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WO2022179567A1 - Cellules tigit modifiées et leur composition - Google Patents

Cellules tigit modifiées et leur composition Download PDF

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Publication number
WO2022179567A1
WO2022179567A1 PCT/CN2022/077702 CN2022077702W WO2022179567A1 WO 2022179567 A1 WO2022179567 A1 WO 2022179567A1 CN 2022077702 W CN2022077702 W CN 2022077702W WO 2022179567 A1 WO2022179567 A1 WO 2022179567A1
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Prior art keywords
cells
cell
tigit
seq
antibody
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Chinese (zh)
Inventor
李宗海
石志敏
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Crage Medical Co Ltd
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Crage Medical Co Ltd
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Priority to CN202280017000.5A priority Critical patent/CN116897202A/zh
Publication of WO2022179567A1 publication Critical patent/WO2022179567A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application relates to a cell with anti-transplant rejection function, and also relates to a method for anti-transplant immune rejection, in particular to a method for anti-NK cell immune rejection.
  • the donor may also be recognized and attacked by immune cells in the recipient, thereby inhibiting or eliminating the foreign body.
  • grafts resulting in a host-versus-graft response (HVGR).
  • HVGR host-versus-graft response
  • By knocking out the MHC molecules in the graft cells it can effectively resist the rejection of the graft by the host T cells, but it may cause the rejection of other immune cells in the host.
  • the deletion of MHC-I molecules in allogeneic cells will lead to the rejection of NK cells in the host and enhance the clearance of allogeneic cells (Nat Biotechnol. 2017; 35(8): 765-772. doi: 10.1038/nbt.3860). Therefore, how to effectively prevent the immune rejection of host NK cells is crucial for the development of allogeneic cell transplantation therapy.
  • the purpose of the present application is to provide a cell against transplant immune rejection and a method for resisting rejection.
  • a genetically engineered cell characterized in that the cell expresses a first protein that can recognize TIGIT.
  • the first protein contains an antibody capable of recognizing TIGIT, preferably the sequence of TIGIT is shown in SEQ ID No: 10.
  • the first protein comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first protein is a CAR comprising:
  • the cell comprises: a knockout of a gene encoding a TIGIT protein and/or low or no expression of endogenous TIGIT molecules.
  • CRISPR/Cas9 technology is used to knock out the TIGIT gene of the cell, and the gRNA used is selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , any one of the sequences shown in 35, 36 or a combination thereof.
  • the cells are selected from the group consisting of T cells, NK cells, cytotoxic T cells, NKT cells, DNT cells, NK92 cells, macrophages, CIK cells, and stem cell-derived immune effector cells or combinations thereof .
  • the cells are autologous or allogeneic T cells, primary T cells or autologous T cells derived from humans.
  • the cell comprises a knockout of a gene encoding a TCR protein and/or low or no expression of endogenous TCR molecules, and/or a knockout and/or endogenous expression of a gene encoding an MHC protein Derived MHC expression is low or not.
  • the endogenous MHC molecule B2M and endogenous TCR are knocked out using CRISPR/Cas9 technology.
  • the gRNA used for knocking out B2M includes the sequences shown in SEQ ID NO: 24, 72, 73 and/or 74
  • the gRNA used for knocking out TCR includes SEQ ID NO: 23, 65, 66, Sequences shown at 67, 68, 69, 70 and/or 71.
  • the antibody recognizing TIGIT comprises:
  • HCDR1 shown in SEQ ID NO: 3 HCDR2 shown in SEQ ID NO: 4
  • HCDR3 shown in SEQ ID NO: 5 LCDR1 shown in SEQ ID NO: 6, and shown in SEQ ID NO: 7 LCDR2, and/or LCDR3 of SEQ ID NO: 8; or
  • the first protein also recognizes a tumor and/or a pathogen; preferably, the tumor expression includes BCMA, CD19, GPC3, CLDN18A2, EGFR, or a combination thereof.
  • the first protein comprises an antibody that recognizes TIGIT and an antibody that recognizes tumor and/or pathogen antigens, and their connection modes include:
  • light chain (or light chain variable region) of an antibody recognizing tumor and/or pathogen antigens heavy chain (or heavy chain variable region) of an antibody recognizing TIGIT—light chain (or light chain variable region) of an antibody recognizing TIGIT chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens;
  • the light chain (or light chain variable region) of an antibody that recognizes TIGIT the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens light chain (or light chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT
  • the first protein is CAR
  • the CAR includes:
  • the antibody recognizing the tumor antigen recognizes CLDN18A2, comprising:
  • HCDR1 described in SEQ ID NO: 13 HCDR2 described in SEQ ID NO: 14, HCDR3 described in SEQ ID NO: 15, LCDR1 described in SEQ ID NO: 16, described in SEQ ID NO: 17
  • the first protein comprises the sequence shown in SEQ ID NO: 48, 50, 52, 90 or 91.
  • the cell further expresses a second protein, chemokine, chemokine receptor, cytokine, siRNA that reduces PD-1 expression, targeting recognition of tumor antigens, and/or pathogen antigens, A protein that blocks the binding of PD-L1 to PD-1, a safety switch, or a combination thereof.
  • a second protein chemokine, chemokine receptor, cytokine, siRNA that reduces PD-1 expression, targeting recognition of tumor antigens, and/or pathogen antigens, A protein that blocks the binding of PD-L1 to PD-1, a safety switch, or a combination thereof.
  • the cells when the cells are co-cultured with host NK cells, the cells are capable of killing host NK cells, or the cells are resistant to killing of the cells by host NK cells, or the cells are capable of killing host NK cells Resists killing of said cells by activated host NK cells.
  • the cells are administered in combination with an agent that enhances their function, preferably in combination with a chemotherapeutic agent; and/or
  • the cells are administered in combination with an agent that ameliorates one or more side effects associated therewith; and/or
  • the cells are administered in combination with cells expressing a second protein that recognizes a different antigen from the first protein.
  • the second protein comprises a CAR comprising:
  • the cells expressing the second protein comprise:
  • the cell expressing the second protein is a cell expressing the second protein:
  • the cells expressing the second protein are selected from the group consisting of T cells, NK cells, cytotoxic T cells, NKT cells, DNT cells, NK92 cells, macrophages, CIK cells, and stem cell-derived immune cells effector cells or a combination thereof.
  • the cells are autologous or allogeneic T cells, primary T cells or autologous T cells derived from humans.
  • a method for increasing the persistence and/or transplantation survival rate of a first immune cell in the presence of a second immune cell of a host comprising:
  • modifying the first immune cell by reducing or inhibiting the expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in the response to self and non-self antigen recognition;
  • the polypeptide in step b) is selected from MHC, TCR, and/or TIGIT.
  • step b) comprises:
  • step b) comprises:
  • the gRNA used for knocking out the TIGIT molecule is selected from the sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and/or 36;
  • the gRNA used for knocking out TCR includes the sequences shown in SEQ ID NO: 23, 65, 66, 67, 68, 69, 70 and/or 71; and/or
  • the gRNA used to knock out the MHC molecule B2M includes the sequences shown in SEQ ID NOs: 24, 72, 73 and/or 74.
  • the first protein comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first protein comprises:
  • the first protein comprises:
  • it also includes step d) encoding a second protein targeting tumor antigens and/or pathogen antigens and/or viral antigens, chemokines, chemokine receptors, cytokines, reducing PD-1
  • the first immune cell is modified by expressed siRNA, a protein that blocks the binding of PD-L1 to PD-1, or a non-endogenous polynucleotide such as a safety switch.
  • the first immune cell is selected from the group consisting of T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells or a combination thereof.
  • the first immune cells are autologous or allogeneic T cells, primary T cells or human-derived autologous T cells.
  • an engineered cell prepared by the above-mentioned method of the present application.
  • a polynucleotide which is a nucleic acid molecule encoding the construction of a cell as described in the present application or encoding a nucleic acid molecule required for administering the method of the present application as described above.
  • a vector comprising the polynucleotide as described in the present application.
  • a virus comprising the vector described in the present application.
  • composition comprising an effective amount of a cell as described in the present application, a polynucleotide as described in the present application, a vector as described in the present application, and a vector as described in the present application virus.
  • a method of treating an inflammatory disorder, viral infection and/or tumor comprising administering to a subject in need thereof a cell as described herein or a combination as described herein thing.
  • This application also relates to:
  • a genetically engineered cell characterized in that the cell expresses the first protein that can recognize TIGIT;
  • the TIGIT amino acid sequence is shown in SEQ ID NO: 10;
  • the first protein contains an antibody or a functional fragment thereof capable of recognizing TIGIT;
  • the first protein contains the heavy chain variable region described in SEQ ID NO: 1 and/or the light chain variable region described in SEQ ID NO: 2;
  • the first protein comprises an antibody or functional fragment thereof recognizing TIGIT, an antibody or functional fragment thereof recognizing tumor antigen or pathogen antigen, a transmembrane region, and an intracellular domain;
  • the antibody recognizing TIGIT or its functional fragment and the antibody recognizing tumor antigen or pathogen antigen are linked through a linking peptide.
  • the cells are derived from human T cells
  • the cells are human primary T cells
  • the cells are allogeneic T cells.
  • the first protein comprises the TCR intracellular domain from the stimulatory domain of the intracellular signaling domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ or TCR ⁇ .
  • the MHC is MHC Class I molecule; more preferably, the MHC class I molecule is HLA; more preferably, the HLA is HLA-I; more preferably, the HLA-I is selected from HLA-A, HLA-B, HLA- One or more of C, B2M; most preferably, the HLA-I includes HLA-A and/or B2M; preferably, the endogenous TCR includes one of the ⁇ and ⁇ chains of TCR or both chains;
  • said being reduced or inhibited is by using TAL nucleases, meganucleases, zinc finger nucleases, Cas9 and Argonaute;
  • the engineered T cells comprise inhibitory nucleic acid molecules or gRNAs targeting genes encoding MHC;
  • the inhibitory nucleic acid molecule comprises a sequence complementary to the MHC-encoding gene and/or the endogenous TCR;
  • the inhibitory nucleic acid comprises an RNA interfering agent
  • the inhibitory nucleic acid comprises siRNA, shRNA or miRNA
  • the gRNA sequence contains the sequence shown in SEQ ID NO: 23 and/or SEQ ID NO: 24;
  • said reduction in MHC and/or endogenous TCR expression, activity and/or signaling is permanent, transient or inducible;
  • the expression, activity and/or signaling of MHC and/or endogenous TCR in said engineered cells is compared to the expression, activity and/or signaling of MHC and/or endogenous TCR in non-engineered T cells and/or a reduction in signaling greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%;
  • the expression of MHC and/or endogenous TCR expressed in the engineered cells is undetectable using immunoblot assays and/or in flow assays.
  • the first protein is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC) , T cell fusion protein (TFP) or a combination thereof;
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • T cell fusion protein T cell fusion protein
  • the first protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain.
  • the antibody that specifically recognizes TIGIT contains the heavy chain variable region described in SEQ ID NO: 1 and/or the light chain variable region described in SEQ ID NO: 2;
  • the amino acid sequence of the first protein shares at least 80%, preferably 90%, and more preferably 95% identity to SEQ ID NO:9.
  • chemokine receptors chemokine receptors, cytokines, siRNAs that reduce PD-1 expression, proteins that block the binding of PD-L1 to PD-1, safety switches, or a combination thereof;
  • the second protein comprises: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), aTCR -T or a combination thereof;
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • the second protein is capable of specifically recognizing Claudin18.2, GPC3, BCMA or CD19.
  • the cells also express NKG2A binding molecules;
  • the NKG2A binding molecule is a cell membrane-bound protein or a secreted protein
  • the NKG2A binding molecule comprises an extracellular domain, a transmembrane domain; or comprises an extracellular domain, a transmembrane domain and an intracellular domain;
  • the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane.
  • said being reduced or inhibited is by using TAL nucleases, meganucleases, zinc finger nucleases, Cas9 and Argonaute;
  • the immune cell comprises an inhibitory nucleic acid molecule or gRNA targeting the gene encoding TIGIT;
  • the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding TIGIT;
  • the inhibitory nucleic acid comprises an RNA interfering agent
  • the inhibitory nucleic acid comprises siRNA, shRNA or miRNA
  • said reduction in TIGIT expression, activity and/or signaling is permanent, transient or inducible
  • the expression, activity and/or signaling of TIGIT in the engineered cell is reduced by greater than or greater than about 50%, 60% compared to the expression, activity and/or signaling of TIGIT in the unengineered cell , 70%, 80%, 90%, 95% or 100%;
  • the expression of TIGIT expressed in the cells is undetectable using immunoblot assays and/or in flow assays.
  • said cells are resistant to killing of said cells by cytokine-activated NK cells in host NK cells;
  • said cells are resistant to killing of said cells by host NK cells expressing NKG2A;
  • the cells are significantly resistant to killing of the cells by host NK cells that low express NKG2A.
  • the cells are administered in combination with an agent that ameliorates one or more side effects associated therewith.
  • a method of increasing the persistence and/or transplantation survival of allogeneic immune cells in the presence of host immune cells comprising:
  • the polypeptide in step b) is selected from MHC and/or endogenous TCR, and the MHC is an MHC class I molecule; more preferably, the MHC I
  • the class molecule is HLA; more preferably, the HLA is HLA-I; more preferably, the HLA-I is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M;
  • the endogenous TCR includes one or both of the ⁇ and ⁇ chains of TCR; more preferably, the HLA-I includes HLA-A and/or B2M;
  • step b) modifies the cell by reducing or inhibiting B2M and TCR expression, activity and/or signaling;
  • said being reduced or inhibited is by using TAL nucleases, meganucleases, zinc finger nucleases, Cas9 and Argonaute;
  • the cell comprises an inhibitory nucleic acid molecule or gRNA targeting a gene encoding an MHC;
  • the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding the MHC;
  • the inhibitory nucleic acid comprises an RNA interfering agent
  • the inhibitory nucleic acid comprises siRNA, shRNA or miRNA
  • the gRNA sequence contains the sequence shown in SEQ ID NO: 23 and/or 24;
  • said reduction in MHC and/or endogenous TCR expression, activity and/or signaling is permanent, transient or inducible;
  • the expression, activity and/or signaling of MHC and/or endogenous TCR in the engineered cell is compared to the expression, activity and/or signaling of MHC and/or endogenous TCR in the unengineered cell /or a reduction in signaling greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%;
  • the expression of MHC expressed in said cells is undetectable using immunoblot assays and/or in flow cytometry.
  • the first protein is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), T cell A fusion protein (TFP) or a combination thereof;
  • the first protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain;
  • the cells transmit signals through the intracellular signaling domain to mediate the inhibition or killing of immune effector cells of the host;
  • the first protein comprises:
  • the antibody that specifically recognizes TIGIT contains the heavy chain variable region described in SEQ ID NO: 1 and/or the light chain variable region described in SEQ ID NO: 2;
  • the amino acid sequence of the first protein shares at least 80%, preferably 90%, and more preferably 95% identity to SEQ ID NO:9.
  • step d) encoding a second protein, chemokine, Chemokine receptors, cytokines, siRNAs that reduce PD-1 expression, proteins that block the binding of PD-L1 to PD-1, or non-endogenous polynucleotides such as safety switches to modify the cells;
  • the second protein comprises: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), aTCR -T or a combination thereof;
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • the second protein is capable of specifically recognizing Claudin18.2, GPC3, BCMA or CD19.
  • step e) modifying the cell with a non-endogenous polynucleotide encoding a ligand or antibody fragment of an NK cell inhibitory receptor ;
  • step e) is further included to modify the cell by a non-endogenous polynucleotide encoding the immune cell NKG2A binding molecule;
  • the NKG2A binding molecule is a cell membrane-bound protein or a secreted protein
  • the NKG2A binding molecule comprises only an extracellular domain, a transmembrane domain; or an extracellular domain, a transmembrane domain and an intracellular domain;
  • the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane.
  • step f) modifying the cell by encoding that TIGIT expression, activity and/or signaling is reduced or inhibited
  • said being reduced or inhibited is by using TAL nucleases, meganucleases, zinc finger nucleases, Cas9 and Argonaute;
  • the cell comprises an inhibitory nucleic acid molecule or gRNA targeting the gene encoding TIGIT;
  • the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding TIGIT;
  • the inhibitory nucleic acid comprises an RNA interfering agent
  • the inhibitory nucleic acid comprises siRNA, shRNA or miRNA
  • said reduction in TIGIT expression, activity and/or signaling is permanent, transient or inducible
  • the expression, activity and/or signaling of TIGIT in the engineered immune cells is reduced by greater than or greater than about 50%, 60%, compared to the expression, activity and/or signaling of TIGIT in non-engineered cells %, 70%, 80%, 90%, 95% or 100%;
  • the expression of TIGIT expressed in the immune cells is undetectable using immunoblotting assays and/or in flow assays.
  • the cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells;
  • the cells are derived from human T cells
  • the cells are human primary T cells
  • the cells are allogeneic T cells.
  • said cells are resistant to killing of said cells by cytokine-activated NK cells in host NK cells;
  • said cells are resistant to killing of said cells by host NK cells expressing NKG2A;
  • the cells are significantly resistant to killing of the cells by host NK cells that low express NKG2A.
  • cells produced by the method are administered in combination with an agent that ameliorates one or more side effects associated therewith.
  • a bispecific antibody construct comprising a first binding domain of human or cynomolgus TIGIT bound to the surface of a target cell and a second binding domain of human CD3 bound to the surface of a T cell.
  • the antibody construct is selected from the following formats: (scFv)2, scFv-single domain mAbs, diabodies and oligomers of these formats.
  • a vector comprising a polynucleotide as defined in item 27.
  • composition comprising an effective amount of the engineered cell of any one of items 1-13 or 24,
  • a pharmaceutically acceptable carrier is also included;
  • the carrier is saline solution, dextrose solution or 5% human serum albumin.
  • a cryoprotectant is also included.
  • a kit comprising the engineered cell according to any one of items 1-13 or 24 or the composition according to item 30 and an additional agent for treating a disease.
  • a method for treating a disease comprising administering the engineered cell as described in any one of items 1-13 or 24 or the composition as described in item 30 or as described in item 31 to a subject in need the described kit,
  • the engineered cell is produced by the method according to any one of items 14-23 prior to administration of the engineered cell;
  • it also comprises administering an additional agent;
  • the disease is selected from inflammatory disorders, infections and tumors;
  • the subject is a human
  • the engineered cells are autologous or allogeneic T cells to the subject.
  • Figure 1 shows the effect of cytokines on the ratio of TIGIT+NK cells
  • Figure 2 shows the percentage of TIGIT+ NK cells in activated NK cells
  • FIG. 3 shows the TIGIT-Z CAR vector map
  • FIG. 4 shows the positive rate of TIGIT-Z CAR-T cells
  • Figure 5 shows the resistance function of TIGIT-UCAR-T cells to NK cells
  • Figure 6 shows the vector diagrams of CAR1, CAR2, CAR3 dual targeting TIGIT & CLDN18A2 and CAR targeting only CLDN18A2;
  • Figure 7 shows the positive rate of UCAR-T1, UCAR-T2, UCAR-T3 dual-targeting TIGIT&CLDN18A2 and CLDN18A2-UCAR-T cells targeting only CLDN18A2;
  • Figure 8 shows the killing effect of UCAR-T1, UCAR-T2, UCAR-T3 dual-targeting TIGIT&CLDN18A2 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 on target cells;
  • Figure 9 shows the positive rate and TIGIT expression of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 dual-targeting TIGIT & CLDN18A2, and CLDN18A2-UCAR-T cells that only target CLDN18A2;
  • Figure 10 shows the changes in the proportions of T cells and NK cells after co-culture of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 targeting TIGIT & CLDN18A2, and CLDN18A2-UCAR-T cells targeting only CLDN18A2 with PBMC cells happening;
  • Figure 11A shows the therapeutic effect of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 dual targeting TIGIT & CLDN18A2, and CLDN18A2-UCAR-T cells targeting only CLDN18A2 on HGC-27-CLDN18A2 xenografts;
  • Figure 11B The survival of peripheral blood CD4+ and CD8+ T cells in the above groups is shown;
  • Figure 12 shows the knockdown efficiency of different gRNAs for TIGIT
  • Figure 13 shows the CAR positive rate of CLDN18A2-UCAR-T, CLDN18A2-UCAR-T-TIGITKO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells;
  • Figure 14A shows the CAR-positive rate and the proportion of TIGIT-positive cells in CLDN18A2-CAR-T, CLDN18A2-CAR-T-TIGIT KO cells;
  • Figure 14B shows CLDN18A2-CAR-T, CLDN18A2-CAR-T-TIGIT KO cells versus target In vitro killing effect of cells;
  • Figure 15 shows the effect of U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN18A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells on HGC- Therapeutic effect of 27-CLDN18A2 xenograft;
  • Figure 16 shows vector maps of TIGIT-BBZ and TIGIT-28Z targeting TIGIT
  • Figure 17 shows the changes in the proportions of T cells and NK cells after TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR cells were co-cultured with NK cells, respectively.
  • any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer within the stated range, as well as, where appropriate, fractions thereof (eg, one tenth of an integer and one percent).
  • TIGIT T cell Ig and ITIM domain
  • TIGIT inhibitory receptor is a member of the poliovirus receptor (PVR)/Nectin family, gene accession number: 201633, human TIGIT amino acid sequence as SEQ ID NO : 10 shown. It consists of an extracellular immunoglobulin variable region (IgV) domain-type transmembrane domain and an intracellular immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT) motif. domain composition.
  • IgV immunoglobulin variable region
  • ITIM intracellular immunoreceptor tyrosine inhibitory motif
  • ITT immunoglobulin tyrosine tail
  • TIGIT is expressed in lymphocytes, especially in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells and natural killer (NK) cells (Eur.J.Immunol.2015.45: 2886–2897). Since CAR-T cells secrete a large amount of cytokines when treating tumor patients, some examples of this application use different combinations of IL-2, IL-15 and IL-12 cytokines to treat NK cells, and detect TIGIT+NK cells change in proportions.
  • Claudin 18 refers to claudin 18 (Claudin 18, CLD18) molecules (Genbank accession numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2): NM_001002026, NP_001002026) that are approximately 27,9/27,72kD intrinsic transmembrane protein.
  • Claudin is an intrinsic membrane protein located in the tight junctions of epithelium and endothelium. Tight junctions organize a web of interconnected chains of particles within the membrane between adjacent cells. In tight junctions, occludin and claudin are the most important transmembrane protein components.
  • transplant immune rejection refers to the fact that after the host has transplanted an allogeneic tissue, organ, or cell transplant, the foreign graft is recognized by the host's immune system as an "alien component" and initiates targeting of the graft. immune response to attack, destruction, and clearance.
  • the present application provides an anti-transplant immune rejection cell and a method for anti-transplant rejection.
  • the term "graft" refers to a biological material or formulation derived from an individual other than the host for implantation into the host.
  • the graft can be from any animal source, such as a mammalian source, preferably from a human.
  • the graft can be derived from the host, such as cells from the host that have been cultured in vitro, or engineered to be re-implanted into the host.
  • the graft may be derived from an allogeneic other individual, such as cells from another human being cultured in vitro, or engineered into a host.
  • the graft may be from a xenogeneic individual, such as an organ from another species (eg, murine, porcine, monkey) implanted into a human.
  • cell refers to cells of animal origin, human or non-human.
  • the term "host” refers to the recipient of the graft, which, in some embodiments, may be an individual, such as a human, to which exogenous cells are engrafted.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Subjects of the present application include, but are not limited to, humans, non-human primates (eg, rhesus monkeys or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • immune effector cell refers to a cell involved in an immune response that produces an immune effector, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages , mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cells can be autologous T cells, xenogeneic T cells, allogeneic T cells.
  • the NK cells may be autologous NK cells or allogeneic NK cells.
  • artificially engineered cells with immune effector cell function refers to a cell or cell line without immune effector that has acquired immune effector cell function after being artificially engineered or stimulated by a stimulus.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells can be PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and natural T cells obtained from infection sites, ascites, pleural effusion, spleen tissue, tumor tissue, and can also be A population of cells with specific phenotypic characteristics obtained by sorting, etc., or a mixed population of cells with different phenotypic characteristics, such as "T cells” can be cells comprising at least one subset of T cells: memory stem-like T cells (stem). cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells (Tem).
  • Tstem memory stem-like T cells
  • Tscm cells central memory T cells
  • effector T cells Tef, Teff
  • Tregs regulatory T cells
  • Tregs effector memory T cells
  • a "T cell” may be a particular subtype of T cell, such as ⁇ T cells.
  • T cells can be obtained from blood collected from an individual using any technique known to those of skill in the art, such as FicollTM separation and/or apheresis.
  • T cells can be of any type and of any developmental stage, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD8+ T cells (e.g. cytotoxic T cells) , tumor infiltrating cells, memory T cells, naive T cells, etc.
  • the T cells may be CD8+ T cells or CD4+ T cells.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • the T cells can be derived from a healthy donor, or from an individual diagnosed with cancer.
  • activation and “activation” are used interchangeably and can refer to the process by which cells change from a resting state to an active state. This process may include responses to phenotypic or genetic changes in antigenic, migratory and/or functionally active states.
  • activation can refer to the process of stepwise activation of T cells. The activation process is co-regulated by the first stimulatory signal and the co-stimulatory signal. Activation of T cells is a dynamic process, and its duration and degree of activation are affected by external stimulation.
  • T cell activation or “T cell activation” refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.
  • peripheral blood mononuclear cells refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.
  • pluripotent stem cell has the potential to differentiate into any of the three germ layers: endoderm (eg, gastric junction, gastrointestinal tract, lung, etc.), mesoderm (eg, muscle, bone, blood, urogenital tissue, etc.) ) or ectoderm (eg epidermal tissue and nervous system tissue).
  • endoderm eg, gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm eg, muscle, bone, blood, urogenital tissue, etc.
  • ectoderm eg epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cell” also encompasses "induced pluripotent stem cell” or "iPSC,” a type of pluripotent stem cell derived from a non-pluripotent cell.
  • the pluripotent stem cells are derived from cells that have the characteristics of pluripotent stem cells by reprogramming somatic cells. Such "iPS” or “iPSC” cells can be generated by induc
  • the term "engineering” refers to the application of the principles and methods of cell biology and molecular biology, through some engineering means, at the overall level of cells or at the level of organelles, to change the genetic material in cells or obtain cells according to people's wishes. A comprehensive science and technology of products.
  • the engineering refers to one or more alterations of nucleic acids, such as nucleic acids within the genome of an organism.
  • the engineering refers to changes, additions and/or deletions of genes.
  • the engineered cell or the engineered cell may also refer to a cell with added, deleted and/or altered genes.
  • the terms “genetic modification”, “genetic modification”, “genetically engineered” or “modified” refer to methods of modifying cells, including but not limited to, by means of gene editing, in the coding or non-coding regions of genes or their expression regulatory regions. ; Or through endonuclease and/or antisense RNA technology; or increase the introduction of exogenous proteins and/or complexes, small molecule inhibitors to change the protein expression level of the gene to cause gene defects.
  • the modified cells are stem cells (eg, hematopoietic stem cells (HSC) or progenitor cells, embryonic stem cells (ES), induced pluripotent stem (iPS) cells), lymphocytes (eg, T cells), which can is obtained from the subject or donor.
  • Cells can be modified to express foreign constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which can be integrated into the cell genome.
  • CARs chimeric antigen receptors
  • TCRs T cell
  • gene silencing refers to the phenomenon that a gene is not expressed or is underexpressed due to various reasons. Gene silencing can be gene silencing at the transcriptional level due to DNA methylation, heterochromatinization, and position effects; it can also be post-transcriptional gene silencing, that is, specific inhibition of target RNA at the post-transcriptional level. Instead, gene inactivation, including antisense RNA, co-suppression, gene repression, RNA interference, and microRNA-mediated translation inhibition, etc.; can also lead to undetectable or low protein expression by increasing protein degradation, including PROTAC, LYTAC , AbTAC, ATTEC, AUTAC and intracellular retention of membrane proteins.
  • TCR silencing refers to no or low expression of endogenous TCR.
  • MHC silencing refers to no or low expression of endogenous MHC.
  • Low expression as used herein means that the protein and/or RNA level of the target gene expressed in the engineered cell is lower than the expression level before the cell engineering treatment.
  • low expression of B2M or TCR or TIGIT refers to a decrease in the expression of B2M or TCR or TIGIT in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • Protein expression or content in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using antibodies specific for B2M or TCR or TIGIT.
  • MHC stands for the histocompatibility complex and is the collective name for all the gene groups that encode the antigens of the biocompatibility complex.
  • MHC antigens are expressed in the tissues of all higher vertebrates, and are called HLA antigens in human cells and are used in transplantation reactions. Rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
  • MHC antigens are divided into NHC class I antigens and MHC class II antigens.
  • HLA human leukocyte antigen
  • HLA Human leukocyte antigen
  • HLA human leukocyte antigen
  • HLA is the gene encoding the major histocompatibility complex in humans, located on chromosome 6 (6p21.31), and is closely related to the function of the human immune system.
  • HLA includes class I, class II and class III gene portions.
  • the antigens expressed by HLA class I and class II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C sites) and MHC-II (encoded by HLA-D region),
  • HLA I Class II is distributed on the surface of almost all cells in the body and is a heterodimer composed of heavy chain ( ⁇ chain) and ⁇ 2 microglobulin (B2M).
  • Class II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes .
  • B2M beta-2 microglobulin, the light chain of an MHC class I molecule.
  • B2M is encoded by the b2m gene located on chromosome 15, as opposed to other MHC genes located as gene clusters on chromosome 6.
  • T cell receptor mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. body.
  • the classic TCR receptor is composed of two peptide chains, ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region. The specificity exists in the V region, and the V region (V ⁇ , V ⁇ ) has three hypervariable regions, CDR1, CDR2, and CDR3.
  • T cells expressing the classic TCR can be stimulated by antigens. Induce specificity of T-cell TCRs for target antigens.
  • TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, ⁇ and ⁇ , and TCR2 is composed of two chains, ⁇ and ⁇ .
  • TCR refers to the constant region of the TCR ⁇ chain.
  • gene editing refers to genetic engineering techniques that utilize site-specific nucleases to insert, knock out, modify or replace DNA at specific locations in the genome of an organism to alter DNA sequences. This technique is sometimes called “gene clipping” or “genome engineering.” Gene editing can be used to achieve precise and efficient gene knockout or gene knock-in.
  • Nuclease-guided genome targeted modification technology usually consists of a DNA recognition domain and a non-specific endonuclease domain.
  • the DNA recognition domain recognizes the target site and locates the nuclease to the genomic region that needs to be edited. Then, the DNA double-strand is cut by the non-specific endonuclease, causing the DNA breakage self-repair mechanism, thereby triggering the mutation of the gene sequence and promoting the occurrence of homologous recombination.
  • the endonuclease may be a Meganuclease, a zinc finger nuclease, a CRISPR/Cas9 nuclease, a MBBBD-nuclease or a TALEN-nuclease.
  • the endonuclease is CRISPR/Cas9 nuclease, TALEN-nuclease.
  • Gene knockout techniques using nucleases include CRISPR/Cas9 technology, ZFN technology, TALE technology and TALE-CRISPR/Cas9 technology, Base Editor technology, guide editing technology and/or homing endonuclease technology.
  • ZFN artificial zinc finger nuclease
  • ZFN zinc finger Nucleases
  • TALE transcription activator-like effector
  • the term "transcription activator-like effector (TALE)" has DNA binding specificity and a module that can specifically recognize bases, and the operation is simple and convenient.
  • the TALE-DNA binding domain is composed of tandem repeat units, most of which contain 34 amino acids.
  • the 12th and 13th amino acids of the unit are designed as variable regions (repeat variable residues, RVD).
  • RVD variat variable residues
  • the RVD of TALE recognizes the 4 bases of DNA sequence with high specificity, and the 13th amino acid directly binds specifically to the base of DNA.
  • a specific TALEDN recognition and binding domain can be constructed at any site, which can be widely used in gene sequence mutation modification and gene targeting.
  • TALE-DNA binding domain sets the DNA target sequence, assemble the TALE-DNA binding domain, fuse the non-specific DNA cleavage domain of Fok I endonuclease, and assemble into TALE nucleases (tanscription activator-like effector nucleases, TALENs).
  • TALENs target and bind to DNA, resulting in DNA double-strand breaks (DNA double-srand breaks, DSBs).
  • CRISPR/Cas9 is the third generation of gene editing technology.
  • An example of this application uses CRISPR/Cas9 technology to prepare UCAR-T cells.
  • CRISPR Clustered regularly interspaced short palindromicrepeats
  • Cas9 CRISPR associated nuclease
  • Cas9 enzymes can be wild-type Cas9 or engineered Cas9.
  • CRISPER/Cas9 system collectively refers to transcripts and other elements involved in the expression of the Cas9 enzyme gene or directing its activity, including sequences encoding the Cas9 gene, tracr (transactivating CRISPR) sequences (eg, tracrRNA or active part tracrRNA), tracr Paired sequences (covering "direct repeats” and partial direct repeats of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also known as "spacers" in the context of endogenous CRISPR systems, i.e. gRNAs ), or other sequences and transcripts from CRISPR loci.
  • tracr transactivating CRISPR sequences
  • tracr Paired sequences covering "direct repeats” and partial direct repeats of tracrRNA processing in the context of endogenous CRISPR systems
  • guide sequences also known as "spacers” in the context of endogenous CRISPR systems, i.e. gRNAs , or other sequences
  • CRISPR systems are characterized by elements that facilitate the formation of a CRISPR complex (also referred to as a protospacer in the context of an endogenous CRISPR system) at the site of a target sequence.
  • target sequence refers to a sequence to which a guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Perfect complementarity is not required, provided that sufficient complementarity is present to cause hybridization and facilitate the formation of a CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • a guide sequence is any ribonucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence.
  • gRNA guide sequence
  • it can be a targeted DNA sequence, or it can also be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA, crRNA and TracrRNA.
  • gRNAs are used to guide, bind or recognize Cas enzymes.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80% when optimally aligned using a suitable alignment algorithm , 85%, 90%, 95%, 97.5%, 99% or more.
  • Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Algorithms based on Burrows-Wheeler Transform (e.g.
  • sgRNA refers to short gRNAs.
  • the CRISPR enzyme comprises one or more heterologous protein domains (eg, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) part of a fusion protein.
  • the CRISPR enzyme fusion protein can comprise any other protein, and optionally a linker sequence between any two domains.
  • protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and one or more protein domains with the following activities: methylase activity, demethylase activity, transcription Activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags .
  • the administered gRNA, tracr pairing sequence, and tracr sequence can be administered alone, or a complete RNA sequence can be administered.
  • the combination of Cas9 protein and gRNA can cut DNA at a specific site.
  • the CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23bp and can target 20bp.
  • the last 3 NGG sequence of its recognition site is called PAM ( protospacer adjacent motif) sequence.
  • CRISPR/Cas transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
  • vectors eg, AAV, adenovirus, lentivirus
  • particles and/or nanoparticles e.g., electroporation.
  • This application simultaneously knocks out the genes TRAC and B2M to inactivate the functions of TCR and MHC molecules to obtain universal T cells or universal CAR-T cells.
  • the exons of the corresponding genes encoding the constant regions of one or both of the alpha and beta chains of B2M, TCR are knocked out using CRISPR/Cas technology, respectively.
  • the gRNA used to knock out the endogenous TCR is selected from the sequence shown in SEQ ID NO: 23, 65, 66, 67, 68, 69, 70 or 71.
  • the endogenous B2M gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequence shown in SEQ ID NO: 24, 72, 73 or 74.
  • the cellular TIGIT gene is knocked out.
  • the TIGIT gene is knocked out using CRISPR/Cas9 technology, and the gRNA used is selected from the sequence shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.
  • “Suppressing” or “suppressing” the expression of B2M or TCR or TIGIT means reducing the expression of B2M or TCR or TIGIT in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • Protein expression or content in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using antibodies specific for B2M or TCR or TIGIT.
  • RNA interfering agent is defined as any agent that interferes with or inhibits the expression of a target gene by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules, short interfering RNAs (siRNAs), shRNAs, or miRNAs that are RNA molecules homologous to the target gene or fragments thereof, and small molecules that interfere or inhibit the expression of the target gene by RNA interference (RNAi). molecular.
  • specific CAR-T cells are constructed first, and then CRISPER/Cas9 technology is used to knock out the endogenous TRAC, B2M and/or TIGIT of the CAR-T cells to construct the corresponding UCAR-T.
  • CRISPER/Cas9 technology knocks out endogenous TRAC, B2M and/or TIGIT and expresses specific CAR simultaneously to construct UCAR-T cells.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be accomplished by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The sequence of these deoxyribonucleotides determines the sequence of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • sequence when used in reference to a nucleotide sequence, the term "sequence" as used herein may include DNA or RNA, and may be single-stranded or double-stranded.
  • sequence identity determines percent identity by comparing two best matched sequences over a comparison window (eg, at least 20 positions), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise Additions or deletions (ie, gaps), eg, 20% or less gaps (eg, 5 to 15%, or 10 to 12%, for the two sequences that best match) compared to the reference sequence (which does not contain additions or deletions) %). Percentages are usually calculated by determining the number of positions in the two sequences at which identical nucleic acid bases or amino acid residues occur to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( i.e. window size) and multiply the result by 100 to yield the percent sequence identity.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems.
  • Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function that it has when overexpressed.
  • endogenous refers to a nucleic acid molecule or polypeptide or the like that is derived from the organism itself.
  • the term "antibody” is used herein in the broadest sense and includes a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and which can specifically bind an antigen or An antibody fragment of an antigenic determinant may be used as long as it exhibits the desired antigen-binding activity.
  • the present invention provides T cells expressing a first protein comprising an antibody that recognizes TIGIT.
  • the invention also provides T cells expressing a first protein comprising a tandem antibody recognizing TIGIT and a tumor antigen.
  • the invention also provides T cells expressing a first protein comprising a tandem antibody recognizing TIGIT and a pathogen antigen.
  • Antibody fragment refers to a fragment of an antibody that can specifically bind to an antigen or antigenic determinant, and non-limiting antibody fragments include Fab, F(ab')2, Fv, CDR, and ScFv.
  • variable region or variable domain refers to a domain of an antibody heavy or light chain that is involved in antibody antigen binding.
  • the heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved FRs and three CDRs.
  • the application provides T cells expressing a first protein comprising a tandem antibody that recognizes TIGIT and a tumor antigen, or TIGIT and a pathogen antigen, the tandem format comprising:
  • light chain (or light chain variable region) of an antibody recognizing tumor and/or pathogen antigens heavy chain (or heavy chain variable region) of an antibody recognizing TIGIT—light chain (or light chain variable region) of an antibody recognizing TIGIT chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens;
  • hypervariable region or “complementarity determining region” or “CDR” refers to an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting regions of residues ("antigen contacts").
  • an antibody contains six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
  • the antibody or its functional fragment that recognizes TIGIT in the present application contains a heavy chain variable region, and the heavy chain variable region comprises 3 CDRs, the heavy chain CDR1 shown in SEQ ID NO:3, and the heavy chain CDR1 shown in SEQ ID NO:4.
  • the heavy chain CDR2 of SEQ ID NO: 5 shows the heavy chain CDR3.
  • the antibody or its functional fragment that recognizes TIGIT in the present application contains a light chain variable region, and the light chain variable region includes 3 CDRs, the light chain CDR1 shown in SEQ ID NO:6, the light chain CDR2 shown in SEQ ID NO:7 , the light chain CDR3 shown in SEQ ID NO:8.
  • the antibody or functional fragment thereof that recognizes TIGIT of the present application contains the heavy chain variable region shown in SEQ ID NO:1 and/or the light chain variable region shown in SEQ ID NO:2.
  • the antibody or functional fragment thereof that recognizes CLDN18A2 of the present application contains a heavy chain variable region, and the heavy chain variable region comprises 3 CDRs, the heavy chain CDR1 shown in SEQ ID NO:13, and the heavy chain CDR1 shown in SEQ ID NO:14.
  • the antibody or its functional fragment that recognizes CLDN18A2 of the present application contains a light chain variable region, and the light chain variable region includes 3 CDRs, the light chain CDR1 shown in SEQ ID NO: 16, and the light chain CDR2 shown in SEQ ID NO: 17. , the light chain CDR3 shown in SEQ ID NO: 18.
  • the antibody or functional fragment thereof that recognizes CLDN18A2 of the present application contains the heavy chain variable region shown in SEQ ID NO: 11 and/or the light chain variable region shown in SEQ ID NO: 12.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments or protein corresponding cDNAs from different sources by gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor, T Cell Antigen Coupler (TAC).
  • chimeric T cell receptor consists of a TCR subunit combined with an antigen binding domain (such as an antibody domain), wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane region (or The TCR subunit is operably linked to the antibody domain.
  • the extracellular, transmembrane, intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ , CD3 ⁇ , CD3z, the alpha chain of TCR, or the beta chain of TCR, and the chimeric T cell is affected by The body can form a complex with TCR/CD3 expressed on T cells.
  • T cell antigen coupler includes three functional domains: (1) antigen binding domain, including single chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) ) or other targeting groups; (2) the extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; (3) the transmembrane domain and the intracellular domain of the CD4 co-receptor, Among them, the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • the first protein chimeric receptor of the present application is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the present application provides a T cell that is resistant to killing by autologous or allogeneic NK cells. Specifically, the present application provides expression of CAR-T cells comprising TIGIT recognition. The application provides T cells expressing endogenous TIGIT knockout comprising a CAR that recognizes TIGIT. The present application provides universal T cells expressing a CAR that recognizes TIGIT and knocking out endogenous TCR and B2M. The present application provides universal T cells expressing a CAR that recognizes TIGIT and knocking out endogenous TIGIT, TCR and B2M.
  • the present application also provides a T cell that can not only resist the killing of autologous or allogeneic NK cells, but also can significantly kill tumor cells.
  • the present application provides T cells expressing CARs comprising dual targets recognizing TIGIT and tumor antigens.
  • the present application provides T cells expressing a CAR containing dual targets recognizing TIGIT and pathogen antigens, and knocking out endogenous TIGIT.
  • the present application provides universal T cells expressing a CAR that contains dual targets that recognize TIGIT and pathogen antigens, and knocks out endogenous TCR and B2M.
  • the present application provides universal T cells expressing a CAR that recognizes dual targets of TIGIT and pathogen antigens and knocks out endogenous TIGIT, TCR and B2M.
  • connection mode of the antibody that recognizes TIGIT and tumor antigen in the antigen-binding region of the above-mentioned dual-target CAR includes:
  • the present application also provides a T cell that can not only resist the killing of autologous or allogeneic NK cells, but also can significantly kill tumor cells.
  • the application provides T cells expressing a CAR that recognizes TIGIT and a CAR that recognizes a tumor antigen.
  • the present application provides T cells that express a CAR that recognizes TIGIT and a CAR that recognizes a tumor antigen, and knocks out endogenous TIGIT.
  • the application provides universal T cells expressing a CAR that recognizes TIGIT and a CAR that recognizes a tumor antigen, and knocks out endogenous TCR and B2M.
  • the present application provides universal T cells expressing a CAR that recognizes TIGIT and a CAR that recognizes tumor antigens and knocks out endogenous TIGIT, TCR and B2M.
  • the present application also provides the combined use of the T cells that can resist autologous or allogeneic NK cell killing provided by the present application and T cells that express a second protein (such as CAR) that recognizes tumor antigens.
  • the T cells that can resist the killing of autologous or allogeneic NK cells provided in this application can promote the survival of T cells expressing a second protein (such as CAR) that recognizes tumor antigens in the presence of autologous or allogeneic immune cells.
  • the endogenous TIGIT of the T cell expressing a second protein (eg, CAR) that recognizes a tumor antigen is knocked out.
  • the T cells expressing a second protein (such as CAR) that recognizes tumor antigens are universal T cells in which endogenous TCR and B2M are knocked out.
  • the T cells expressing a second protein (such as CAR) that recognizes tumor antigens are universal T cells knocked out of endogenous TIGIT, TCR and B2M.
  • chimeric antigen receptor includes an extracellular domain, a transmembrane region, and an intracellular signaling domain.
  • Intracellular signaling domains include functional signaling domains of stimulatory molecules and/or costimulatory molecules (referred to as intracellular signaling domains and costimulatory signaling domains, respectively), and in one aspect, the stimulatory molecules are associated with T cell receptor complex-bound delta chain; in one aspect, the cytoplasmic signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (ie, CD137), CD27, and /or CD28. In certain embodiments, groups of polypeptides are linked to each other.
  • a CAR targeting TIGIT comprises the sequence set forth in SEQ ID NO: 9, and a CAR targeting CLDN18A2 comprises the sequence set forth in SEQ ID NO: 21, 75, 76 or 77.
  • a CAR targeting both TIGIT and CLDN18A2 comprises the antigen binding domain shown in SEQ ID NO: 48, 50 or 52; or the CAR shown in SEQ ID NO: 54, 56 or 58.
  • the engineered T cells targeting both TIGIT and CLDN18A2 comprise the amino acid sequence set forth in SEQ ID NO: 54, 56 or 58; or comprise the nucleotide set forth in SEQ ID NO: 53, 55 or 57 sequence.
  • the term "primary signaling domain” modulates the initial activation of the TCR complex in a stimulatory manner.
  • the primary signaling domain is initiated by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.).
  • Primary signaling domains that act in a stimulatory manner may contain immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs.
  • ITAM-containing primary signaling domains examples include, but are not limited to, those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ) and the sequence of CD66d, in a special case of the CAR of the present application, the intracellular signaling domain in any one or more of the CARs of the present application comprises an intracellular signaling sequence, such as the primary signaling domain of CD3 ⁇ .
  • signaling domain refers to a functional portion of a protein that acts by transmitting messages within a cell to regulate a cell via a defined signaling pathway by producing a second messenger or by acting as an effector in response to such a messenger activity.
  • the intracellular signaling domain may include the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or functional fragments or derivatives thereof.
  • costimulatory molecule refers to binding to a cell stimulatory signaling molecule, such as TCR/CD3, in combination with a signal that results in T cell proliferation and/or up- or down-regulation of key molecules. is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, including but not limited to cell proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137 ).
  • the intracellular signaling domain (or referred to as the domain) can be selected from any of the costimulatory domains of Table 1.
  • the domains can be modified such that the identity to the reference domain can range from about 50% to about 100%.
  • Any one of the domains of Table 1 can be modified such that the modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or up to about 100% identity.
  • the intracellular signaling domain can be designed to contain several possible costimulatory signaling domains, it can contain a single costimulatory domain, such as a delta chain (first generation CAR), or it can further contain a A costimulatory domain such as delta chain with CD28 or 4-1BB (second generation CAR), or it may further comprise two costimulatory domains, such as delta chain with CD28/OX40 or CD28/4-1BB (third generation CAR) CAR).
  • the signaling pathways used by these costimulatory molecules are all synergistic with the master T cell receptor activation signal.
  • the signals provided by these costimulatory signaling domains can synergize with main effector activating signals derived from one or more ITAM motifs (eg, the CD3zeta signaling domain) and can accomplish the requirements for T cell activation.
  • the signaling domain of a CAR targeting TIGIT, targeting CLDN18A2, and/or dual targeting TIGIT and CLDN18A2 includes CD3 ⁇ .
  • CD3 ⁇ is a human CD3 ⁇ molecule comprising the sequence shown in SEQ ID NO:46.
  • the signaling domain of a CAR targeting TIGIT, targeting CLDN18A2, and/or dual targeting TIGIT and CLDN18A2 includes the CD28 intracellular domain.
  • the CD28 intracellular domain comprises the sequence set forth in SEQ ID NO:44.
  • CD3 ⁇ (also known as CD3Zeta) is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • a "CD3 ⁇ domain” is defined as the amino acid residues from the cytoplasmic domain of the ⁇ chain sufficient to functionally transmit the initial signals required for T cell activation.
  • the cytoplasmic domain of ⁇ comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, functional orthologs thereof - from non-human species such as mouse, rodent, monkey, ape, etc. Valence residues.
  • CD3 ⁇ is also known as the T cell receptor T3 ⁇ chain or CD247.
  • CD3delta refers primarily to human CD3delta and its isoforms, as known from Swissprot entry P20963, including proteins having substantially the same sequence.
  • the full T-cell receptor T3delta chain is not required, and any derivative thereof comprising the signaling domain of the T-cell receptor T3delta chain is suitable, including any functional equivalents thereof.
  • CD3 ⁇ is used interchangeably with “CD3z” and “CD3Z” in this application.
  • a CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signal derived from a stimulatory molecule conduction domain.
  • the CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a stimulatory derived domain The functional signaling domain of the molecule.
  • the CAR comprises an optional leader sequence at the amino acid of the CAR protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.
  • the leader sequence comprises the sequence shown in SEQ ID NO:38.
  • the application provides chimeric antigen receptors comprising the extracellular, transmembrane, and intracellular domains described herein.
  • the extracellular domain of a CAR is derived from mouse or humanized or human monoclonal antibodies.
  • antigen-binding regions can be designed for chimeric antigen receptors, including single-chain variable fragments (scFvs) derived from antibodies, fragments antigen-binding regions (Fabs) selected from libraries, single-domain fragments, or binding to their cognate receptors. natural ligands of the body.
  • the extracellular domain may comprise scFv, Fab or natural ligands, and any derivatives thereof.
  • An extracellular domain can refer to a molecule other than an intact antibody, which can comprise a portion of the intact antibody and can bind to the antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, linear antibodies; single-chain antibody molecules (eg, scFv); multispecific antibodies.
  • the extracellular domain such as a scFv, Fab or natural ligand, can be part of a CAR that determines antigen specificity.
  • the extracellular domain can bind any complementary target.
  • Extracellular domains can be derived from antibodies of known variable region sequences.
  • the extracellular domain can be obtained from antibody sequences obtained from available mouse hybridomas. Alternatively, the extracellular domain can be obtained from whole exo-cleavage sequencing of tumor cells or primary cells such as tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the binding specificity of the extracellular domain of the CAR can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity can be determined by the light and heavy chain CDRs.
  • the present application provides expression of CAR-T cells comprising TIGIT recognition. In a specific embodiment, the present application also provides CAR-T cells expressing dual targets that recognize TIGIT and tumor antigens. In a specific embodiment, the present application also provides expression of CAR-T cells comprising dual targets that recognize TIGIT and pathogen antigens.
  • the extracellular domain of the CAR includes an antigen binding region and a linker fragment (also referred to as a hinge, spacer, or linker).
  • Linker fragments can be considered part of the CAR used to provide flexibility to the extracellular domain.
  • the different linking fragments can lead to whether the CAR on the cell surface can be activated by the target antigen, or to a very low degree of activation, or after activation, T cells will experience significant exhaustion and loss of function, which may be due to antibodies in the extracellular domain.
  • the spatial structure formed by the antibody fragment has different degrees of complementarity with the spatial structure of the target antigen. For example, whether the length of an immunoglobulin-derived linker fragment needs to be optimized depends on where the ectodomain targets the target epitope.
  • the composition and length of the linker fragment of the CAR polypeptide are adjustable.
  • the spatial conformation of immune synaptic binding between T cells and target cells defines the distance at which membrane-distal epitopes on target molecules cannot be functionally bridged with CARs, and even CARs with short linking fragments cannot make the synaptic distance reach the signal Approximate value that can be conducted.
  • membrane-proximal CAR target epitopes were only observed in the context of long-linked fragment CARs.
  • Linking fragments can be adjusted according to the ectodomain used.
  • Linked fragments can be of any length.
  • the CAR includes a hinge domain, which is a CD8 ⁇ hinge, preferably, the CD8 ⁇ hinge domain includes the amino acids of SEQ ID NO:40.
  • the transmembrane (TM) domain (or domain) of a CAR can anchor the CAR to the plasma membrane of cells.
  • the native transmembrane portion of CD28 can be used in CAR. In other cases, the native transmembrane portion of CD8 ⁇ can also be used in the CAR.
  • CD8 may be a protein that is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • a “CD8 nucleic acid molecule” can be a polynucleotide encoding a CD8 polypeptide, and in some cases, the transmembrane domain can be the native transmembrane portion of CD28, and “CD28” can refer to NCBI reference number: NP_006130 or its stimulatory activity A fragment of at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to the protein.
  • a "CD28 nucleic acid molecule” can be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion can comprise a CD8 ⁇ region.
  • the TM is the CD28 transmembrane domain.
  • the CD28 transmembrane region comprises the amino acids of SEQ ID NO:42.
  • the pharmaceutically acceptable carrier can be one of those conventionally used and is limited only by chemical physical considerations, such as solubility and non-reactivity with the active agent, and route of administration.
  • the pharmaceutically acceptable carriers described herein, such as adjuvants, excipients and diluents, are well known to those skilled in the art and are readily available to the public.
  • a pharmaceutically acceptable carrier is one that is innocuous under the conditions of use and has no toxic side effects.
  • suitable dosage forms for the pharmaceutical compositions of the present application Methods of preparing administrable (eg, parenterally) compositions are known or apparent to those skilled in the art.
  • the engineered cells of the present application can be administered to a subject in any suitable manner.
  • the CAR materials of the present application are administered by injection (eg, subcutaneous, intravenous, intratumoral, intraarterial, intramuscular, intradermal, interperitoneal, or intrathecal).
  • the CAR material of the present application is administered intravenously.
  • Suitable pharmaceutically acceptable carriers for injectable CAR materials of the present application may include any isotonic carrier, for example, physiological saline (about 0.90% w/v NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 mOsm/L NaCl per liter of water. g NaCl), normal temperature or electrolyte solution.
  • the pharmaceutically acceptable carrier is supplemented with human serum protein.
  • an “effective amount” or “therapeutically effective amount” refers to a dose sufficient to prevent or treat a disease (cancer) in a subject. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose will also depend on the active substance selected, the method of administration, the timing and frequency of administration, the presence, nature and extent of adverse side effects that may accompany administration of the particular active substance, and the desired physiological effect. According to the judgment of the prescribing doctor or those skilled in the art, one or more rounds, or multiple administrations of the CAR materials of the present application may be required. By way of example and not limitation of the present application, when the CAR material of the present application is a host cell, an exemplary dose of the host cell may be at least one million cells ( 1 x 106 cells/dose).
  • Embodiments of the present application also include depletion of mammalian lymphocytes prior to administration of the CAR material of the present application, including but not limited to non-myeloablative lymphoid depletion chemotherapy, myeloablative lymphoid depletion chemotherapy, total body irradiation, and the like.
  • treatment refers to interventions that attempt to modify the disease process, either prophylactically or clinically.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, improving or relieving the condition, relieving or improving the prognosis, etc.
  • the engineered T cells provided in the present application can inhibit tumor cell proliferation, and/or inhibit tumor cell proliferation and increase tumor volume in vivo.
  • prevention refers to interventions that are attempted prior to the development of a disease such as rejection of a cell transplant.
  • the present application provides cells, nucleic acids, expression vectors, host cells, or compositions of the present application for use in the treatment or prevention of tumors.
  • the engineered T cells provided in this application can be used to treat, prevent or ameliorate autoimmune diseases or inflammatory diseases, especially inflammatory diseases associated with autoimmune diseases, such as arthritis (eg, rheumatoid arthritis, chronic progressive arthritis (arthritis chronicaprogrediente and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including ankylosing spondylitis), Reiter's syndrome Symptoms, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, enthesitis, hypersensitivity reactions (including airway hypersensitivity reactions and skin hypersensitivity reactions) and allergies.
  • arthritis eg, rheumatoid arthritis, chronic progressive arthritis (arthritis chronicaprogrediente and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarth
  • the engineered T cells provided herein are used for the treatment and prevention of autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE) ), lupus nephritis, inflammatory muscle disease (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis , Stephen Johnson syndrome, spontaneous sprue, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine eye disease, Graves disease, Sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes
  • the engineered T cells provided in this application can be used to treat, prevent or ameliorate asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema, and other obstructive or inflammatory diseases of the airways.
  • the engineered T cells of the present application can be used as the sole active ingredient or in combination with other drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory or other cytotoxic or anticancer agents (eg, as adjuvants thereof or in combination with them). ), for example, to treat or prevent diseases associated with immune disorders.
  • drugs such as immunosuppressive or immunomodulatory agents or other anti-inflammatory or other cytotoxic or anticancer agents (eg, as adjuvants thereof or in combination with them).
  • the antibodies of the present application can be used in combination with the following drugs: DMARDs, such as gold salts, sulfasalazine, antimalarial drugs, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, other Crolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors such as cyclosporine A or FK 506; modulators of lymphocyte recycling such as FTY720 and FTY720 analogs; mTOR inhibitors such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; Ascomycetes with immunosuppressive properties corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homolog
  • the tumor described herein can be any tumor, including acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (eg, medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal, eye, intrahepatic bile duct, joint, neck, gallbladder or pleura, nose, nasal cavity or middle ear, oral cavity, vulvar, chronic lymphocytic leukemia (CLL), chronic myeloid Cell cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (such as head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer Cancer, leukemia, liver cancer, lung cancer (eg, non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor,
  • Tumor antigen refers to an antigen that is new or overexpressed during the development, progression of a hyperproliferative disease.
  • the hyperproliferative disorder of the present application refers to cancer.
  • the tumor antigens described in this application can be solid tumor antigens or hematological tumor antigens.
  • the tumor antigens of this application include but are not limited to: Thyroid Stimulating Hormone Receptor (TSHR); CD171; CS-1; C-type Lectin-like Molecule-1; Ganglioside GD3; Tn Antigen; CD19; CD20; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet
  • the pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
  • an autologous or allogeneic cell such as a T cell, genetically engineered to express TIGIT-CAR for resistance to NK cell killing, thereby providing an increase in autologous or allogeneic primary immune cells in the presence of a host
  • an autologous or allogeneic cell such as a T cell
  • the "host” is the recipient of the "first immune cell”, such as a subject, a patient, etc.; after the "first immune cell” has been engineered and transplanted into the host, the Any of the other immune cells other than the first immune cell is referred to as the "second immune cell".
  • the "first immune cell” and the "second immune cell” may be cells from the same individual, or may be allogeneic cells.
  • the first immune cell is genetically engineered to express TIGIT-CAR. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, and gene editing techniques are used to knock out endogenous TIGIT. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, and gene editing techniques are used to knock out endogenous B2M and TCR. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, and gene editing techniques are used to knock out endogenous TIGIT, B2M, and TCR.
  • the first immune cell is genetically engineered to express TIGIT-CAR, the cell is also genetically engineered to express at least one chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or a combination thereof). In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or a combination thereof), and using gene editing technology to knock out endogenous TIGIT.
  • CAR chimeric receptor
  • the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or a combination thereof), and gene editing technology was used to knock out endogenous B2M and TCR.
  • the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or a combination thereof), and using gene editing technology to knock out endogenous TIGIT, B2M and TCR.
  • the first immune cell is genetically engineered to express a dual-targeted CAR that recognizes TIGIT and a tumor antigen. In some embodiments, the first immune cell is genetically engineered to express a CAR that recognizes dual targets of TIGIT and tumor antigens, and gene editing technology is used to knock out endogenous IGIT. In some embodiments, the first immune cell is genetically engineered to express a dual-target CAR that recognizes TIGIT and tumor antigens, and gene editing technology is used to knock out endogenous B2M and TCR. In some embodiments, the first immune cell is genetically engineered to express a CAR that recognizes dual targets of TIGIT and tumor antigens, and gene editing technology is used to knock out endogenous TIGIT, B2M, and TCR.
  • a chimeric antigen receptor targeting the inhibitory receptor TIGIT on the surface of NK cells is simultaneously expressed on TRAC and B2M gene double knockout T cells. This strategy can avoid the attack of TRAC and B2M gene double knockout T cells by host NK cells, and prolong the survival time of universal T cells in the host.
  • Density gradient centrifugation was performed using Ficoll-Paque (GE bioscience), PBMCs were isolated from peripheral blood of donors, and NK cells were isolated using NK cell isolation kit (purchased from Miltenyi). TIGIT was detected on the surface of 36% of NK cells using APC-TIGIT (Invitrogen) antibody by flow cytometry (see Figure 1A).
  • IL-2 250U/ml
  • IL-15 150U/ml
  • IL-12 10ng/ml
  • APC-TIGIT Invitrogen antibody detects the percentage of TIGIT+NK cells.
  • the results of flow cytometry showed that the proportion of TIGIT+NK cells under the action of a single cytokine IL-12 and IL-15 was 35.5% (see Figure 1B) and 37.5% (see Figure 1C), respectively; a single cytokine IL-15 -2 or the combination of two or three cytokines can significantly increase the proportion of TIGIT+NK cells, and the corresponding proportions of TIGIT+NK cells are shown in Figure 1D-1H, which are 69.7% (IL-2), 68.9%, respectively. %(IL-15+IL-12), 67.1%(IL-2+IL-12), 74.5%(IL-2+IL-15) and 67.5%(IL-2+IL-15+IL-12) .
  • PBMCs peripheral blood of 5 donors by density gradient centrifugation using Ficoll-Paque (GE bioscience), and NK cells were isolated by NK cell isolation kit (purchased from Miltenyi).
  • IL-2 500U/ml
  • IL-15 150U/ml
  • FIG. 2 shows the results of flow cytometry.
  • the percentages of TIGIT+NK cells in the NK cells of the 5 donors after activation by cytokines were 86.1%, 76.2%, 74.1%, 38.3% and 55%, respectively.
  • the proportion of TIGIT+NK cells exceeded 70%.
  • TIGIT protein is expressed on resting NK cells and activated NK cells.
  • Engineered T cells containing a CAR targeting TIGIT were constructed and their resistance to NK cells was observed.
  • the expression vector was constructed using conventional molecular biology methods in the art (Fig. 3). Design and construct a single chain antibody comprising CD8 ⁇ signal peptide (SEQ ID NO: 38), anti-TIGIT (the amino acid sequence of VH is shown in SEQ ID NO: 1, the amino acid sequence of VL is shown in SEQ ID NO: 2), CD8 ⁇ Hinge region (amino acid sequence shown in SEQ ID NO: 40), CD28 transmembrane region (amino acid sequence shown in SEQ ID NO: 42), T cell activator CD3 ⁇ (amino acid sequence shown in SEQ ID NO: 46) Chimeric antigen receptor carrier TIGIT-CAR, and the lentivirus formed by its packaging was named PRRL-TIGIT.
  • PBMCs Mononuclear cells
  • GE bioscience Ficoll-Paque
  • T cells were obtained by adding anti-CD3/CD28 magnetic beads to activate in vitro.
  • the above lentivirus PRRL-TIGIT was infected with T cells and cultured and expanded to the required number to obtain TIGIT CAR-T cells.
  • TIGIT CAR-T cells After 48 hours of in vitro expansion of TIGIT CAR-T cells, the cell density was adjusted to 2*10 ⁇ 7/mL. Double knockout of TRAC and B2M in TIGIT CAR-T cells.
  • the sgRNA sequences targeting TRAC and B2M were synthesized in vitro according to the reagent instructions (GeneArt TM Precision gRNA Synthesis Kit, Thermo Tisher).
  • Cas 9 enzyme purchased from NEB
  • sgRNA were incubated at room temperature at a ratio of 1:4 for 10 minutes to obtain an RNP complex
  • the nucleic acid sequence of TRAC-gRNA gRNA for targeting TRAC knockout
  • the nucleic acid sequence of B2M-gRNA gRNA for targeted knockout of B2M
  • SEQ ID NO: 24 the nucleic acid sequence of B2M-gRNA (gRNA for targeted knockout of B2M) is shown in SEQ ID NO: 24.
  • the final concentration of Cas 9 enzyme is 2 ⁇ M
  • TRAC single-gene knockout TRAC and B2M double-gene knockout were performed on UTD cells (T cells not transfected with virus), respectively.
  • the UTD cells with single knockout of TRAC, TIGIT CAR-T cells with double knockout of B2M and TRAC, and UTD cells with double knockout of B2M and TRAC were expanded in vitro, and the cell density was adjusted to 1*10 ⁇ 7/ on the 4th day after electroporation. mL, the cells were labeled with anti-PE-HLA-ABC antibody (Invitrogen) and PE-B2M antibody (Invitrogen).
  • TRAC and B2M double negative cells (the sorting kit was purchased from Miltenyi), that is, more than 99% of TRAC-/-UTD cells with TRAC deletion, TIGIT CAR-T cells with double deletion of TRAC and B2M (TIGIT-UCAR-T cells) were obtained.
  • CAR-T cells were labeled with Biotin-labeled goat anti-human Fab antibody (Jackson), and then labeled with PE-Streptavidin secondary antibody.
  • the CAR expression of TIGIT-UCAR-T cells was detected by flow cytometry.
  • the experimental results are shown in Figure 4. As shown, the positive rate can reach more than 80%.
  • the anti-TIGIT scFv is fully human and can be labeled with a universal anti-human Fab antibody.
  • TIGIT-UCAR-T cells we used Biotin-labeled goat anti-human Fab antibody (Jackson) to label CAR-positive T cells. Then PE-Streptavidin secondary antibody was used for labeling, and the labeled cells were sorted by anti-PE magnetic beads through a sorting column, and TIGIT-UCAR-T cells were collected. After screening, the positive proportion of TIGIT-UCAR-T cells reached more than 95%.
  • Density gradient centrifugation was performed with Ficoll-Paque (GE bioscience) to separate PBMCs from the peripheral blood of donors, and NK cells in PBMC cells were separated with NK cell separation kit (purchased from Miltenyi), and the cell density was adjusted to 1* 10 ⁇ 6/ml or 2*10 ⁇ 6/ml.
  • the U-UTD cells and TRAC-/-UTD cells constructed in Example 2 were selected as negative and positive controls, respectively, and the cell concentration was adjusted to 1*10 ⁇ 6/ml. According to the ratio of NK cells to T cells 1:1 or 2:1, the cells were seeded in 24-well plates and incubated in an incubator for 0hr, 24hr and 48hr, respectively.
  • APC-CD56 (Invitrogen) antibody was used to label NK cells
  • APC-HLA-ABC antibody (Invitrogen) was used to label NK cells in the other mixed culture groups. Changes in the proportion and number of cells.
  • the single chain antibody of CLDN18A2 (VH sequence is shown in SEQ ID NO: 11, VL sequence is shown in SEQ ID NO: 12, HCDR1 sequence is shown in SEQ ID NO: 13, HCDR2 sequence is shown in SEQ ID NO: 14 shown, the HCDR3 sequence is shown in SEQ ID NO: 15, the LCDR1 sequence is shown in SEQ ID NO: 16, the LCDR2 sequence is shown in SEQ ID NO: 17, and the LCDR3 sequence is shown in SEQ ID NO: 18), TIGIT’s Single chain antibody (VH sequence is shown in SEQ ID NO: 1, VL sequence is shown in SEQ ID NO: 2, HCDR1 sequence is shown in SEQ ID NO: 3, HCDR2 sequence is shown in SEQ ID NO: 4, HCDR3 sequence is shown in As shown in SEQ ID NO: 5, LCDR1 sequence is shown in SEQ ID NO: 6, LCDR2 sequence is shown in SEQ ID NO: 7, LCDR3 sequence is shown in SEQ ID NO: 8), G4S (SEQ ID NO: 60
  • tandem fragment 1 SEQ ID NO: 48
  • tandem fragment 2 SEQ ID NO: 50
  • tandem fragment 3 SEQ ID NO: 52
  • CD8 ⁇ Signal peptide SEQ ID NO:38
  • CD8 hinge region SEQ ID NO:40
  • CD28 transmembrane region SEQ ID NO:42
  • intracellular domain SEQ ID NO:44
  • T cell activator CD3 ⁇ SEQ ID NO: 46
  • constructed CAR1 fragment SEQ ID NO: 54
  • CAR2 fragment SEQ ID NO: 56
  • CAR3 fragment SEQ ID NO: 58
  • lentiviral plasmid PRRLsin-CLDN18A2-CAR expressing the chimeric antigen receptor of CLDN18A2 ( Figure 6), including single-chain antibody of CLDN18A2 (SEQ ID NO: 11, 12), CD8 ⁇ signal peptide (SEQ ID NO: 38), CD8 hinge region (SEQ ID NO:40), CD28 transmembrane region (SEQ ID NO:42) and intracellular domain (SEQ ID NO:44), T cell activator CD3 ⁇ (SEQ ID NO:46); slow packaging formation Viral PRRL-CLDN18A2-CAR.
  • Single-chain antibody of CLDN18A2 SEQ ID NO: 11, 12
  • CD8 ⁇ signal peptide SEQ ID NO: 38
  • CD8 hinge region SEQ ID NO:40
  • CD28 transmembrane region SEQ ID NO:42
  • intracellular domain SEQ ID NO:44
  • T cell activator CD3 ⁇ SEQ ID NO:46
  • CLDN18A2-CAR-T targeting CLDN18A2 was prepared with reference to the method described in Example 2; CAR-T1 (expressing CAR1), CAR-T2 (expressing CAR2), and CAR-T3 (expressing CAR3) cells dual targeting TIGIT and CLDN18A2 were prepared.
  • Preparation of TRAC and B2M double knockout cells with reference to the method described in Example 2: U-UTD as negative control (only TRAC and B2M knockout T cells), CLDN18A2-UCAR-T targeting only CLDN18A2, dual targeting UCAR-T1 (CAR1 expressing), UCAR-T2 (CAR2 expressing), UCAR-T3 (CAR3 expressing) cells of TIGIT and CLDN18A2.
  • Example 5 Killing detection of target cells by dual-target UCAR-T cells targeting TIGIT and tumor antigens
  • human CLDN18A2 (SEQ ID NO: 19, 20) were transfected into pancreatic cancer cells BXPC-3 (ATCC, USA) and gastric cancer cells HGC-27 (Chinese Academy of Sciences), which do not express endogenous CLDN18A2, respectively, by lentivirus.
  • BXPC-3 ATCC, USA
  • HGC-27 Choinese Academy of Sciences
  • BXPC-3-A2 and HGC-27-A2 stably transfected cell lines expressing CLDN18A2 were constructed.
  • LDH Cytotoxicity Detection Kit
  • Example 6 Resistance of dual-target UCAR-T cells targeting TIGIT and tumor antigens to PBMC cells
  • Dual-target UCAR-T cells targeting TIGIT and tumor antigens were co-cultured with allogeneic PBMC cells to observe whether they could resist the killing of NK cells in PBMC cells.
  • TIGIT and CLDN18A2 dual-target UCAR-T cells were prepared according to Example 4, and the positive rate of CAR-T cells and the expression of TIGIT were detected ( Figure 9).
  • Some U-UTD and CLDN18A2-UCAR-T cells expressed TIGIT protein on the surface, but UCAR-T1, UCAR-T2, and UCAR-T3 cells could not detect TIGIT protein.
  • Example 7 Therapeutic effect of dual-target UCAR-T cells targeting TIGIT and tumor antigens on transplanted tumors
  • Dual-target UCAR-T targeting TIGIT and tumor antigens can resist allogeneic NK cells and maintain a high survival rate.
  • HGC-27-A2 xenograft tumors (marked as D0) were subcutaneously inoculated into the NPG of immunodeficient mice, and each mouse was inoculated with 5 ⁇ 10 6 HGC-27-A2 cells.
  • the tumor volume was measured and grouped on the 15th day, and then transplanted.
  • the tumor volume was about 90mm3, divided into 5 groups (U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3), 5 in each group; 1 ⁇ 10 6 U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells.
  • mice 14 days after UCAR-T cell treatment, the peripheral blood of mice was collected to detect the density of human CD4+ and CD8+ T cells in the peripheral blood of mice.
  • TIGIT Since some T cells also express TIGIT, in order to avoid the killing of CAR-T cells by CAR-T cells, the TIGIT gene of CAR-T cells was further knocked out on the basis of TRAC/B2M double knockout.
  • TIGIT gene we designed 12 pairs of gRNAs 1-12 (SEQ ID NOs: 25-36) targeting the TIGIT gene.
  • the sgRNA sequences targeting TIGIT were synthesized in vitro according to the reagent instructions (GeneArt TM Precision gRNA Synthesis Kit, Thermo Tisher).
  • T cells were activated by coating magnetic beads with CD3-CD28 antibody, and the T cells on the 5th day of activation were electroporated using a Maxcyte electroporator.
  • the electroporation system was 0.5 ⁇ M Cas9+2 ⁇ M TIGIT sgRNA.
  • APC-TIGIT antibody was used to detect the knockout efficiency of each gRNA. Refer to Example 2 for specific experimental operations.
  • gRNA6 and gRNA10 had the highest knockout efficiencies, with knockdown efficiencies of 76% and 72%, respectively. And gRNA11, 5, 7, and 8 can also knock out TIGIT.
  • TIGIT&CLDN18A2 dual-target UCAR-T In order to avoid TIGIT&CLDN18A2 dual-target UCAR-T from killing its own cells, it is necessary to knock out the TIGIT gene of UCAR-T cells on the basis of TRAC/B2M knockout.
  • T cells were activated by coating magnetic beads with CD3-CD8 antibody, and 48 hours later, they were infected with lentiviruses PRRL-CLDN18A2-CAR, PRRL-CAR1, PRRL-CAR2, and PRRL-CAR3. After 24 hours, the medium was centrifuged. Continue to culture until the 6th day, using Maxcyte electroporator to double knockout TRAC&B2M or triple knockout TRAC&B2M&TIGIT gene.
  • TIGIT sgRNA is shown in SEQ ID NO: 30
  • TRAC-gRNA is shown in SEQ ID NO: 23
  • B2M-gRNA is shown in SEQ ID NO: 24.
  • TRAC and B2M double knockout CLDN18A2-UCAR-T cells TRAC&B2M&TIGIT triple knockout cells U-UTD-TIGIT KO, CLDN18A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR- T2-TIGIT KO, UCAR-T3-TIGIT KO cells.
  • the specific experimental operation is described in Example 2.
  • the above CAR-T cells were stained with PE-labeled anti-CLDN18A2 scFv antibody, and the positive rate of each CAR-T cell was detected by flow cytometry.
  • the positive rate of CAR-T cells is shown in Figure 13.
  • the TIGIT gene knockout efficiencies of UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, and UCAR-T3-TIGIT KO cells were 84.3%, 85.2%, and 84.7%, respectively.
  • Example 9 The effect of TIGIT gene knockout on the killing activity of CAR-T cells in vitro
  • Knockout of endogenous TIGIT gene may affect the killing of target cells by CAR-T cells.
  • the electroporation system is 1.5 ⁇ M Cas9+6 ⁇ M TIGIT sgRNA, wherein the nucleic acid sequence of TIGIT sgRNA is shown in SEQ ID NO: 30, and CLDN18A2-CAR-T is obtained. -TIGIT KO cells.
  • LDH Cytotoxicity Detection Kit
  • Example 10 Therapeutic effect of dual-target UCAR-T-TIGIT KO cells targeting TIGIT and tumor antigens on transplanted tumors
  • NPG mice were subcutaneously inoculated with HGC-27-A2 xenografts, and each mouse was inoculated with 5 ⁇ 10 6 HGC-27-A2 cells. On the 20th day, the tumor volume was measured and grouped.
  • the transplanted tumor volume was about 200 mm3, and the patients were divided into 6 groups (U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN18A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO , UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO), 5 in each group; 2 ⁇ 10 6 U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN18A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells.
  • Example 11 Dual-target U-CAR T-TIGIT KO cells targeting TIGIT and tumor antigens enhance the therapeutic effect on transplanted tumors in the presence of NK cells
  • HGC-27-A2 xenograft tumor was measured about 100 mm 3 and divided into 6 groups, of which 5 ⁇ 10 6 PBMC cells were administered to the 1st, 3rd, 4th, 5th and 6th groups.
  • group 1 received 5 ⁇ 10 5 U-UTD-TIGIT KO cells via tail vein
  • group 2 received 5 ⁇ 10 5 CLDN18A2-UCAR T-TIGIT KO cells via tail vein
  • group 3 received 5 ⁇ 10 5 via tail vein.
  • CLDN18A2-UCAR T-TIGIT KO group 4 were given 5 ⁇ 10 5 U-CAR-T1-TIGIT KO cells by tail vein, group 5 were given 5 ⁇ 10 5 UCAR-T2-TIGIT KO cells, group 6 were given tail vein 5 ⁇ 10 5 UCAR-T3-TIGIT KO cells.
  • TIGIT-BBZ-CAR T and TIGIT-28Z-CAR T cells were constructed with reference to Example 2.
  • the fragments shown in Table 4 were inserted for the construction of TIGIT-targeting CAR-T cells.
  • CD8 ⁇ signal peptide SEQ ID NO: 38
  • anti-TIGIT single chain antibody the amino acid sequence of VH is shown in SEQ ID NO: 1, the amino acid sequence of VL is shown in SEQ ID NO: 2
  • CD8 ⁇ hinge region Amino acid sequence shown in SEQ ID NO: 40
  • CD8 transmembrane region amino acid sequence shown in SEQ ID NO: 86
  • CD28 transmembrane region amino acid sequence shown in SEQ ID NO: 42
  • CD28 intracellular domain amino acid sequence shown in SEQ ID NO: 44
  • CD137 intracellular domain amino acid sequence shown in SEQ ID NO: 88
  • T cell activator CD3 ⁇ amino acid sequence shown in SEQ ID NO: 46
  • TIGIT-BBZ and TIGIT-28Z in Table 4 were inserted into the lentiviral vector, and the lentiviral plasmids PRRL-TIGIT-BBZ and PRRL-TIGIT-28Z (see Figure 16) were constructed, and then transfected respectively.
  • 293T cells were packaged to obtain lentiviruses TIGIT-BBZ and TIGIT-28Z, which were respectively infected with T cells to obtain TIGIT-targeted TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells.
  • TRAC and B2M in TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells were respectively knocked out to prepare TRAC and B2M double-negative TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells.
  • TRAC-/-UTD was prepared by single-gene knockout of TRAC in UTD cells (T cells not transfected with virus) as a positive control.
  • UTD cells with double deletion of TRAC and B2M (U-UTD cells) served as negative controls.
  • NK cells Adjust the cell concentration to 1*10 ⁇ 6/ml. According to the ratio of NK cells to T cells 1:1, the cells were seeded in 24-well plates and incubated in an incubator for 0hr, 24hr and 48hr respectively.
  • APC-CD56 (Invitrogen) antibody was used to label NK cells
  • APC-HLA-ABC antibody Invitrogen was used to label NK cells in the other mixed culture groups. Changes in the proportion of cells.
  • TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells were respectively knocked out with reference to Example 9, and TIGIT-BBZ-UCAR T-TIGIT KO cells, TIGIT-28Z-UCAR T-TIGIT KO cells were prepared cell.
  • Example 13 CAR-T cells targeting TIGIT enhance the resistance of CAR-T cells targeting tumor antigens to NK cells and their antitumor activity
  • CAR T cells targeting tumor antigen CLDN18A2 we tested in vitro whether CAR-T cells targeting TIGIT could enhance the survival and antitumor effect of CAR-T cells targeting tumor antigens in the presence of NK cells. active.
  • Example 14 CAR-T cells targeting TIGIT enhance the resistance of CAR-T cells targeting tumor antigens to NK cells and their in vivo antitumor activity
  • NPG mice were subcutaneously inoculated with HGC-27-A2, each inoculated with 5 ⁇ 10 6 cells.
  • the volume of HGC-27-A2 xenograft tumor was measured about 100 mm 3 and divided into 5 groups, of which 5 ⁇ 10 6 PBMC cells were administered to the 1st, 3rd, 4th and 5th groups, respectively.
  • 5 ⁇ 10 5 U-UTD-TIGIT KO cells were injected into the tail vein of group 1
  • 5 ⁇ 10 5 CLDN18A2-UCAR T-TIGIT KO cells were injected into the tail vein of group 2
  • 5 ⁇ 10 5 were injected into the tail vein of group 3.

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Abstract

La présente invention concerne une cellule immunitaire modifiée plus durable et/ou présentant un taux élevé de survie après transplantation dans un organisme hôte et utilisée pour l'immunothérapie, ainsi qu'un procédé de préparation à cet effet. La présente invention concerne une cellule génétiquement modifiée, caractérisée en ce que la cellule exprime une première protéine capable de reconnaître un TIGIT. La présente invention concerne également un procédé permettant d'augmenter la durabilité et/ou le taux de survie à la transplantation d'une première cellule immunitaire en présence d'une seconde cellule immunitaire hôte.
PCT/CN2022/077702 2021-02-24 2022-02-24 Cellules tigit modifiées et leur composition Ceased WO2022179567A1 (fr)

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