Then, they were stained with anti-human CD69-PE antibody (BioLegend310906, 1:50 dilution) and EGFR-APC antibody (transduction label, BioLegend352906) at 4°C in the dark for 30 minutes. Finally, the cells were resuspended in PBS + 2% FBS and analyzed by Beckman CytoFLEX LX flow cytometer. FlowJo v10.8 software was used for analysis: a gating strategy was first used to exclude debris (FSC-A / SSC-A) and dead cells (FVD506+), and CAR-Treg was identified by EGFR-FITC.

[0119] The above results are as follows Figure 5 As shown, G83FCAR-Treg cells can be specifically activated by the target. After activation of G83FCAR-Treg cells using magnetic beads coated with CD212 protein, 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, remaining at the same level as in the resting group, indicating that G83FCAR-Treg cells can be specifically activated by the target.

[0120] Example 3: In vitro verification of CAR-Treg inhibitory function

[0121] The specific procedures for the verification experiment are as follows: First, mononuclear cells were isolated from the blood of healthy donors using human PBMC separation medium (Ficoll-Paque PLUS, Cytiva 17-1440-03). After obtaining CD4+CD25-Teff cells using the Miltenyi CD4+ T cell sorting kit (Miltenyi Biotec 130-096-533) combined with CD25 microbead negative selection (Miltenyi 130-092-983), the cells were labeled with 5 μM CFSE (Thermo Fisher C34554) at 37℃ for 15 minutes, centrifuged (300g×5min) and washed three times. Subsequently, the Teff cells were pre-activated by co-incubating 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. Meanwhile, CAR-Treg cells were treated via three different pathways: ① Activation group A: stimulated with Gibco CD3 / CD28 magnetic beads (bead-to-cell ratio 3:1); ② Target activation group B: co-incubated with streptavidin magnetic beads (M-280 Streptavidin Beads, Invitrogen 11205D) coated with IL12RB1-Fc fusion protein (R&D Systems 6579-RB) (20 μg protein / mg magnetic beads, bead-to-cell ratio 2:1); ③ Unactivated group C: no stimulants were added. After treatment, the CAR-Treg cells were washed with PBS (300g × 5min × 3 times) and then co-cultured with pre-activated Teff cells at an effector-to-target ratio of 1:1 (2 × 10^5 cells / well) in TexMACS medium (Miltenyi 170-076-307) for 72 hours. The medium contained 300 IU / mL IL-2 (PeproTech 200-02). After cell collection, CFSE signal attenuation was detected by Beckman CytoFLEX flow cytometry. The inhibition rate was calculated as [(Teff alone PI - co-culture PI) / Teff alone PI] × 100%.

[0122] The above results are as follows Figure 6 As shown, the inhibitory function of G83FCARTreg cells is higher than that of Treg cells that do not express CAR. Furthermore, the inhibitory function of CARreg is directly proportional to the amount added, indicating that the inhibitory function has a significant dose-response effect; the more cells added, the higher the inhibitory function.

[0123] Example 4: In vivo verification of CAR-Treg inhibitory function

[0124] The CAR-Treg cell therapy mouse model of GvHD was established as follows: 12 mice arrived at the animal facility and, after x days of acclimatization, were divided into two groups: a control group (Control, N=7) and a treatment group (CAR, N=5). All mice were administered human PBMCs intraperitoneally on day 0 (D0) at a dose of 5 × 10⁻⁶. 6 Cells / mice were used to establish a GvHD model. Female NOG mice aged 5.6-8 weeks were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Mice were housed in the SPF-grade animal facility of Shanghai Paisi New Biomedical Technology Co., Ltd. This study was conducted entirely under the supervision of IACUC of Shanghai Paisi New Biomedical Technology Co., Ltd., IACUC approval number P08240919AR1.

[0125] Mice in the treatment group were administered G83F-CAR via tail vein injection on days 7 and 14, at doses of 2 × 10⁻⁶. 6 and 1.3×10 6 Cells / mouse, administration volume was 0.2 mL / mouse; control mice were given an equal volume of PBS via tail vein injection at the same time point.

[0126] Starting from day 7, mouse weight was recorded using an electronic balance, and GvHD symptoms were scored according to the table below until the end of the study on day 59.

[0127]

[0128] The results, as shown in Figures 7-8, indicate that mice in the control group began to develop the disease from day 20 onwards, exhibiting a gradual decrease in body weight and a gradual increase in GvHD scores. Mice in the drug-treated group experienced a slight decrease in body weight at the beginning of drug administration, but the rate of weight loss gradually decreased from day 20 onwards, and then began to gain weight after day 40. The GvHD score peaked on day 25 and then gradually decreased, reaching almost zero by the endpoint. In conclusion, G83F-CAR has a significant therapeutic effect on human PBMC-induced GvHD in mice.

[0129] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A p40 subunit mutant, characterized in that, The p40 subunit mutant has a mutation at position 83 compared to the wild-type p40 subunit, the mutation being G83F, and the amino acid sequence of the wild-type p40 subunit is shown in SEQ ID No.

1.

2. The p40 subunit mutant according to claim 1, characterized in that, The amino acid sequence of the p40 subunit mutant is shown in SEQ ID No.

2.

3. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor contains the p40 subunit mutant as described in claim 1 or 2.

4. The chimeric antigen receptor according to claim 3, characterized in that, The chimeric antigen receptor comprises one or more of the following: a guide peptide, an antigen-binding domain, a hinge domain, a transmembrane domain, or an intracellular signaling domain.

5. The chimeric antigen receptor according to claim 3, characterized in that, The P40 subunit mutant is the antigen-binding domain of the chimeric antigen receptor.

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

7. The chimeric antigen receptor according to claim 6, characterized in that, The cleavage peptide is selected from one or more of P2A, E2A, F2A or T2A.

8. An isolated polynucleotide, characterized in that, The polynucleotide encodes the P40 subunit mutant of claim 1 or 2 or the chimeric antigen receptor of any one of claims 3-7.

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

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

11. A CAR-Treg cell, characterized in that, The CAR-Treg cells comprise the P40 subunit mutant of claim 1 or 2 or the chimeric antigen receptor of any one of claims 3-7.

12. Use of the polynucleotide of claim 8, the nucleic acid construct of claim 9, the viral vector of claim 10, or the CAR-Treg cell of claim 11 in the preparation of graft-versus-host disease therapeutics.

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