CN116897202A - TIGIT engineered cells and compositions thereof - Google Patents

Abstract

Methods for developing engineered immune cells for immunotherapy with greater persistence in host organisms and/or high transplantation survival rates and methods for their preparation. A genetically engineered cell, characterized in that the cell expresses a first protein capable of recognizing TIGIT, and is involved in increasing the persistence and/or transplantation survival rate of the first immune cell in the presence of the host's second immune cell. method.

Description

TIGIT engineered cells and compositions thereof

Related patent applications

This patent application claims priority from the Chinese patent application with application number 202110205868.5 submitted on February 24, 2021.

Technical field

The present application relates to a cell with the function of resisting transplant rejection, and also relates to a method of resisting transplant immune rejection, and in particular to a method of resisting NK cell immune rejection.

Background technique

Due to the immunogenetic differences between the donor and the recipient, when performing an exogenous donor transplant, as an exogenous graft, the donor may also be recognized and attacked by immune cells in the recipient's body, thereby inhibiting or eliminating the foreign source. graft, producing a host-versus-graft response (HVGR). By knocking out MHC molecules in transplant cells, the rejection of the graft by host T cells can be effectively resisted, but it may cause rejection by other immune cells in the host. For example, in allogeneic cell transplantation, the loss of MHC-I class molecules in allogeneic cells will lead to NK cell rejection in the host body and enhance the clearance of allogeneic cells (Nat Biotechnol. 2017; 35(8):765-772. doi:10.1038/nbt.3860). Therefore, how to effectively prevent immune rejection of host NK cells is crucial to the development of allogeneic cell transplantation therapy.

Detailed description of the invention

The purpose of this application is to provide a cell that resists transplant immune rejection and a method for resisting rejection.

In a first aspect of the present application, a genetically engineered cell is provided, characterized in that the cell expresses a first protein capable of recognizing TIGIT.

In a specific embodiment, the first protein contains an antibody capable of recognizing TIGIT, preferably the sequence of TIGIT is shown in SEQ ID No: 10.

In a specific embodiment, the first protein includes a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.

In a specific embodiment, the first protein is a CAR, and the CAR includes:

(i) Antibodies that recognize TIGIT, CD28 or the transmembrane region of CD8, CD3δ;

(ii) Antibodies that recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ;

(iii) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD137 and CD3δ; and/or

(iv) An antibody that recognizes TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ.

In a specific embodiment, the cells comprise: knockout of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules.

In a specific embodiment, CRISPR/Cas9 technology is used to knock out the TIGIT gene of the cell, and the gRNA used is selected from SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , any one of the sequences shown in 35, 36 or a combination thereof.

In a specific embodiment, 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 .

In a specific embodiment, the cells are autologous or allogeneic T cells, primary T cells, or autologous T cells derived from humans.

In a specific embodiment, the cells include knockout of the gene encoding TCR protein and/or low expression or no expression of endogenous TCR molecules, and/or knockout and/or endogenous expression of the gene encoding MHC protein. The source of MHC has low or no expression.

In a specific embodiment, CRISPR/Cas9 technology is used to knock out endogenous MHC molecule B2M and endogenous TCR.

In a specific embodiment, the gRNA used to knock out B2M includes the sequences shown in SEQ ID NO: 24, 72, 73 and/or 74, and the gRNA used to knock out TCR includes SEQ ID NO: 23, 65, 66, Sequences shown in 67, 68, 69, 70 and/or 71.

In a specific embodiment, the antibody that recognizes TIGIT includes:

(i) HCDR1 represented by SEQ ID NO: 3, HCDR2 represented by SEQ ID NO: 4, HCDR3 represented by SEQ ID NO: 5, LCDR1 represented by SEQ ID NO: 6, and represented by SEQ ID NO: 7 LCDR2, and/or LCDR3 shown in SEQ ID NO: 8; or

(ii) The heavy chain variable region shown in SEQ ID NO: 1 and/or the light chain variable region shown in SEQ ID NO: 2; or

(iii) scFv shown in SEQ ID NO:78.

In a specific embodiment, the first protein also recognizes tumors and/or pathogens; preferably, the tumor expression includes BCMA, CD19, GPC3, CLDN18A2, EGFR or a combination thereof.

In a specific embodiment, the first protein includes an antibody that recognizes TIGIT and an antibody that recognizes tumor and/or pathogen antigens, and their connection method includes:

(i) Light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT—Heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes TIGIT region)—the heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes tumor and/or pathogen antigens—the light chain/heavy chain (or heavy chain) of an antibody that recognizes tumor and/or pathogen antigens Light chain variable region/heavy chain variable region);

(ii) The light chain (or light 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 TIGIT - The light chain (or light chain variable region) of an antibody that recognizes TIGIT chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens; and/or

(iii) 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,

Wherein, the first protein is a CAR, and the CAR includes:

(i) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, CD3δ;

(ii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ;

(iii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137, and CD3δ; and/or

(iv) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ.

In a specific embodiment, the antibody that recognizes a tumor antigen recognizes CLDN18A2, including:

(i) 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, and SEQ ID NO: 17 LCDR2, and/or LCDR3 described in SEQ ID NO: 18; or

(ii) The heavy chain variable region described in SEQ ID NO: 11 and/or the light chain variable region described in SEQ ID NO: 12; or

(iii) scFv shown in SEQ ID NO: 82.

In a specific embodiment, the first protein includes the sequence shown in SEQ ID NO: 48, 50, 52, 90 or 91.

In a specific embodiment, the cells also express a second protein targeting recognition of tumor antigens and/or pathogen antigens, chemokines, chemokine receptors, cytokines, siRNA that reduces PD-1 expression, Proteins, safety switches, or combinations thereof that block the binding of PD-L1 to PD-1.

In a specific embodiment, when the cells are co-cultured with host NK cells, the cells can kill host NK cells, or the cells can resist the killing of the cells by host NK cells, or the cells can Resist killing of the cells by activated host NK cells.

In a specific embodiment, 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 than the first protein.

In a specific embodiment, the second protein includes a CAR, and the CAR includes:

(i) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, CD3δ;

(ii) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, and CD3δ;

(iii) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137, and CD3δ; and/or

(iv) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ.

In a specific embodiment, the cells expressing the second protein include:

(i) Knockout of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules; or

(ii) Knockout of genes encoding TCR and/or MHC proteins and/or low or no expression of endogenous TCR and/or MHC molecules; or

(iii) Knockout of genes encoding TIGIT, TCR and MHC proteins and/or low or no expression of endogenous TIGIT, TCR and MHC molecules.

In a specific embodiment, the cell expressing the second protein:

(i) Use CRISPR/Cas9 technology to knock out TIGIT molecules;

(ii) Use CRISPR/Cas9 technology to knock out TCR and/or MHC molecule B2M; or

(iii) Use CRISPR/Cas9 technology to knock out TIGIT, TCR and MHC molecule B2M.

In a specific embodiment, 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 combinations thereof.

In a specific embodiment, the cells are autologous or allogeneic T cells, primary T cells, or autologous T cells derived from humans.

According to a second aspect of the present application, a method for increasing the persistence and/or transplantation survival rate of a first immune cell in the presence of a host's second immune cell is provided, including:

a) Provide first immune cells;

b) optionally modifying said first immune cell by reducing or inhibiting expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in response to self- and non-self antigen recognition;

c) modifying the first immune cell with a polynucleotide encoding a first protein targeting TIGIT.

In a specific embodiment, the polypeptide in step b) is selected from MHC, TCR, and/or TIGIT.

In a specific embodiment, step b) includes:

(i) Use CRISPR/Cas9 technology to knock out TIGIT molecules;

(ii) Use CRISPR/Cas9 technology to knock out TCR and/or MHC molecule B2M; or

(iii) Use CRISPR/Cas9 technology to knock out TIGIT, TCR and MHC molecule B2M.

In a specific embodiment, step b) includes:

(i) The gRNA used to knock 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;

(ii) The gRNA used to knock out TCR includes the sequences shown in SEQ ID NO: 23, 65, 66, 67, 68, 69, 70 and/or 71; and/or

(iii) The gRNA used to knock out the MHC molecule B2M includes the sequence shown in SEQ ID NO: 24, 72, 73 and/or 74.

In a specific embodiment, the first protein includes a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.

In a specific embodiment, the first protein includes:

(i) an antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; and/or

(ii) an antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137 and CD3δ; and/or

(iii) an antibody that recognizes TIGIT, optionally an antibody that recognizes tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ;

(iv) An antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, and CD3δ.

In a specific embodiment, the first protein includes:

(i) scFv of TIGIT shown in SEQ ID NO: 78;

(ii) scFv of CLDN18A2 shown in SEQ ID NO: 82;

(iii) The tandem sequence of the TIGIT antibody and CLDN18A2 antibody shown in SEQ ID NO: 48, 50 or 52; or

(iv) The first protein shown in SEQ ID NO: 9, 54, 56, 58, 90 or 91.

In a specific embodiment, 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 with expressed siRNA, a protein that blocks the binding of PD-L1 to PD-1, or a safety switch and other non-endogenous polynucleotides.

In a specific embodiment, 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 combinations thereof.

In a specific embodiment, the first immune cell is an autologous or allogeneic T cell, a primary T cell, or an autologous T cell derived from a human.

According to the third aspect of the present application, an engineered cell is provided, which is prepared by the method of the present application.

According to a fourth aspect of the present application, a polynucleotide is provided, which encodes a nucleic acid molecule for constructing the cell described in the present application or encodes a nucleic acid molecule required for administering the method of the present application.

According to the fifth aspect of the present application, a vector is provided, which contains the polynucleotide as described in the present application.

According to the sixth aspect of the present application, a virus comprising the vector described in the present application is provided.

According to the seventh aspect of the present application, there is provided a composition comprising an effective amount of cells as described in the present application, polynucleotides as described in the present application, vectors as described in the present application, of viruses.

According to an eighth aspect of the present application, there is provided a method of treating inflammatory conditions, viral infections and/or tumors, comprising administering cells as described in the present application or a combination as described in the present application to a subject in need things.

This application also relates to:

1. A genetically engineered cell, characterized in that the cell expresses the first protein capable of recognizing TIGIT;

Preferably, the TIGIT amino acid sequence is shown in SEQ ID NO: 10;

Preferably, the first protein contains an antibody or functional fragment thereof capable of recognizing TIGIT;

Preferably, 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;

Preferably, the first protein includes an antibody that recognizes TIGIT or a functional fragment thereof, an antibody that recognizes a tumor antigen or a pathogen antigen or a functional fragment thereof, a transmembrane region, and an intracellular domain;

Preferably, the antibody or functional fragment thereof that recognizes TIGIT and the antibody that recognizes a tumor antigen or pathogen antigen are connected through a connecting peptide.

2. The cell according to item 1, characterized in that the cell is an immune effector cell or an artificially modified cell having the function of an immune effector cell.

3. The cell according to item 1 or 2, characterized in that the cell is selected from the group consisting of T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells;

Preferably, the cells are derived from human T cells;

Preferably, the cells are human primary T cells;

More preferably, the cells are allogeneic T cells.

4. The cell according to any one of items 1 to 3, characterized in that the first protein is also connected to a cell activation signal,

Preferably, the first protein comprises a TCR intracellular domain derived from a stimulatory domain of an intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα or TCRβ.

5. The cell according to any one of items 1 to 4, characterized by reducing MHC and/or endogenous TCR expression, activity and/or signaling in the cell; preferably, the MHC is MHC Class I molecules; 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- C. One or more of B2M; most preferably, the HLA-I includes HLA-A and/or B2M; preferably, the endogenous TCR includes one of the α and β chains of TCR or two chains;

Preferably, said being reduced or inhibited is carried out by using TAL nuclease, meganuclease, zinc finger nuclease, Cas9 and Argonaute;

Preferably, the engineered T cells comprise inhibitory nucleic acid molecules or gRNA targeting genes encoding MHC;

Preferably, the inhibitory nucleic acid molecule comprises a sequence complementary to the MHC-encoding gene and/or the endogenous TCR;

Preferably, the inhibitory nucleic acid comprises an RNA interference agent;

Preferably, the inhibitory nucleic acid comprises siRNA, shRNA or miRNA;

Preferably, the gRNA sequence contains the sequence shown in SEQ ID NO: 23 and/or SEQ ID NO: 24;

Preferably, the reduction in expression, activity and/or signaling of MHC and/or endogenous TCR is permanent, transient or inducible;

Preferably, the expression, activity and activity of MHC and/or endogenous TCR in the engineered cells are compared with the expression, activity and/or signaling of MHC and/or endogenous TCR in non-genetically engineered T cells. and/or the reduction in signaling is greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%;

Preferably, the expression of MHC and/or endogenous TCR expressed in the engineered cells is undetectable using immunoblot assay and/or flow cytometric detection.

6. The cell according to any one of items 1 to 5, characterized in that 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 combination thereof;

Preferably, the first protein includes an extracellular domain, a transmembrane region and an intracellular signaling domain.

7. The cell according to any one of items 1-6, characterized in that the first protein includes:

(i) An antibody or functional fragment thereof that specifically recognizes TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; and/or

(ii) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137 and CD3δ; and/or

(iii) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of CD137 and CD3δ;

(iv) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, and CD3δ;

Preferably, 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;

Preferably, the amino acid sequence of the first protein shares at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NO:9.

8. The cell according to any one of items 1 to 7, characterized in that the cell also expresses a second protein, a chemokine, a chemokine targeting a tumor antigen, and/or a pathogen antigen, and/or a viral antigen. Chemical factor receptors, cytokines, siRNA that reduces PD-1 expression, proteins that block the binding of PD-L1 to PD-1, safety switches, or combinations thereof;

Preferably, the second protein includes: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), aTCR -T or its combination;

Preferably, the second protein can specifically recognize Claudin18.2, GPC3, BCMA or CD19.

9. The cell according to any one of items 1-8, characterized in that the cell also expresses a ligand or antibody fragment of an NK cell inhibitory receptor;

Preferably, the cells further express an NKG2A binding molecule;

Preferably, the NKG2A binding molecule is a cell membrane-binding protein or a secreted protein;

Preferably, the NKG2A binding molecule contains an extracellular domain and a transmembrane region; or contains an extracellular domain, a transmembrane region and an intracellular region;

Preferably, the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane.

10. The cell according to any one of items 1-9, characterized in that the endogenous TIGIT expression, activity and/or signaling of the cell is reduced or inhibited;

Preferably, said being reduced or inhibited is carried out by using TAL nuclease, meganuclease, zinc finger nuclease, Cas9 and Argonaute;

Preferably, the immune cell comprises an inhibitory nucleic acid molecule or gRNA targeting the gene encoding TIGIT;

Preferably, the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding TIGIT;

Preferably, the inhibitory nucleic acid comprises an RNA interference agent;

Preferably, the inhibitory nucleic acid comprises siRNA, shRNA or miRNA;

Preferably, the reduction in expression, activity and/or signaling of TIGIT is permanent, transient or inducible;

Preferably, the expression, activity and/or signaling of TIGIT in the engineered cells is reduced by greater than or greater than about 50%, 60% compared to the expression, activity and/or signaling of TIGIT in the non-genetically engineered cells. , 70%, 80%, 90%, 95% or 100%;

Preferably, the expression of TIGIT expressed in the cells is undetectable using immunoblotting assays and/or flow cytometry.

11. The cell according to any one of items 1 to 10, characterized in that when the cell is co-cultured with host NK cells, the cell can kill host NK cells.

12. The cell according to any one of items 1-11, characterized in that when the cell is co-cultured with host NK cells, the cell can resist killing of the cell by host NK cells;

Preferably, the cell is resistant to killing of the cell by NK cells activated by cytokines in the host NK cells;

Preferably, the cells are resistant to killing of the cells by host NK cells expressing NKG2A;

Preferably, the cells are significantly resistant to killing of the cells by host NK cells with low expression of NKG2A.

13. The cell according to any one of items 1 to 12, characterized in that the cell is administered in combination with an agent that enhances its function, preferably in combination with a chemotherapy drug;

and/or the cells are administered in combination with an agent that ameliorates one or more side effects associated therewith.

14. Methods to increase the persistence of allogeneic immune cells and/or transplant survival in the presence of host immune cells, including:

a) Provide allogeneic cells;

b) modifying said cell by reducing or inhibiting expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in response to self- and non-self antigen recognition;

c) modifying the cell with a polynucleotide encoding a first protein targeting TIGIT.

15. The method according to item 14, characterized in that 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; Preferably, the endogenous TCR includes one or both chains of α and β chains of TCR; more preferably, the HLA-I includes HLA-A and/or B2M;

Preferably, step b) modifies said cells by reducing or inhibiting B2M and TCR expression, activity and/or signaling;

Preferably, said being reduced or inhibited is carried out by using TAL nuclease, meganuclease, zinc finger nuclease, Cas9 and Argonaute;

Preferably, the cell comprises an inhibitory nucleic acid molecule or gRNA targeting a gene encoding MHC;

Preferably, the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding MHC;

Preferably, the inhibitory nucleic acid comprises an RNA interference agent;

Preferably, the inhibitory nucleic acid comprises siRNA, shRNA or miRNA;

Preferably, the gRNA sequence contains the sequence shown in SEQ ID NO: 23 and/or 24;

Preferably, the reduction in expression, activity and/or signaling of MHC and/or endogenous TCR is permanent, transient or inducible;

Preferably, the expression, activity and signaling of MHC and/or endogenous TCR in the engineered cells are compared to the expression, activity and/or signaling of MHC and/or endogenous TCR in cells that are not genetically engineered. /or the reduction in signaling is greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100%;

Preferably, expression of the MHC expressed in said cells is undetectable using immunoblotting assays and/or flow cytometry.

16. The method according to item 14 or 15, characterized in that 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 Fusion protein (TFP) or combination thereof;

Preferably, the first protein includes an extracellular domain, a transmembrane region and an intracellular signaling domain;

Preferably, the cell transmits signals through an intracellular signaling domain to mediate the suppression or killing of the host's immune effector cells;

Preferably, the first protein includes:

(i) An antibody or functional fragment thereof that specifically recognizes TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; and/or

(ii) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137 and CD3δ; and/or

(iii) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of CD137 and CD3δ;

(iv) Antibodies or functional fragments thereof that specifically recognize TIGIT, CD28 or the transmembrane region of CD8, and CD3δ;

Preferably, 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;

Preferably, the amino acid sequence of the first protein shares at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NO:9.

17. The method according to any one of items 14-16, further comprising step d) encoding a second protein, a chemokine targeting a tumor antigen, and/or a pathogen antigen, and/or a viral antigen, Non-endogenous polynucleotides such as chemokine receptors, cytokines, siRNA that reduces PD-1 expression, proteins that block the binding of PD-L1 to PD-1, or safety switches are used to modify the cells;

Preferably, the second protein includes: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), aTCR -T or its combination;

Preferably, the second protein can specifically recognize Claudin18.2, GPC3, BCMA or CD19.

18. The method according to any one of items 14-17, further comprising step e) modifying the cell with a non-endogenous polynucleotide encoding a ligand or antibody fragment of an NK cell inhibitory receptor ;

Preferably, it also includes step e) modifying the cell by encoding a non-endogenous polynucleotide encoding the immune cell NKG2A binding molecule;

Preferably, the NKG2A binding molecule is a cell membrane-binding protein or a secreted protein;

Preferably, the NKG2A binding molecule only contains an extracellular domain and a transmembrane region; or contains an extracellular domain, a transmembrane region and an intracellular region;

Preferably, the NKG2A binding molecule is an NKG2A antibody or antibody fragment bound to the cell membrane.

19. The method according to any one of items 14-18, further comprising step f) modifying the cell by encoding that TIGIT expression, activity and/or signaling is reduced or inhibited;

Preferably, said being reduced or inhibited is carried out by using TAL nuclease, meganuclease, zinc finger nuclease, Cas9 and Argonaute;

Preferably, the cell comprises an inhibitory nucleic acid molecule or gRNA targeting the gene encoding TIGIT;

Preferably, the inhibitory nucleic acid molecule comprises a sequence complementary to the gene encoding TIGIT;

Preferably, the inhibitory nucleic acid comprises an RNA interference agent;

Preferably, the inhibitory nucleic acid comprises siRNA, shRNA or miRNA;

Preferably, the reduction in expression, activity and/or signaling of TIGIT is permanent, transient or inducible;

Preferably, the expression, activity and/or signaling of TIGIT in the engineered immune cells is reduced by greater than or greater than about 50%, 60 %, 70%, 80%, 90%, 95% or 100%;

Preferably, the expression of TIGIT expressed in the immune cells is undetectable using immunoblot assay and/or flow cytometric detection.

20. The method according to any one of items 14 to 19, wherein 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;

Preferably, the cells are derived from human T cells;

Preferably, the cells are human primary T cells;

More preferably, the cells are allogeneic T cells.

21. The method according to any one of items 14 to 20, characterized in that when the cells prepared by the method are co-cultured with host NK cells, the cells can kill host NK cells.

22. The method according to any one of items 14-21, characterized in that when the cells prepared by the method are co-cultured with host NK cells, the cells can resist the killing of the cells by host NK cells;

Preferably, the cell is resistant to killing of the cell by NK cells activated by cytokines in the host NK cells;

Preferably, the cells are resistant to killing of the cells by host NK cells expressing NKG2A;

Preferably, the cells are significantly resistant to killing of the cells by host NK cells with low expression of NKG2A.

23. The method according to any one of items 14 to 22, characterized in that the cells prepared by the method are used in combination with agents that enhance their functions, preferably in combination with chemotherapy drugs;

Or the cells prepared by the method are administered in combination with an agent that improves one or more side effects associated therewith.

24. An engineered cell produced by the method of any one of items 14-23.

25. A bispecific antibody construct comprising a first binding domain that binds to human or macaque TIGIT on the surface of a target cell and a second binding domain that binds to human CD3 on the surface of a T cell.

26. The antibody construct of item 25, wherein the second binding domain binds to human CD3ε and common marmoset, cottontop tamarin or squirrel monkey CD3ε;

Preferably, the antibody construct is selected from the following formats: (scFv)2, scFv-single domain mAb, diabodies and oligomers of these formats.

27. A polynucleotide encoding an antibody construct as described in Item 25 or 26 or a construct encoding a cell as described in any one of Items 1-13 or encoding a method for administering any one of Items 14-23 Required construct.

28. A vector comprising a polynucleotide as defined in item 27.

29. A virus that infects the vector according to item 28.

30. A composition comprising an effective amount of the engineered cells of any one of items 1-13 or 24,

Preferably, a pharmaceutically acceptable carrier is also included;

Preferably, the carrier is saline solution, dextrose solution or 5% human serum albumin.

Preferably, a cryoprotectant is also included.

31. A kit, characterized by comprising the engineered cells according to any one of items 1-13 or 24 or the composition according to item 30 and additional agents for treating diseases.

32. A method of treating a disease, comprising administering to a subject in need an engineered cell as described in any one of items 1-13 or 24 or a composition as described in item 30 or as described in item 31 The above-mentioned kit,

Preferably, before administering said engineered cells, said engineered cells are produced by a method according to any one of items 14-23;

Preferably, it also includes administering an additional pharmaceutical agent;

Preferably, the disease is selected from inflammatory conditions, infections and tumors;

Preferably, the subject is a human;

Preferably, wherein said engineered cells are autologous or allogeneic T cells to said subject.

It should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described below (such as embodiments) can be combined with each other to constitute a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows the effect of cytokines on the proportion of TIGIT+NK cells;

Figure 2 shows the percentage of TIGIT+NK cells among activated NK cells;

Figure 3 shows the TIGIT-Z CAR vector map;

Figure 4 shows the TIGIT-Z CAR-T cell positivity rate;

Figure 5 shows the resistance function of TIGIT-UCAR-T cells to NK cells;

Figure 6 shows the vector map 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 only targeting CLDN18A2;

Figure 10 shows the changes in the ratio of T cells and NK cells after co-culture of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 dual-targeting TIGIT&CLDN18A2, and CLDN18A2-UCAR-T cells only targeting CLDN18A2, with PBMC cells. Condition;

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 transplanted tumors; Figure 11B The survival status of peripheral blood CD4+ and CD8+ T cells in each of the above groups is shown;

Figure 12 shows the knockdown efficiency of different gRNAs of TIGIT;

Figure 13 shows the CAR positive rate of CLDN18A2-UCAR-T, CLDN18A2-UCAR-T-TIGITKO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, and UCAR-T3-TIGIT KO cells;

Figure 14A shows the CAR-positive rate and TIGIT-positive cell proportion of CLDN18A2-CAR-T and CLDN18A2-CAR-T-TIGIT KO cells; Figure 14B shows the target response of CLDN18A2-CAR-T and CLDN18A2-CAR-T-TIGIT KO cells. Cell killing effect in vitro;

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- The therapeutic effect of 27-CLDN18A2 transplanted tumors;

Figure 16 shows the vector map of TIGIT-BBZ and TIGIT-28Z targeting TIGIT;

Figure 17 shows the changes in the ratio of T cells and NK cells after TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR cells were co-cultured with NK cells respectively.

Detailed ways

After extensive and in-depth research, the applicant unexpectedly found that expressing TIGIT-targeting chimeric antigen receptors on the surface of immune effector cells or artificially modified cells with immune effector cell functions can destroy most NK in the host body. cells, thereby largely avoiding the attack of host NK cells on allogeneic immune effector cells or artificially modified cells with immune effector cell functions, prolonging the survival time of allogeneic T cells in the host body, and improving the anti-tumor effect . On this basis this application was completed.

the term

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, where suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise stated, the practice of this application will employ traditional techniques in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are all within the technical scope of the art. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J. Gaited., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Harries & S.J. Higginseds.1984); Transcription And Translation (B.D.Hames &S.J.Higginseds.1984); Culture Of Animal Cells (R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols.154 and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" ( D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In the disclosure, various aspects of the claimed subject matter are presented in scope format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, descriptions of ranges should be deemed to have specifically disclosed all possible subranges as well as individual values within such ranges. For example, where a range of values is provided, it will be understood that every intervening value between the upper and lower limits of the range, as well as any other stated or intermediate value within the stated range, is included in the claimed claim. Within the subject matter, the upper and lower limits of the stated range also belong to the scope of the claimed subject matter. The upper and lower limits of such smaller ranges may independently be included within the stated smaller ranges and are within the scope of the claimed subject matter unless the upper and lower limits of the stated ranges are expressly excluded. Where a range is stated to include one or both of the limits, the claimed subject matter also includes a range excluding one or both of the stated limits. This applies regardless of the width of the range.

The term approximately as used herein refers to the usual error range for each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, descriptions of "about X" include descriptions of "X". For example, "about" or "including" may mean within 1 or more than 1 based on the actual standard deviation in the field. Alternatively "about" or "comprising" may mean a range of up to 10% (i.e. ±10%). For example, approximately 5uM may include any number between 4.5uM and 5.5uM.

Unless otherwise indicated, any concentration range, percentage range, ratio range or integer range stated herein is to be understood to include any integer within the stated range and, where appropriate, fractions thereof (e.g., one-tenth of an integer and one-tenth of an integer). One percent) value.

To facilitate a better understanding of this application, relevant terms are defined as follows:

The term "TIGIT (T cell Ig and ITIM domain)" or "TIGIT inhibitory receptor" is a member of the poliovirus receptor (PVR)/Nectin family. Gene accession number: 201633. The human TIGIT amino acid sequence is as SEQ ID NO. :10 shown. It consists of an extracellular immunoglobulin variable region (IgV) domain-type transmembrane region and an intracellular region with classical immunoreceptor tyrosine inhibitory motifs (ITIM) and immunoglobulin tyrosine tail (ITT) motifs. Structural domain composition. 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 embodiments of this application use different combinations of IL-2, IL-15 and IL-12 cytokines to treat NK cells and detect TIGIT+NK cells. Changes in proportions.

The term "CLDN18A2" refers to Claudin 18 (CLD18) molecules (Genbank accession numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2): NM_001002026, NP_001002026) with a molecular weight of approximately 27,9/27,72kD intrinsic transmembrane protein. Claudins are intrinsic membrane proteins located in the tight junctions of epithelium and endothelium. Tight junctions are networks of interconnected chains of particles within tissue membranes between adjacent cells. In tight junctions, occludin and claudin are the main transmembrane protein components. Due to their strong intercellular adhesive properties, they create a primary barrier that prevents and controls paracellular transport of solutes and limits lateral diffusion of membrane lipids and proteins to maintain cell polarity. Proteins that form tight junctions are critically involved in tissue epithelial tissue structure.

The term "transplant immune rejection" means that after the host undergoes allogeneic tissue, organ, or cell transplantation, the foreign graft is recognized by the host's immune system as an "alien component" and initiates a response to the transplant. Immunological response of attack, destruction and clearance. The present application provides a cell that resists transplantation immune rejection and a method that resists transplantation rejection.

The term "graft" refers to a biological material or preparation derived from an individual other than the host and intended for implantation into the host. The graft may be from any animal source, such as mammalian source, preferably from humans. In some embodiments, the graft may be from a host, for example, cells from the host may be cultured in vitro or modified and re-implanted into the host. In some embodiments, the transplant may be from another allogeneic individual, such as cells from other people that have been cultured in vitro, or modified and implanted into the host. In some embodiments, the transplant may be from a heterogeneous individual, such as an organ from another species (such as mouse, pig, monkey) implanted into a human.

The term "cell" refers to human or non-human cells of animal origin.

The term "host" refers to a recipient of a graft, which in some embodiments may be an individual, such as a human, to whom exogenous cells are implanted.

The term "individual" refers to any animal, such as a mammal or marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (eg, rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

The term "immune effector cells" refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, and macrophages. , mast cells, etc. In some embodiments, the immune effector cells are T cells, NK cells, or NKT cells. In some embodiments, the T cells can be autologous T cells, xenogeneic T cells, or allogeneic T cells. In some embodiments, the NK cells may be autologous NK cells or allogeneic NK cells.

The term "artificially modified cells with immune effector cell functions" refers to cells or cell lines that do not have immune effectors that have acquired immune effector cell functions after being artificially modified or stimulated by stimulants. For example, 293T cells have been artificially modified to function as immune effector cells; such as stem cells, which have been induced in vitro to differentiate into immune effector cells.

The "T cells" described herein 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, or can also be obtained through Cell populations with specific phenotypic characteristics obtained by sorting, etc., or mixed cell populations with different phenotypic characteristics, such as "T cells" can be cells containing at least one T cell subpopulation: memory stem cell-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). In some cases, a "T cell" may be a specific subtype of T cell, such as a gamma delta T cell. In some cases, T cells may be obtained from blood collected from an individual using any technique known to those skilled in the art, such as Ficoll™ isolation and/or apheresis. T cells can be any type of T cell and can be at 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 (such as cytotoxic T cells) , tumor infiltrating cells, memory T cells, naive T cells, etc. T cells may be CD8+ T cells or CD4+ T cells. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps. The T cells can be derived from healthy donors, or from individuals diagnosed with cancer.

The terms "activation" and "activation" are used interchangeably and can refer to the process by which cells transition from a quiescent to an active state. The process may include responses to phenotypic or genetic changes in antigen, migration and/or functional activity states. For example, the term "activation" can refer to the process of progressive activation of T cells. This activation process is jointly regulated by the first stimulatory signal and the co-stimulatory signal. The activation of T cells is a dynamic process, and its duration and degree of activation are affected by external stimuli. "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. Using CD3/CD28 magnetic beads, in vitro antigen stimulation or in vivo antigen stimulation will affect the degree and duration of T cell activation. In one embodiment, the engineered T cells are co-incubated with tumor cells containing specific target antigens or activated after viral infection.

The term "peripheral blood mononuclear cell (PBMC)" refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.

The term "pluripotent stem cells" have the potential to differentiate into any of the three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.) ) or ectoderm (such as epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cells" also encompasses "induced pluripotent stem cells" or "iPSCs," which are a type of pluripotent stem cells derived from non-pluripotent cells. In one embodiment, the pluripotent stem cells are derived from cells transformed by reprogramming somatic cells with characteristics of pluripotent stem cells. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by exogenously applying certain proteins.

The term "engineering" refers to the application of the principles and methods of cell biology and molecular biology to change the genetic material in cells or obtain cells according to people's wishes at the overall cell level or organelle level through some engineering means. A comprehensive science and technology of products. In one embodiment, engineering refers to one or more changes in a nucleic acid, such as within the genome of an organism. In one embodiment, engineering refers to changes, additions and/or deletions of genes. In one embodiment, the engineered cells or the engineered cells may also refer to cells with added, deleted and/or altered genes.

The terms "genetic modification", "gene modification", "genetic engineering" or "modified" refer to methods of modifying cells, including but not limited to gene editing methods in the coding or non-coding regions of genes or their expression regulatory regions. ; Or through endonuclease and/or antisense RNA technology; or by introducing exogenous proteins and/or complexes, small molecule inhibitors to change the protein expression level of the gene to cause gene defects. In some embodiments, 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 a subject or donor. Cells can be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), which can be integrated into the cell genome.

The term "gene silencing" refers to the phenomenon of non-expression or low expression of genes due to various reasons. Gene silencing can be gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and position effects; it can also be post-transcriptional gene silencing, that is, specific inhibition of target RNA at the level after gene transcription. Rather, it is gene inactivation, including antisense RNA, co-suppression, gene repression, RNA interference, and microRNA-mediated translation inhibition; it can also lead to undetectable or low protein expression by increasing protein degradation, including PROTAC, LYTAC , AbTAC, ATTEC, AUTAC and membrane protein intracellular retention technology, etc.

The "TCR silencing" refers to the absence or low expression of endogenous TCR.

The "MHC silencing" refers to the non-expression or low expression of endogenous MHC.

"Low expression" as used in this application means that the protein and/or RNA level of the target gene expressed in the engineered cells is lower than the expression level before the cell engineering treatment. In specific embodiments, low expression of B2M or TCR or TIGIT means that the expression of B2M or TCR or TIGIT in cells is reduced 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%. The expression or content of the protein in the cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies for B2M or TCR or TIGIT.

The term "MHC" stands for histocompatibility complex, which is the collective name for all gene groups encoding biocompatibility complex antigens. MHC antigens are expressed in the tissues of all higher vertebrates and are called HLA antigens in human cells. In transplantation reactions plays an important role in mediated rejection 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.

The term "Human leukocyte antigen" (HLA) is the gene encoding the human major histocompatibility complex, 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 parts. 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, and HLA-C sites) and MHC-II (encoded by the HLA-D region). HLA I Class II is distributed on the surface of almost all cells in the body. It is a heterodimer composed of heavy chain (α chain) and β2 microglobulin (B2M). Class II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes. .

The term "B2M" stands for beta-2 microglobulin, the light chain of an MHC class I molecule. In humans, B2M is encoded by the b2m gene located on chromosome 15, opposite other MHC genes located as a gene cluster on chromosome 6. Studies have shown that when the B2M gene is mutated, hematopoietic grafts from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice, indicating that the defective expression of MHC I molecules makes the cells vulnerable to host immune system Repulsion (Bix et al. 1991).

The term "T cell receptor (TCR)" 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, α and β. Each peptide chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region. Its antigen Specificity exists in the V region, and the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3. In one aspect, T cells expressing classic TCR can use methods such as antigen stimulation on T cells to Induces T cell TCR specificity for target antigen. TCR is divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, γ and δ, and TCR2 is composed of two chains, α and β. The term "TRAC" refers to the constant region of the TCRα chain.

The term "gene editing" refers to genetic engineering technology that uses site-specific nucleases to insert, knock out, modify or replace DNA at specific locations in the genome of an organism to change the DNA sequence. This technique is sometimes called "gene editing" 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 genome region that needs to be edited. Then non-specific endonuclease cuts the DNA double strands, causing the DNA break self-repair mechanism, thereby triggering mutations in the gene sequence and promoting homologous recombination. The endonuclease may be a Meganuclease, a zinc finger nuclease, a CRISPR/Cas9 nuclease, an MBBBD-nuclease or a TALEN-nuclease. In a preferred embodiment, the endonuclease is CRISPR/Cas9 nuclease, TALEN-nuclease. Gene knockout technologies using nucleases include CRISPR/Cas9 technology, ZFN technology, TALE technology and TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology.

The term "artificial zinc finger nucleases (ZFN)" technology is the first generation of nuclease site-directed modification technology, which uses zinc finger motifs (motifs) that can specifically recognize triplet DNA fragments instead of bases as recognition The basic unit of a specific DNA sequence. The most classic zinc finger nuclease fuses a non-specific endonuclease FokI with a zinc finger-containing domain, and its purpose is naturally to cleave specific sequences.

The term "transcription activator-like effector (TALE)" has DNA binding specificity, has a module that can specifically recognize bases, and is simple and convenient to operate. The TALE-DNA binding domain is composed of tandem repeating 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). TALE's RVD recognizes 4 bases of the DNA sequence with high specificity, and the 13th amino acid directly binds specifically to the bases of DNA. It can construct a specific TALEDN recognition binding domain at any position based on the DNA sequence, and can be widely used for gene sequence mutation modification and gene targeting. Set 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 DNA and generate DNA double-strand breaks (DSBs).

CRISPER/Cas9 is the third generation of gene editing technology. One embodiment of the present application uses CRISPR/Cas9 technology to prepare UCAR-T cells. The term "CRISPR (Clustered regularly interspaced short palindromicrepeats)" refers to clustered regularly interspaced short palindromic repeats. The term "Cas9 (CRISPR associated nuclease)" refers to CRISPR-associated nuclease, an RNA-guided technology that uses Cas9 nuclease to edit targeted genes. Cas9 enzyme can be wild-type Cas9 or artificially modified Cas9. The "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 (e.g., tracrRNA or active portion tracrRNA), tracr Pairing sequence (covering "direct repeats" and partial direct repeats for tracrRNA processing in the context of the endogenous CRISPR system), guide sequence (also called "spacer" in the context of the endogenous CRISPR system, i.e. gRNA ), or other sequences and transcripts from CRISPR loci. CRISPR systems are characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence (also called the protospacer in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, where hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. Complete complementarity is not required, provided that sufficient complementarity is present to cause hybridization and promote the formation of a CRISPR complex. After the CRISPR complex is formed, under the action of the cas9 enzyme, it can cut specific sites in the genome and introduce gene mutations; it can also regulate gene expression, such as activation or inhibition. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

Generally speaking, a guide sequence (gRNA) is any ribonucleotide sequence with sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. Whenever the gRNA sequence is involved in this application, it can be a targeted DNA sequence, or it can be a complete Cas9 guide sequence formed by the ribonucleotide corresponding to the DNA, crRNA, and TracrRNA. gRNA is used to guide, bind or recognize Cas enzyme. In some embodiments, the degree of complementarity between a 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. Any suitable algorithm for aligning sequences may be used to determine the optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Algorithms based on the Burrows-Wheeler Transform (e.g. Burrows Wheeler Aligner), ClustalW, Clustal (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The term "sgRNA" refers to short gRNA.

In some embodiments, the CRISPR enzyme is comprised of one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, in addition to the CRISPR enzyme). 9, 10 or more domains) as part of a fusion protein. CRISPR enzyme fusion proteins can contain any other protein, and optionally connecting sequences between any two domains. Examples of 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 Activating activity, transcription repressor activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza virus hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag .

When performing gene editing, the gRNA, tracr pairing sequence, and tracr sequence can be given individually or as a complete RNA sequence.

Cas9 protein combines with gRNA to cut DNA at specific sites. The CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23 bp and can target 20 bp. The last 3 NGG sequences of its recognition site are called PAM ( protospacer adjacent motif) sequence.

CRISPR/Cas transgenes can be delivered via vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.

This application simultaneously knocks out the genes TRAC and B2M to inactivate the functions of TCR and MHC molecules, and obtain universal T cells or universal CAR-T cells. In one embodiment, CRISPER/Cas technology is used to knock out the exons of the corresponding coding genes in the constant regions of one or both chains of B2M and TCR α and β chains respectively. In one embodiment, 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. In one embodiment, CRISPR/Cas9 technology is used to knock out the endogenous B2M gene, and the gRNA used is selected from the sequence shown in SEQ ID NO: 24, 72, 73 or 74.

In one embodiment, the cellular TIGIT gene is knocked out. For example, CRISPR/Cas9 technology is used to knock out the TIGIT gene, 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.

"Inhibiting" 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%. The expression or content of the protein in the cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies for B2M or TCR or TIGIT.

The term "RNA interference agent" is defined as any agent that interferes with or inhibits the expression of a target gene by RNA interference (RNAi). Such RNA interference agents include, but are not limited to, nucleic acid molecules that are homologous to RNA molecules of the target gene or its fragments, short interfering RNA (siRNA), shRNA or miRNA, and small RNA interference or inhibition of the expression of the target gene through RNA interference (RNAi). molecular.

In one embodiment of the present application, specific expression-specific CAR-T cells are first constructed, 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. In one embodiment, CRISPER/Cas9 technology is first used to knock out endogenous TRAC, B2M and/or TIGIT to construct universal T cells, and then express specific CAR to construct UCAR-T cells. In one embodiment, CRISPER/Cas9 technology knocks out endogenous TRAC, B2M and/or TIGIT and expresses specific CAR simultaneously to construct UCAR-T cells.

The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved 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.

The terms "nucleic acid molecule encoding", "encoding DNA sequence" and "encoding DNA" refer to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. The order of these deoxyribonucleotides determines the order of the amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

When used to refer to a nucleotide sequence, the term "sequence" as used herein may include DNA or RNA, and may be single-stranded or double-stranded.

As used herein, the term sequence "identity" determines percent identity by comparing two best-matched sequences over a comparison window (eg, at least 20 positions), where the portion of the polynucleotide or polypeptide sequence in the comparison window may include Additions or deletions (i.e., gaps), e.g., 20% or less gaps (e.g., 5 to 15%, or 10 to 12 %). The percentage is usually calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of positions that are correctly matched, dividing the number of positions that are correctly matched by the total number of positions in the reference sequence ( i.e. window size) and multiply the result by 100 to yield a percentage of sequence identity.

The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide that contains expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in vitro expression system. Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

The term "vector" as used herein is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.

The term "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 of overexpression.

The term "endogenous" refers to a nucleic acid molecule or polypeptide etc. that comes 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 (e.g., bispecific antibodies) and those that can specifically bind to an antigen or An antibody fragment of an antigenic determinant is sufficient as long as it displays the desired antigen-binding activity. The invention provides T cells expressing a first protein comprising an antibody that recognizes TIGIT. In specific embodiments, the invention also provides T cells expressing a first protein comprising a tandem antibody that recognizes TIGIT and a tumor antigen. In specific embodiments, the invention also provides T cells expressing a first protein comprising a tandem antibody that recognizes TIGIT and a pathogen antigen.

"Antibody fragment" refers to an antibody fragment that can specifically bind to an antigen or antigenic determinant. Non-limiting antibody fragments include Fab, F(ab')2, Fv, CDR, and ScFv.

The term "variable region or variable domain" refers to the 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 natural antibodies generally have similar structures, with each domain containing four conserved FRs and three CDRs. In specific embodiments, 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, in a tandem format including:

(i) Light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT—Heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes TIGIT region)—the heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes tumor and/or pathogen antigens—the light chain/heavy chain (or heavy chain) of an antibody that recognizes tumor and/or pathogen antigens Light chain variable region/heavy chain variable region);

(ii) The light chain (or light 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 TIGIT - The light chain (or light chain variable region) of an antibody that recognizes TIGIT chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens; and/or

(iii) Light chain (or light chain variable region) of an antibody that recognizes TIGIT - Heavy chain (or heavy chain variable region) of an antibody that recognizes tumors and/or pathogens - Light chain of an antibody that recognizes tumors and/or pathogens (or light chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT.

The term "hypervariable region" or "complementarity determining region" or "CDR" refers to sequences in an antibody variable domain that are hypervariable and/or form structurally defined loops ("hypervariable loops") and/or contain regions that contact the antigen. Regions of residues ("antigenic contacts"). Typically, antibodies contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplarily, the antibody or functional fragment thereof that recognizes TIGIT in this application contains a heavy chain variable region, and the heavy chain variable region contains 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 and the heavy chain CDR3 shown in SEQ ID NO:5. The antibody or functional fragment thereof that recognizes TIGIT in this application contains a light chain variable region. The light chain variable region contains 3 CDRs, the light chain CDR1 shown in SEQ ID NO: 6, and 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 in 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. Exemplarily, the antibody or functional fragment thereof that recognizes CLDN18A2 in this application contains a heavy chain variable region, and the heavy chain variable region contains 3 CDRs, the heavy chain CDR1 shown in SEQ ID NO: 13, and the heavy chain CDR1 shown in SEQ ID NO: 14 The heavy chain CDR2 and the heavy chain CDR3 shown in SEQ ID NO:15. The antibody or functional fragment thereof that recognizes CLDN18A2 in this application contains a light chain variable region. The light chain variable region contains 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 in 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.

The term "chimeric receptor" refers to a fusion molecule formed by connecting DNA fragments from different sources or cDNA corresponding to proteins using genetic recombination technology, including an extracellular domain, a transmembrane region and an intracellular domain. Chimeric receptors include, but are not limited to: chimeric antigen receptors (CAR), chimeric T cell receptors, and T cell antigen couplers (TAC).

The term "chimeric T cell receptor" consists of a TCR subunit combined with an antigen-binding domain (such as an antibody domain), where the TCR subunit includes at least part of the TCR extracellular domain, transmembrane region (or is the transmembrane domain) and the stimulation domain of the TCR intracellular signaling domain; the TCR subunit and the antibody domain are effectively connected. In specific embodiments, the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε, CD3γ, CD3z, the α chain of TCR, or the β chain of TCR, and the chimeric T cell is The body can form a complex with TCR/CD3 expressed on T cells.

The term "T cell antigen coupler (TAC)" includes three functional domains: (1) Antigen-binding domain, including single-chain antibodies, designed ankyrin repeat protein (DARPin) ) or other targeting groups; (2) extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor and TCR receptor close to each other; (3) the transmembrane domain and the intracellular region of the CD4 co-receptor, Among them, the intracellular domain-linked protein kinase LCK catalyzes the phosphorylation of the immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.

In some embodiments, the first protein chimeric receptor of the application is a chimeric antigen receptor (CAR).

The present application provides a T cell capable of resisting killing by autologous or allogeneic NK cells. Specifically, the present application provides CAR-T cells expressing CAR-T cells that recognize TIGIT. The application provides T cells that express a CAR that recognizes TIGIT and have endogenous TIGIT knockout. 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.

This application also provides a T cell that not only resists killing by autologous or allogeneic NK cells, but can also significantly kill tumor cells. Specifically, the present application provides T cells expressing a CAR that recognizes dual targets of TIGIT and tumor antigens. This application provides T cells that express a CAR containing dual targets that recognize TIGIT and pathogen antigens, and knock out endogenous TIGIT. This application provides a universal T cell that expresses a CAR that recognizes dual targets of TIGIT and pathogen antigens and knocks out endogenous TCR and B2M. The present application provides a universal T cell that expresses a CAR that recognizes dual targets of TIGIT and pathogen antigens and knocks out endogenous TIGIT, TCR and B2M.

The connection methods of the antibodies that recognize TIGIT and tumor antigens in the antigen-binding region of the above-mentioned CAR that recognizes dual targets include:

(i) Light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT—Heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes TIGIT region)—the heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes a tumor antigen—the light chain/heavy chain (or light chain variable region/heavy chain) of an antibody that recognizes a tumor antigen variable region);

(ii) The light chain (or light chain variable region) of an antibody that recognizes a tumor antigen—the heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT—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 a tumor antigen; and/or

(iii) 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 a tumor antigen - the light chain (or light chain variable region) of an antibody that recognizes a tumor antigen region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT.

This application also provides a T cell that not only resists killing by autologous or allogeneic NK cells, but can also significantly kill tumor cells. Specifically, the present application provides T cells expressing a CAR comprising 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 have knocked out endogenous TIGIT. The present application provides universal T cells that express a CAR that recognizes TIGIT and a CAR that recognizes tumor antigens, and have the endogenous TCR and B2M knocked out. The present application provides universal T cells that express a CAR that recognizes TIGIT and a CAR that recognizes tumor antigens, and have knocked out endogenous TIGIT, TCR, and B2M.

This application also provides a combination of the T cells provided in this application that can resist killing by autologous or allogeneic NK cells and T cells that express a second protein (such as CAR) that recognizes tumor antigens. The T cells provided in this application that can resist killing by autologous or allogeneic NK cells 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. In a specific embodiment, the endogenous TIGIT of the T cells expressing a second protein that recognizes the tumor antigen (eg, CAR) is knocked out. In a specific embodiment, the T cells expressing a second protein (such as CAR) that recognizes tumor antigens are universal T cells in which endogenous TCR and B2M have been knocked out. In specific embodiments, the T cells expressing a second protein (eg, CAR) that recognizes tumor antigens are universal T cells with endogenous TIGIT, TCR, and B2M knockout.

The term "chimeric antigen receptor" (CAR) includes extracellular domains, transmembrane regions, and intracellular signaling domains. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a costimulatory molecule (referred to as an intracellular signaling domain and a costimulatory signaling domain, respectively). In one aspect, the stimulatory molecule is associated with T cell receptor complex-bound delta chain; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and /or CD28. In certain embodiments, groups of polypeptides are linked to each other. Exemplarily, the CAR targeting TIGIT includes the sequence shown in SEQ ID NO: 9, and the CAR targeting CLDN18A2 includes the sequence shown in SEQ ID NO: 21, 75, 76 or 77. A CAR that simultaneously targets TIGIT and CLDN18A2 includes the antigen-binding domain shown in SEQ ID NO: 48, 50, or 52; or includes the CAR shown in SEQ ID NO: 54, 56, or 58. In specific embodiments, 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 sequence set forth in SEQ ID NO: 53, 55 or 57 sequence.

The term "primary signaling domain" regulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signaling domain is initiated by, for example, the 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.). The primary signaling domain that acts in a stimulatory manner may contain an immunoreceptor tyrosine activation motif or a signaling motif of an ITAM. Examples of ITAM-containing primary signaling domains that are particularly useful in this application include, but are not limited to, those derived from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ) and the sequence of CD66d. In a special example of the CAR of the present application, the intracellular signaling domain in any one or more CARs of the present application includes an intracellular signaling sequence, such as the primary signaling domain of CD3ξ.

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell, serves to regulate the cell via a defined signaling pathway, by producing a second messenger or acting as an effector by responding 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.

The term "costimulatory molecule" refers to a signal that binds to a cell-stimulating signaling molecule, such as TCR/CD3, and the combination results in T cell proliferation and/or up- or down-regulation of key molecules. It is a relevant binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, including but not limited to cell proliferation. Costimulatory molecules are non-antigen receptor cell surface molecules 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, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137 ).

The intracellular signaling domain (or structural region) can be selected from any costimulatory domain in Table 1. In some embodiments, a domain may be modified such that the identity to a reference domain may be from about 50% to about 100%. Any of the domains of Table 1 may be modified such that the modified form may comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or up to about 100% identity. Table 1. Costimulatory domains

In some cases, 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 a delta chain with CD28 or 4-1BB (second generation CAR), or it can further comprise two costimulatory domains, such as a delta chain with CD28/OX40 or CD28/4-1BB (third generation CAR) CAR). The signaling pathways used by these costimulatory molecules all work synergistically with primary T cell receptor activation signals. The signals provided by these costimulatory signaling domains can synergize with primary effector activation signals derived from one or more ITAM motifs (e.g., CD3zeta signaling domain) and can fulfill the requirements for T cell activation. Exemplarily, the signaling domain of a CAR targeting TIGIT, targeting CLDN18A2, and/or dual targeting TIGIT and CLDN18A2 includes CD3δ. In a specific embodiment, CD3δ is a human CD3δ molecule, comprising the sequence shown in SEQ ID NO:46. Exemplarily, the signaling domain of a CAR targeting TIGIT, targeting CLDN18A2, and/or dual targeting TIGIT and CLDN18A2 includes the CD28 intracellular domain. In specific embodiments, the CD28 intracellular domain includes the sequence shown in SEQ ID NO:44.

The term "CD3ξ (also known as CD3Zeta)" is defined as the protein provided by GenBan accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. "CD3 ξ domain" is defined as the amino acid residues from the cytoplasmic domain of the ξ chain that are sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ξ includes residues 52 to 164 of GenBan accession number BAG36664.1, its functional orthologs—from non-human species such as mice, rodents, monkeys, apes, etc. Valence residue. CD3ξ is also known as the T cell receptor T3δ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition by several intracellular signaling pathways with primary effector activation of T cells. As used herein, CD3 delta refers primarily to human CD3 delta and its isoforms, as known from Swissprot entry P20963, including proteins with essentially identical sequences. The full T cell receptor T3 delta chain is not required to be part of a chimeric antigen receptor, and any derivative thereof containing the signaling domain of the T cell receptor T3 delta chain is suitable, including any functional equivalents thereof. "CD3δ" is used interchangeably with "CD3z" and "CD3Z" in this application.

In the present application, in one aspect, a CAR includes an extracellular domain, a transmembrane region, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR includes an extracellular domain, a transmembrane region, and an intracellular signaling domain containing a functional signaling domain derived from a costimulatory molecule and a functional signaling derived from a stimulatory molecule conductive domain. In one aspect, the CAR includes an extracellular domain, a transmembrane region, and an intracellular signaling domain that includes at least two functional signaling domains derived from one or more costimulatory molecules and an intracellular signaling domain derived from a stimulatory molecule. Functional signaling domain of a molecule. In one aspect, the CAR contains an optional leader sequence within the amino acids of the CAR protein. In one aspect, the CAR also contains 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. In one embodiment, the leader sequence includes the sequence shown in SEQ ID NO:38.

The present application provides chimeric antigen receptors comprising an extracellular domain, a transmembrane region, and an intracellular domain as described herein. Commonly, the extracellular domain of a CAR is derived from mouse or humanized or human monoclonal antibodies.

Chimeric antigen receptors can be designed with a variety of antigen-binding regions, including single-chain variable fragments (scFv) derived from antibodies, fragmented antigen-binding regions (Fab) selected from libraries, single-domain fragments, or conjugated to their homologous receptors. body’s natural ligand. In some embodiments, the extracellular domain may comprise scFv, Fab or natural ligands, as well as any derivatives thereof. An extracellular domain may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and which may bind to the antigen to which the intact antibody binds. Examples of 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); and those formed from antibody fragments Multispecific antibodies. Extracellular domains, such as scFv, Fab or natural ligands, can be part of the CAR that determines antigen specificity. The extracellular domain can bind any complementary target. The extracellular domain can be derived from antibodies of known variable region sequences. The extracellular domain can be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, the extracellular domain can be obtained from whole ecto-cleavage sequencing of tumor cells or primary cells such as tumor-infiltrating lymphocytes (TILs).

In some embodiments, 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 light chain CDRs and heavy chain CDRs. The present application provides CAR-T cells expressing CAR-T cells that recognize TIGIT. In specific embodiments, the present application also provides CAR-T cells expressing dual targets that recognize TIGIT and tumor antigens. In specific embodiments, the present application also provides CAR-T cells expressing dual targets that recognize TIGIT and pathogen antigens.

In some embodiments, the extracellular domain of the CAR includes an antigen-binding region and a linker segment (also called a hinge, spacer, or linker). The linker fragment can be considered as the part of the CAR that provides flexibility to the extracellular domain. In some cases, different linking fragments can lead to whether the CAR on the cell surface can be activated by the target antigen, or the degree of activation is very low, or the T cells will undergo significant exhaustion and loss of function after activation. This may be due to the presence of antibodies in the extracellular domain. Or the spatial structure formed by the antibody fragment has a different degree of complementarity with the spatial structure of the target antigen. For example, whether the length of linker fragments derived from immunoglobulins needs to be optimized depends on where the extracellular domain targets the target epitope.

The composition and length of the linker segments of CAR polypeptides are adjustable. The spatial conformation of the immunological synaptic binding between T cells and target cells limits the distance at which the membrane-distal epitope on the target molecule cannot functionally bridge with the CAR. Even the use of short connecting fragments of CAR cannot make the synaptic distance reach the signal. An approximation that can be conducted. Likewise, signal output from membrane-proximal CAR target epitopes was only observed in the context of long linker fragment CARs. The linker fragment can be adjusted depending on the extracellular domain used. Connection segments can be of any length. Exemplarily, in one embodiment of the present application, the CAR includes a hinge domain, which is a CD8α hinge. Preferably, the CD8α hinge domain includes the amino acid of SEQ ID NO:40.

The transmembrane (TM) domain (or structural region) of CAR can anchor the CAR to the plasma membrane of the cell. The native transmembrane portion of CD28 can be used in CARs. In other cases, the native transmembrane portion of CD8α can also be used in CARs. "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" may be a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane domain may be the native transmembrane portion of CD28. "CD28" may refer to the same entity as NCBI Reference Number: NP_006130 or which has stimulatory activity. A fragment having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to a protein. A "CD28 nucleic acid molecule" may be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion may comprise a CD8 alpha region. Exemplarily, in one embodiment of the present application, TM is the CD28 transmembrane region. In a preferred embodiment, it is the human CD28 transmembrane region. Preferably, the CD28 transmembrane region comprises the amino acids of SEQ ID NO:42.

With regard to pharmaceutical compositions, the pharmaceutically acceptable carrier may be one of those conventionally used and is limited only by chemical and physical considerations, such as solubility and non-reactivity with the active agent, and the 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. Preferably, the pharmaceutically acceptable carrier is one that is harmless and has no toxic side effects under the conditions of use. The pharmaceutical compositions of the present application are available in a variety of suitable dosage forms. Methods of preparing administrable (eg, parenteral) 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. Preferably, the CAR materials of the present application are administered by injection (eg, subcutaneous, intravenous, intratumoral, intraarterial, intramuscular, intradermal, interperitoneal or intrathecal). Preferably, the CAR materials of the present application are administered intravenously. Suitable pharmaceutically acceptable carriers for the 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% w/v NaCl in water, or about 9.0% w/v NaCl in water). g NaCl), room temperature or electrolyte solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum protein.

"Effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat a disease (cancer) in an individual. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the disease 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 time and frequency of administration, the presence, nature and extent of adverse side effects that may accompany the administration of the particular active substance and the desired physiological effect. Depending on the judgment of the prescribing physician or those skilled in the art, one or more rounds, or multiple administrations of the CAR material of this 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× ¹⁰ cells/dose).

Embodiments of the present application also include removing mammalian lymphocytes before administering the CAR material of the present application, including but not limited to non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

The term "treatment" refers to interventions that attempt to modify the course of a disease, either preventatively or in the clinical pathological process. 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 down the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc. In specific embodiments, the engineered T cells provided by the present application can inhibit tumor cell proliferation, and/or inhibit tumor cell proliferation and increase tumor volume in vivo.

The term "prevention" refers to interventions that attempt to prevent disease (such as rejection of cell transplantation) before it occurs.

The present application provides cells, nucleic acids, expression vectors, host cells, or compositions of the present application for use in treating or preventing tumors.

The engineered T cells provided by the present application can be used to treat, prevent or improve autoimmune diseases or inflammatory diseases, especially inflammatory diseases related to autoimmune diseases, such as arthritis (eg, rheumatoid arthritis, chronic progressive arthritis (arthritis chronicaprogrediente and degenerative arthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including ankylosing spondylitis), Reiter syndrome symptoms, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, enthesitis, hypersensitivity (including airway hypersensitivity and skin hypersensitivity) and allergies. The engineered T cells provided by this application are used for the treatment and prevention of autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red blood 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 , Stevens-Johnson syndrome, spontaneous sprue, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), endocrine ophthalmopathy, Graves' disease, tuberculosis Arthritis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes mellitus (type I diabetes), uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, periprosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome, including, for example, idiopathic nephrotic syndrome or minimal change nephropathy), multiple myeloma, other types of tumors, skin and Inflammatory diseases of the cornea, myositis, loosening of bone implants, metabolic disorders (such as obesity, atherosclerosis and other cardiovascular diseases including dilated cardiomyopathy, myocarditis, type II diabetes and dyslipidemia) and autoimmunity Thyroid diseases (including Hashimoto's thyroiditis), primary vasculitis of small and medium vessels, large vessel vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjogren's syndrome, Behcet's disease diseases, atopic and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathies, immune renal, liver and thyroid diseases, inflammation and atherosclerosis, autoinflammatory febrile syndromes, immunohematology disorders and bullous diseases of the skin and mucous membranes.

The engineered T cells provided in this application can be used to treat, prevent or improve 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 immunosuppressants or immunomodulators or other anti-inflammatory agents or other cytotoxic agents or anti-cancer agents (for example, as adjuvants or combinations thereof ) administration, for example, to treat or prevent diseases associated with immune disorders. For example, the antibodies of the present application can be used in combination with DMARDs such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, others Crolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors, such as cyclosporine A or FK 506; regulators 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, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxysperguanidin or its immunosuppressive homologues , analogs or derivatives; immunosuppressive monoclonal antibodies, for example, directed against leukocyte receptors, for example, MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. Monoclonal antibodies to CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds.

The tumor described herein may 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 cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia Cell carcinoma, 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 (such as non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin lymphoma, B-chronic lymphoma B-cell leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell lymphoma, acute lymphoblastic leukemia (ALL), Burkitt lymphoma , ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer. Preferably, the tumor is characterized by BCMA expression, and more preferably is multiple myeloma characterized by BCMA expression.

"Tumor antigens" refer to antigens that are newly emerged or overexpressed during the occurrence and development of hyperproliferative diseases. In certain aspects, the hyperproliferative disorder of the present application refers to cancer.

The tumor antigens described in this application may be solid tumor antigens or hematological tumor antigens.

Tumor antigens in 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 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor α (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; casein Amidase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4 , EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe ); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor vascular endothelial marker 1 (TEM1/CD248); tumor vasculature Endothelial marker 7 related (TEM7R); Claudin 6, CLDN18A2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa light chain); CSPG4 ;EGP2, EGP40; FAP; FAR; FBP; Embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Fibronectin ; Tenascin; carcinoembryonic variant of tumor necrotic zone; G protein-coupled receptor class C group 5 - member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose portion of globoHglycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2) ; TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin Binds cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant ; Human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N- Acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytoma disease viral oncogene neuroblastoma-derived homolog source (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells ( SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase-anchored protein 4 (AKAP-4); synovial sarcoma X Breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2) ; CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); Contains EGF-like module mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is CLDN18A2, GPC3, BCMA or CD19.

The pathogen antigen is selected from: viral, bacterial, fungal, protozoan, or parasite antigens; the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.

This article provides an autologous or allogeneic cell, such as a T cell, that is genetically engineered to express TIGIT-CAR for resisting NK cell killing, thereby providing the first immune cell to increase the number of autologous or allogeneic cells in a host. 2. Methods of persistence and/or transplant survival in the presence of immune cells. For clarity, the "host" is the recipient of the "first immune cell", such as a subject, a patient, etc.; after the "first immune cell" is engineered and transplanted into the host, the "first immune cell" is removed from the host's body. Any immune cells other than "first immune cells" are called "second immune cells". The "first immune cell" and the "second immune cell" may be cells from the same individual, or may be allogeneic cells. In some embodiments, 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 endogenous TIGIT is knocked out using gene editing technology. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, and endogenous B2M and TCR are knocked out using gene editing technology. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, and endogenous TIGIT, B2M, and TCR are knocked out using gene editing technology.

In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, said cell is also genetically engineered to express at least one non-TIGIT-targeting chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or combination thereof). In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, said cell is also genetically engineered to express at least one non-TIGIT-targeting chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or its combination), and use gene editing technology to knock out endogenous TIGIT. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, said cell is also genetically engineered to express at least one non-TIGIT-targeting chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or a combination thereof), and use gene editing technology to knock out endogenous B2M and TCR. In some embodiments, the first immune cell is genetically engineered to express TIGIT-CAR, said cell is also genetically engineered to express at least one non-TIGIT-targeting chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR or combination thereof), and use gene editing technology to knock out endogenous TIGIT, B2M and TCR.

In some embodiments, the first immune cell is genetically engineered to express a dual-target CAR that recognizes TIGIT and a tumor antigen. In some embodiments, the first immune cell is genetically engineered to express a dual-target CAR that recognizes TIGIT and a tumor antigen, and endogenous IGIT is knocked out using gene editing technology. In some embodiments, the first immune cell is genetically engineered to express a dual-target CAR that recognizes TIGIT and a tumor antigen, and endogenous B2M and TCR are knocked out using gene editing technology. In some embodiments, the first immune cell is genetically engineered to express a dual-target CAR that recognizes TIGIT and a tumor antigen, and endogenous TIGIT, B2M, and TCR are knocked out using gene editing technology.

This application includes, for example, Chinese patent application publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106 397593A, CN106467573A, CN104140974A, CN 108884459A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN10950 3715A , CN 109908176A, CN109880803A, CN 110055275A, CN110123837A, CN 110438082A, CN 110468105A International patent application publication numbers WO2017186121A1, WO2018006882A1, WO2015172339A 8. WO2018/018958A1, WO2014180306A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186 121A1, WO2018045811A1, WO2018108106A1 , WO 2018/219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/141270, WO2019/149279, WO2019/170147A1, WO 2019/210863 , those CAR-T cells disclosed in WO2019/219029 and preparation method thereof.

When constructing CAR-T cells, 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 prevent host NK cells from attacking T cells with double knockout of TRAC and B2M genes and prolong the survival time of universal T cells in the host body.

The present application will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, 2002, or according to the manufacturer. Suggested conditions.

Example

Example 1. Cytokine activation leads to an increase in the proportion of TIGIT+NK cells

Ficoll-Paque (GE bioscience) was used to perform density gradient centrifugation to isolate PBMC from donor peripheral blood, and NK cells were isolated using an NK cell isolation kit (purchased from Miltenyi). Using flow cytometry using APC-TIGIT (Invitrogen) antibody, 36% of NK cells expressed TIGIT on their surface (see Figure 1A).

Table 2

In vitro, IL-2 (250U/ml), IL-15 (150U/ml), and IL-12 (10ng/ml) were added to the above isolated NK cell culture medium separately or jointly according to Table 2 and incubated for 24 hours. APC-TIGIT (Invitrogen) antibody detects the percentage of TIGIT+NK cells.

The flow cytometry results are shown in Figure 1. The proportion of TIGIT+NK cells under the action of single cytokine IL-12 and IL-15 were 35.5% (see Figure 1B) and 37.5% (see Figure 1C) respectively; the single cytokine IL -2 or a combination of two or three cytokines can significantly increase the proportion of TIGIT+NK cells. The corresponding proportions of TIGIT+NK cells are shown in Figures 1D-1H, which are 69.7% (IL-2) and 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) .

Ficoll-Paque (GE bioscience) was used to perform density gradient centrifugation to isolate PBMC from the peripheral blood of five donors, and NK cells were isolated using an NK cell isolation kit (purchased from Miltenyi). IL-2 (500U/ml) and IL-15 (150U/ml) were jointly added to the NK cell culture medium and cultured. After 10 days of culture, a flow cytometer was used to detect the percentage of TIGIT+NK cells using APC-TIGIT (Invitrogen) antibody. .

The flow cytometry results are shown in Figure 2. The percentages of TIGIT+NK cells in the NK cells of the five donors after cytokine activation were 86.1%, 76.2%, 74.1%, 38.3% and 55% respectively. The proportion of TIGIT+NK cells exceeded 70% in 3 out of 5 cases.

The above results suggest that TIGIT protein is expressed on resting NK cells and activated NK cells.

Example 2. Preparation of CAR-T cells

1. TIGIT-CAR-T cell preparation

Engineered T cells containing TIGIT-targeting CAR were constructed to observe their resistance to NK cells.

The expression vector was constructed using conventional molecular biology methods in this field (Figure 3). Design and construct a single-chain antibody containing CD8α signal peptide (SEQ ID NO: 38), anti-TIGIT (the amino acid sequence of VH is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 2), CD8α 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 activating factor CD3δ (amino acid sequence shown in SEQ ID NO: 46) The vector of chimeric antigen receptor TIGIT-CAR, and the lentivirus formed by its packaging was named PRRL-TIGIT.

Ficoll-Paque (GE bioscience) was used to perform density gradient centrifugation to isolate mononuclear cells (PBMC) from human peripheral blood. Anti-CD3/CD28 magnetic beads were added for activation in vitro to obtain T cells. Infect T cells with the above lentivirus PRRL-TIGIT and culture and expand to the required number to obtain TIGIT CAR-T cells.

2. Preparation of TIGIT-targeting TRAC and B2M double-negative CAR-T cells

(1) Preparation of gene knockout cells

After 48 hours of in vitro expansion of TIGIT CAR-T cells, adjust the cell density to 2*10^7/mL. TRAC and B2M double gene knockout was performed on 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) and sgRNA were incubated at room temperature for 10 minutes at a ratio of 1:4 to obtain an RNP complex, in which the nucleic acid sequence of TRAC-gRNA (gRNA for targeted knockout of TRAC) is as SEQ ID NO: As shown in 23, the nucleic acid sequence of B2M-gRNA (gRNA for targeted knockout of B2M) is shown in SEQ ID NO: 24. Mix 1*10^6 TIGIT CAR-T cells with the RNP complex (the final concentration of Cas 9 enzyme is 2 μM), and use a maxcyte electroporation instrument to introduce the RNP complex containing TRAC-gRNA and B2M-gRNA into the TIGIT CAR -in T cells. On the 4th day after electroporation, flow cytometry was used to detect gene knockout.

According to the above method, TRAC single gene knockout, TRAC and B2M double gene knockout were performed on UTD cells (T cells not transfected with virus).

(2) Screening and identification of TRAC negative cells or TRAC/B2M double negative cells

Expand UTD cells with single TRAC knockout, TIGIT CAR-T cells with double knockout of B2M and TRAC, and UTD cells with double knockout of B2M and TRAC in vitro. Adjust the cell density 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). The labeled cells were sorted by anti-PE magnetic beads on a sorting column, and TRAC-negative cells were collected. 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 and TIGIT CAR-T cells with double deletion of TRAC and B2M (TIGIT-UCAR) were obtained. -T cells), TRAC and B2M double-deleted UTD cells (U-UTD cells).

Biotin-labeled goat anti-human Fab antibody (Jackson) was used to label CAR-T cells, and then PE-Streptavidin secondary antibody was used for labeling. Flow cytometry was used to detect the CAR expression of TIGIT-UCAR-T cells. 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 universal anti-human Fab antibodies. To sort 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 on a sorting column, and TIGIT-UCAR-T cells were collected. The positive rate of TIGIT-UCAR-T cells after screening reached more than 95%.

Example 3. Detection of resistance of TIGIT-UCAR-T cells to NK cells in vitro

Use Ficoll-Paque (GE bioscience) to perform density gradient centrifugation to isolate PBMC from the donor's peripheral blood. Use an NK cell isolation kit (purchased from Miltenyi) to isolate NK cells from the PBMC cells. Adjust the cell density 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. Inoculate NK cells and T cells in a 24-well plate at a ratio of 1:1 or 2:1, and incubate in the incubator for 0hr, 24hr and 48hr respectively. The NK cell + TRAC-/-UTD group used APC-CD56 (Invitrogen) antibody to label NK cells, and other mixed culture groups used APC-HLA-ABC antibody (Invitrogen) to label NK cells. T cells and NK were detected at different time points of co-incubation. Changes in the proportion and number of cells.

The experimental results are shown in Figure 5. When co-cultured with NK cells, the proportion of U-UTD cells gradually decreased over time, while the proportion of TIGIT-UCAR-T cells gradually increased. When the NK:T ratio was 2:1 and 1:1, compared with U-UTD cells, TIGIT-UCAR-T cells had a higher survival ratio and number.

The above results show that CAR-T cells targeting TIGIT can effectively resist killing by NK cells.

Example 4 Preparation of dual-target UCAR-T cells targeting TIGIT and tumor antigens

In order to observe whether CAR-T cells targeting TIGIT can enhance their anti-tumor activity in the presence of NK cells, we took the tumor antigen CLDN18A2 as an example and constructed CAR-T cells targeting both TIGIT and CLDN18A2. Observation Its impact on anti-tumor activity (targeting tumor antigens, such as CLDN18A2) and on NK cell clearance (targeting non-tumor antigens, such as TIGIT).

According to the different tandem connection methods of dual-targeting antigens and hinge domain connection methods (see Table 3), we constructed three different forms of TIGIT and CLDN18A2 dual-target CAR vectors.

table 3

Exemplarily, 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 As 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, 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 As shown in SEQ ID NO: 5, the LCDR1 sequence is shown in SEQ ID NO: 6, the LCDR2 sequence is shown in SEQ ID NO: 7, the LCDR3 sequence is shown in SEQ ID NO: 8), G4S (SEQ ID NO: 60 ), (G4S)3 (SEQ ID NO: 62), linker1 (SEQ ID NO: 64).

Using conventional molecular biology methods in this field, tandem fragment 1 (SEQ ID NO: 48), tandem fragment 2 (SEQ ID NO: 50), and tandem fragment 3 (SEQ ID NO: 52) (see Table 3) were separated from CD8α 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), and T cell activating factor CD3δ The CAR1 fragment (SEQ ID NO: 54), CAR2 fragment (SEQ ID NO: 56), and CAR3 fragment (SEQ ID NO: 58) constructed from (SEQ ID NO: 46) were then inserted into the vector PRRLsin respectively to construct the vector PRRLsin. -CAR1, PRRLsin-CAR2, PRRLsin-CAR3 (Figure 6), and are packaged to form lentivirus PRRL-CAR1, PRRL-CAR2, and PRRL-CAR3 respectively.

The lentiviral plasmid PRRLsin-CLDN18A2-CAR expressing the chimeric antigen receptor of CLDN18A2 was constructed (Figure 6), containing the single-chain antibody of CLDN18A2 (SEQ ID NO: 11, 12), CD8α 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 activating factor CD3δ (SEQ ID NO: 46); packaging forms slow Viral PRRL-CLDN18A2-CAR.

CLDN18A2-CAR-T targeting CLDN18A2 was prepared according 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. Cells with double knockout of TRAC and B2M were prepared according to the method described in Example 2: U-UTD as a negative control (T cells with only knockout of TRAC and B2M), CLDN18A2-UCAR-T that only targets CLDN18A2, dual-targeting UCAR-T1 (expressing CAR1), UCAR-T2 (expressing CAR2), and UCAR-T3 (expressing CAR3) cells of TIGIT and CLDN18A2.

Take 1×10 ⁶ of each UCAR-T cell mentioned above and add PE-labeled anti-anti-anti-CLDN18A2scFv antibody for staining. Flow cytometry was used to detect the positive rate of each UCAR-T cell (Figure 7). The average fluorescence intensities of CLDN18A2-CAR, CAR1, CAR2, and CAR3 on T cells were 21520, 13355, 15973, and 5338, respectively.

Example 5 Detection of killing of target cells by dual-target UCAR-T cells targeting TIGIT and tumor antigens

Conventional molecular biology techniques were used to transfect human CLDN18A2 (SEQ ID NO: 19, 20) with lentivirus into pancreatic cancer cells BXPC-3 (ATCC, USA) and gastric cancer cells HGC-27 (Chinese Academy of Sciences), which do not express endogenous CLDN18A2. Cell bank), select positive single clones by limiting dilution method, and construct BXPC-3-A2 and HGC-27-A2 stably transduced cell lines expressing CLDN18A2.

Inoculate 75 μL of 2×10 ⁵ /mL BXPC-3-A2 and HGC-27-A2 cells (target cells) in a 96-well plate respectively; add effector cells according to the effect-to-target ratio of 3:1, 1:1 or 1:3. U-UTD, CLDN18A2-CAR-T, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells, and set up effector cell spontaneous LDH control wells, target cell spontaneous LDH control wells, and target cell maximum LDH control Wells, volume correction control wells and culture medium background control, each with 4 duplicate wells; 18 hours later, Cytotoxicity Detection Kit (LDH, Roche) was used to detect LDH release, and the killing of target cells by cells in each group was calculated (Figure 8).

The results show that there is no significant difference in the killing of target cells by three different forms of TIGIT&CLDN18A2 dual-target UCAR-T cells (UCAR-T1, UCAR-T2, and UCAR-T3) compared with CLDN18A2-UCAR-T. The dual-target CAR -T cells will not reduce the killing of target cells.

Example 6 Resistance of dual-target UCAR-T cells targeting TIGIT and tumor antigens on 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 killing by 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 TIGIT expression were detected (Figure 9). Some U-UTD and CLDN18A2-UCAR-T cells express TIGIT protein on their surface, while UCAR-T1, UCAR-T2, and UCAR-T3 cells cannot detect TIGIT protein. 1×10 ⁶ U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, and UCAR-T3 cells were co-cultured with 5×10 ⁶ PBMC cells respectively, and PE-anti-HLA-ABC antibody (labeled U -UTD and UCAR-T cells) and APC-anti CD56 antibody (marking NK cells in PBMC cells) to detect U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, The proportion of UCAR-T3 cells and the proportion of NK cells (Figure 10).

The results showed that the proportion of three forms of TIGIT and CLDN18A2 dual-target UCAR-T cells after co-culture with PBMC cells for 48 hours was higher than that of U-UTD and CLDN18A2-UCAR-T; and after co-culture, UCAR The proportion of NK cells in the -T1, UCAR-T2, and UCAR-T3 groups was lower than that in the U-UTD and CLDN18A2-UCAR-T groups. The results show that TIGIT and CLDN18A2 dual-target UCAR-T cells can effectively resist NK cell killing in PBMC cells, and UCAR-T2 has the best resistance to NK cells.

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. We further tested the anti-tumor effect of dual-target UCAR-T cells in vivo.

HGC-27-A2 transplanted tumors (recorded as D0) were inoculated subepithelially in the NPG of immunodeficient mice. Each mouse was inoculated with 5×10 ⁶ HGC-27-A2 cells. On the 15th day, the tumor volume was measured and grouped into groups for transplantation. The tumor volume was approximately 90mm3 and divided into 5 groups (U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, and UCAR-T3), with 5 animals in each group; 1×10 ⁶ U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells. After injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumors were measured and recorded with vernier calipers, the tumor volume was calculated, the tumor growth curve was drawn based on the tumor volume, and the tumor growth curves were compared between the groups. Difference (tumor volume: V=1/2×long diameter×short diameter ² ). (Figure 11A).

The results showed that the therapeutic effects of UCAR-T2 and UCAR-T1 treatment groups on transplanted tumors were significantly different from those of U-UTD.

Fourteen days after UCAR-T cell treatment, the peripheral blood of the mice was taken and the density of human CD4+ and CD8+ T cells in the peripheral blood of the mice was detected.

The results are shown in Figure 11B. The density of human CD4+ and CD8+ T cells in the CLDN18A2-UCAR-T cell treatment group was higher than that in the TIGIT&CLDN18A2 dual-target UCAR-T cell treatment group. This result may be due to the fact that some UCAR-T cells themselves also express the TIGIT protein, causing the dual-target UCAR-T cells to kill themselves, thereby reducing the number of human CD4+ and CD8+ T cells to a certain extent, so the anti-tumor The effect has decreased

Example 8 TIGIT gRNA screening

Since some T cells also express TIGIT, in order to prevent CAR-T cells from killing their own CAR-T cells, the TIGIT gene of CAR-T cells was further deleted on the basis of TRAC/B2M double knockout. For the TIGIT gene, we designed 12 pairs of gRNA1-12 targeting the TIGIT gene (SEQ ID NO: 25-36). The sgRNA sequence targeting TIGIT was synthesized in vitro according to the reagent instructions (GeneArt ^TM Precision gRNA Synthesis Kit, Thermo Tisher).

CD3-CD28 antibody-coated magnetic beads were used to activate T cells, and T cells on the 5th day of activation were electroporated using a Maxcyte electroporation instrument. The electroporation system is 0.5μMCas9+2μM TIGIT sgRNA. On the 4th day after electroporation, APC-TIGIT antibody was used to detect the knockdown efficiency of each gRNA. The specific experimental operations are described in Example 2.

The results are shown in Figure 12: gRNA6 and gRNA10 had the highest knockout efficiencies, with knockout efficiencies of 76% and 72% respectively. And gRNA11, 5, 7, and 8 can also knock out TIGIT.

In order to avoid the killing of own cells by TIGIT&CLDN18A2 dual-target UCAR-T, it is necessary to knock out the TIGIT gene of UCAR-T cells on the basis of TRAC/B2M knockout. On day 0, CD3-CD8 antibody-coated magnetic beads were used to activate T cells. 48 hours later, they were infected with lentivirus PRRL-CLDN18A2-CAR, PRRL-CAR1, PRRL-CAR2, and PRRL-CAR3. After 24 hours, the cells were centrifuged and the medium was replaced. Continue to culture until the 6th day, use Maxcyte electroporation instrument to double knockout TRAC&B2M or triple knockout TRAC&B2M&TIGIT gene. The electroporation system is 1μM cas9 (Kaika Biotech) + 2μM TRAC sgRNA + 2μM B2M sgRNA, 2.5μM Cas9 (Kaika Biotech). +2μM TRAC sgRNA+2μM B2M sgRNA+6μM TIGIT sgRNA; the nucleic acid sequence of TIGIT sgRNA is shown in SEQ ID NO: 30, the nucleic acid sequence of TRAC-gRNA is shown in SEQ ID NO: 23, and the nucleic acid sequence of B2M-gRNA is shown as SEQ ID NO:24 is shown. Thus, CLDN18A2-UCAR-T cells with TRAC and B2M double gene knockout, and TRAC&B2M&TIGIT gene 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. Specific experimental operations are described in Example 2.

PE-labeled anti-anti-anti-CLDN18A2scFv antibody was used to stain the above CAR-T cells, and flow cytometry was used to detect the positive rate of each CAR-T cell. The positive rate of CAR-T cells is shown in Figure 13. Among them, the TIGIT gene knockout efficiencies in 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 Effect of TIGIT gene knockout on the killing activity of CAR-T cells in vitro

Knocking out the endogenous TIGIT gene may affect the killing of target cells by CAR-T cells. In order to evaluate the effect of knocking out the TIGIT gene on the in vitro killing activity of CAR-T, we compared the in vitro killing activity of CLDN18A2-CAR-T and CLDN18A2-CAR-T-TIGIT KO cells on target cells.

CD3-CD28 antibody-coated magnetic beads were used to activate T cells, and 48 hours later, the activated T cells were infected with PRRL-CLDN18A2-CAR lentivirus. MOI=10. Centrifuge and change the medium 24 hours after infection. Continue to culture until day 6, and use Maxcyte electroporation instrument to electroporate CLDN18A2-CAR-T cells. The electroporation system is 1.5 μM Cas9 + 6 μM TIGIT sgRNA, in which the TIGIT sgRNA nucleic acid sequence is shown in SEQ ID NO: 30, and CLDN18A2-CAR-T is obtained. -TIGIT KO cells. 72 hours after electroporation, 1×10 ⁶ cells were taken, and PE-anti-anti-CLDN18A2scFv antibody and APC-TIGIT antibody were used to detect the positive rate of CAR-T cells and the proportion of TIGIT-positive cells respectively (Figure 14A). The specific experimental operations are described in Example 2.

The results showed that the positive rates of CLDN18A2-CAR-T and CLDN18A2-CAR-T-TIGIT KO cells were 80.6% and 82.1%, respectively. The TIGIT knockout efficiency of CLDN18A2-CAR-T-TIGIT KO cells was 83%.

Inoculate 75 μL of 2×10 ⁵ /mL BXPC-3-A2 and HGC-27-A2 cells (target cells) in a 96-well plate respectively; add effects according to the effect-to-target ratio of 3:1, 1:1 or 1:3. Cells UTD, CLDN18A2-CAR-T, CLDN18A2-CAR-T-TIGIT KO cells, and set effector cell spontaneous LDH control wells, target cell spontaneous LDH control wells, target cell maximum LDH control wells, volume correction control wells, and culture medium background Control, 4 duplicate wells each; 18 hours later, Cytotoxicity Detection Kit (LDH, Roche) was used to detect LDH release, and the killing of target cells by cells in each group was calculated (Figure 14B).

The results showed that knocking out the endogenous TIGIT gene did not affect the killing of target cells by CAR-T cells.

Example 10 Therapeutic effect of dual-target UCAR-T-TIGIT KO cells targeting TIGIT and tumor antigens on transplanted tumors

In order to explore whether endogenous TIGIT expression affects the tumor treatment effect of dual-target UCAR-T in Example 7, we deleted the TIGIT gene in UCAR-T cells by referring to Example 9 to observe the dual-target effect in the case of TIGIT knockout. The therapeutic effect of UCAR-T on transplanted tumors.

On day 0, NPG mice were subcutaneously inoculated with HGC-27-A2 transplanted tumors, and each mouse was inoculated with 5×10 ⁶ HGC-27-A2 cells. On the 20th day, the tumor volume was measured and divided into groups. The transplanted tumor volume was about 200mm3 and 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 animals 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. After injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumors were measured and recorded with vernier calipers, the tumor volume was calculated, the tumor growth curve was drawn based on the tumor volume, and the tumor growth curves were compared between the groups. Difference (tumor volume: V=1/2×long diameter×short diameter ² ). (Figure 15).

The results showed that compared with the control group, UCAR-T3-TIGIT KO with endogenous TIGIT knockout had a significantly improved therapeutic effect on transplanted tumors.

Knocking out the TIGIT gene prevents UCAR-T cells from killing their own UCAR-T cells, significantly improving their anti-tumor effect. Moreover, the therapeutic effect of UCAR-T3-TIGIT KO on HGC-27-A2 transplanted tumors compared to the control group was significantly better than the therapeutic effect in Example 7 compared to the control group.

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

In order to explore the in vivo anti-tumor effect of TIGIT and tumor antigen dual-target U-CAR T cells in the presence of NK cells, we compared CLDN18A2-UCAR T-TIGIT KO+NK, UCAR-T1-TIGIT KO+NK, and UCAR- Therapeutic effects of T2-TIGIT KO+NK and UCAR-T3-TIGIT KO on HGC-27-A2 transplanted tumors.

On day 0, NPG mice were subcutaneously inoculated with HGC-27-A2, and each mouse was inoculated with 5 × 10 ⁶ cells. On the 13th day, the volume of the HGC-27-A2 transplanted tumor was measured to be approximately 100 mm ³ and divided into 6 groups, of which 5 × 10 ⁶ PBMC cells were administered to groups 1, 3, 4, 5, and 6. On day 14, group 1 was injected with 5×10 ⁵ U-UTD-TIGIT KO cells into the tail vein, group 2 was injected with 5×10 ⁵ CLDN18A2-UCAR T-TIGIT KO cells into the tail vein, and group 3 was injected with 5×10 ⁵ into the tail vein. CLDN18A2-UCAR T-TIGIT KO, group 4 was injected with 5×10 ⁵ U-CAR-T1-TIGIT KO cells through the tail vein, group 5 was given 5×10 ⁵ UCAR-T2-TIGIT KO cells, and group 6 was given tail vein injection 5×10 ⁵ UCAR-T3-TIGIT KO cells. After injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumors were measured and recorded with vernier calipers, the tumor volume was calculated, the tumor growth curve was drawn based on the tumor volume, and the tumor growth curves were compared between the groups. Difference (tumor volume: V=1/2×long diameter×short diameter ² ).

The results showed that in the presence of NK cells, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO and UCAR-T3-TIGIT KO had better therapeutic effects on HGC-27-A2 transplanted tumors than CLDN18A2-UCAR T-TIGIT KO.

The above experimental data show that in the presence of NK cells, the anti-tumor effect of dual-target CAR-T cells targeting TIGIT and tumor antigens is better than that of CAR-T cells targeting tumor antigens alone.

Example 12 Resistance effect of TIGIT-targeted CAR-T cells on NK cells

1. Preparation of TIGIT-BBZ-CAR T and TIGIT-28Z-CAR T cells. Refer to Example 2 to construct the CAR vectors of TIGIT-BBZ and TIGIT-28Z. The fragments shown in Table 4 were inserted respectively to construct TIGIT-targeting CAR-T cells.

Table 4 Chimeric Antigen Receptor

Among them, CD8α signal peptide (SEQ ID NO: 38), anti-TIGIT single chain antibody (the amino acid sequence of VH is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 2), CD8α hinge region ( The amino acid sequence is shown in SEQ ID NO: 40), the CD8 transmembrane region (the amino acid sequence is shown in SEQ ID NO: 86) or the CD28 transmembrane region (the amino acid sequence is shown in SEQ ID NO: 42), the CD28 intracellular domain (Amino acid sequence is shown in SEQ ID NO: 44) or CD137 intracellular domain (amino acid sequence is shown in SEQ ID NO: 88), T cell activating factor CD3δ (amino acid sequence is shown in SEQ ID NO: 46)

Insert the nucleic acid fragments of TIGIT-BBZ and TIGIT-28Z in Table 4 into the lentiviral vector to construct lentiviral plasmids PRRL-TIGIT-BBZ and PRRL-TIGIT-28Z (see Figure 16), and then transfer them into transfections respectively. 293T cells are packaged to obtain lentivirus TIGIT-BBZ and TIGIT-28Z, which are infected with T cells respectively to obtain TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells targeting TIGIT.

2. Preparation of TIGIT-targeting TRAC and B2M double-negative CAR-T cells and detection of their resistance to NK cells

Refer to Example 2 to knock out TRAC and B2M in TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells respectively to prepare TRAC and B2M double-negative TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells. TRAC single gene knockout was performed on UTD cells (T cells not transfected with virus) to prepare TRAC-/-UTD as a positive control. UTD cells with double deletion of TRAC and B2M (U-UTD cells) were used as negative control.

Adjust the cell concentration to 1*10^6/ml. Inoculate NK cells and T cells in a 24-well plate at a ratio of 1:1, and incubate them in an incubator for 0hr, 24hr and 48hr respectively. The NK cell + TRAC-/-UTD group used APC-CD56 (Invitrogen) antibody to label NK cells, and other mixed culture groups used APC-HLA-ABC antibody (Invitrogen) to label NK cells. T cells and NK were detected at different time points of co-incubation. Changes in the proportion of cells.

The results are shown in Figure 17. When co-cultured with NK cells, the proportion of TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells gradually increased with the prolongation of co-culture time, while the proportion of NK cells gradually decreased. This shows that CAR-T cells targeting TIGIT can effectively resist killing by NK cells.

3. Preparation of TIGIT-BBZ-UCAR T-TIGIT KO cells and TIGIT-28Z-UCAR T-TIGIT KO cells with TIGIT gene knockout

Refer to Example 9 to knock out the TIGIT gene in TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells respectively, and prepare TIGIT-BBZ-UCAR T-TIGIT KO cells and TIGIT-28Z-UCAR T-TIGIT KO. cell.

Example 13 CAR-T cells targeting TIGIT enhance the resistance of CAR-T cells targeting tumor antigens to NK cells and their anti-tumor activity

Taking CAR T cells targeting the tumor antigen CLDN18A2 as an example, we tested in vitro whether CAR-T cells targeting TIGIT can enhance the survival and anti-tumor effects of CAR-T cells targeting tumor antigens in the presence of NK cells. active.

Adjust the density of CLDN18A2-UCAR T-TIGIT KO, TIGIT-BBZ-UCAR-TIGIT KO, TIGIT-28Z-UCAR-TIGIT KO cells, NK cells and HGC-27-A2 cells to 1×10 ⁶ /ml. Co-culture according to the following ratio, add 500 μl for each cell. Co-culture times were 0hr, 24hr and 48hr.

HGC-27-A2+CLDN18A2-UCAR T-TIGIT KO+U-UTD-TIGIT KO+NK

HGC-27-A2+CLDN18A2-UCART-TIGIT KO+TIGIT-BBZ-UCAR-TIGITKO+NK

HGC-27-A2+CLDN18A2-UCAR T-TIGIT KO+TIGIT-28Z-UCAR-TIGIT KO+NK

The changes in the proportion and number of HGC-27-A2 tumor cells and CLDN18A2-UCAR T-TIGIT KO cells in the above co-culture system were detected. The results showed that compared with the control group (1), the proportion and number of CLDN18A2-UCAR T-TIGIT KO cells in the co-culture systems (2) and (3) were higher, while the proportion and number of HGC-27-A2 cells were significantly higher. decline.

The above experimental results show that CAR-T cells targeting TIGIT can significantly improve the survival of CAR-T cells targeting tumor antigens in the presence of NK cells, thereby improving their anti-tumor activity.

Example 14 TIGIT-targeting CAR-T cells enhance the resistance of CAR-T cells targeting tumor antigens to NK cells and their in vivo anti-tumor activity

In order to confirm that combined targeting of TIGIT-UCAR T-TIGIT KO can enhance the anti-tumor effect of universal CAR T cells in the presence of NK cells, taking CAR T targeting CLDN18A2 as an example, CLDN18A2-UCAR T-TIGIT KO and CLDN18A2 were compared -UCAR T-TIGIT KO+NK, CLDN18A2-UCAR T-TIGIT KO+TIGIT-BBZ-UCAR-TIGIT KO+NK, CLDN18A2-UCAR T-TIGIT KO+TIGIT-28Z-UCAR-TIGIT KO+NK versus HGC-27 -Therapeutic effect of A2 transplanted tumors.

On day 0, NPG mice were subcutaneously inoculated with HGC-27-A2, and each mouse was inoculated with 5 × 10 ⁶ cells. On the 13th day, the volume of the HGC-27-A2 transplanted tumor was measured to be approximately 100 mm ³ and divided into 5 groups. Groups 1, 3, 4, and 5 were each given 5 × 10 ⁶ PBMC cells. On day 14, 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, and 5×10 ⁵ cells were injected into the tail vein of group 3. CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ U-UTD-TIGIT KO cells, group 4 was given 5×10 ⁵ CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ TIGIT-BBZ-UCAR-TIGIT-KO cells , Group 5 was given 5×10 ⁵ CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ TIGIT-28Z-UCAR-TIGIT-KO cells through the tail vein. After injection, the body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumors were measured and recorded with vernier calipers, the tumor volume was calculated, the tumor growth curve was drawn based on the tumor volume, and the tumor growth curves were compared between the groups. Difference (tumor volume: V=1/2×long diameter×short diameter ² ).

The results showed that the therapeutic effect of groups 4 and 5 on HGC-27-A2 transplanted tumors was significantly better than that of group 3, indicating that CAR-T cells targeting TIGIT can enhance CAR-T targeting tumor antigens in the presence of NK cells. Antitumor functions of cells in vivo.

All documents mentioned in this application are herein incorporated by reference to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

sequence:

Claims (40)

A genetically engineered cell, characterized in that the cell expresses a first protein capable of recognizing TIGIT. The cell of claim 1, wherein the first protein contains an antibody capable of recognizing TIGIT, preferably the sequence of TIGIT is shown in SEQ ID No: 10. The cell of claim 1 or 2, wherein the first protein includes a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof. The cell according to any one of claims 1 to 3, wherein the first protein is a CAR, and the CAR includes: (i) Antibodies that recognize TIGIT, CD28 or the transmembrane region of CD8, CD3δ; (ii) Antibodies that recognize TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; (iii) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD137, and CD3δ; and/or (iv) An antibody that recognizes TIGIT, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ. The cell according to any one of claims 1 to 4, characterized in that the cell contains: knockout of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules. The cell according to claim 5, characterized in that CRISPR/Cas9 technology is used to knock out the TIGIT gene of the cell, and the gRNA used is selected from SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, Any one of the sequences shown in 32, 33, 34, 35 and 36 or a combination thereof. The cell according to any one of claims 1 to 6, wherein the cell is 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 cell according to any one of claims 1 to 7, characterized in that the cell is an autologous or allogeneic T cell, a primary T cell or an autologous T cell derived from a human. The cell according to any one of claims 1 to 8, characterized in that the cell includes, Knockout of genes encoding TCR proteins and/or low or no expression of endogenous TCR molecules, and/or Knockout of genes encoding MHC proteins and/or low or no expression of endogenous MHC. The cell according to claim 9, characterized in that CRISPR/Cas9 technology is used to knock out endogenous MHC molecule B2M and endogenous TCR. The cell of claim 10, wherein the gRNA used to knock out B2M includes the sequences shown in SEQ ID NO: 24, 72, 73 and/or 74, and the gRNA used to knock out TCR includes SEQ ID NO: 23 , 65, 66, 67, 68, 69, 70 and/or the sequences shown in 71. The cell according to any one of claims 2-11, wherein the antibody that recognizes TIGIT includes: (i) HCDR1 represented by SEQ ID NO: 3, HCDR2 represented by SEQ ID NO: 4, HCDR3 represented by SEQ ID NO: 5, LCDR1 represented by SEQ ID NO: 6, and represented by SEQ ID NO: 7 LCDR2, and/or LCDR3 shown in SEQ ID NO: 8; or (ii) The heavy chain variable region shown in SEQ ID NO: 1 and/or the light chain variable region shown in SEQ ID NO: 2; or (iii) scFv shown in SEQ ID NO:78. The cell according to any one of claims 1 to 12, wherein the first protein also recognizes tumors and/or pathogens; preferably, the tumor expression includes BCMA, CD19, GPC3, CLDN18A2, EGFR or a combination thereof . The cell of claim 13, wherein the first protein includes an antibody that recognizes TIGIT and an antibody that recognizes tumor and/or pathogen antigens, and their connection includes: (i) Light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT—Heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes TIGIT region)—the heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes tumor and/or pathogen antigens—the light chain/heavy chain (or heavy chain) of an antibody that recognizes tumor and/or pathogen antigens Light chain variable region/heavy chain variable region); (ii) The light chain (or light 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 TIGIT - The light chain (or light chain variable region) of an antibody that recognizes TIGIT chain variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigens; and/or (iii) 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, Wherein, the first protein is a CAR, and the CAR includes: (i) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, CD3δ; (ii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; (iii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137, and CD3δ; and/or (iv) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ. The cell of claim 13 or 14, wherein the antibody that recognizes a tumor antigen recognizes CLDN18A2 and includes: (i) 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, and SEQ ID NO: 17 LCDR2, and/or LCDR3 described in SEQ ID NO: 18; or (ii) The heavy chain variable region described in SEQ ID NO: 11 and/or the light chain variable region described in SEQ ID NO: 12; or (iii) scFv shown in SEQ ID NO: 82. The cell of any one of claims 13-15, wherein the first protein includes the sequence shown in SEQ ID NO: 48, 50, 52, 90 or 91. The cell according to any one of claims 1 to 16, characterized in that the cell also expresses a second protein, a chemokine, a chemokine receptor, and a cytokine that target recognition of tumor antigens and/or pathogen antigens. , siRNA that reduces PD-1 expression, proteins that block the binding of PD-L1 to PD-1, safety switches, or combinations thereof. The cell according to any one of claims 1 to 17, wherein when the cell is co-cultured with host NK cells, the cell can kill the host NK cell, or the cell can resist the host NK cell's attack on the host NK cell. Killing of the cells, or the cells are resistant to killing of the cells by activated host NK cells. The cell according to any one of claims 1 to 18, characterized in that the cell is administered in combination with an agent that enhances its function, preferably in combination with a chemotherapy drug; 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 than the first protein. The cell of claim 19, wherein the second protein includes a CAR, and the CAR includes: (i) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, CD3δ; (ii) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, and CD3δ; (iii) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137, and CD3δ; and/or (iv) Antibodies that recognize tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ. The cell of claim 19 or 20, wherein the cells expressing the second protein include: (i) Knockout of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules; or (ii) Knockout of genes encoding TCR and/or MHC proteins and/or low or no expression of endogenous TCR and/or MHC molecules; or (iii) Knockout of genes encoding TIGIT, TCR and MHC proteins and/or low or no expression of endogenous TIGIT, TCR and MHC molecules. The cell of claim 21, wherein the cell expressing the second protein: (i) Use CRISPR/Cas9 technology to knock out TIGIT molecules; (ii) Use CRISPR/Cas9 technology to knock out TCR and/or MHC molecule B2M; or (iii) Use CRISPR/Cas9 technology to knock out TIGIT, TCR and MHC molecule B2M. The cell according to any one of claims 19 to 22, wherein 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, and macrophages. , CIK cells, and stem cell-derived immune effector cells, or combinations thereof. The cell of claim 23, wherein the cell is an autologous or allogeneic T cell, a primary T cell or an autologous T cell derived from a human. Methods to increase persistence and/or transplant survival of primary immune cells in the presence of host secondary immune cells include: a) Provide first immune cells; b) optionally modifying said first immune cell by reducing or inhibiting expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in response to self- and non-self antigen recognition; c) modifying the first immune cell with a polynucleotide encoding a first protein targeting TIGIT. The method of claim 25, wherein the polypeptide in step b) is selected from the group consisting of MHC, TCR, and/or TIGIT. The method of claim 26, wherein step b) includes: (i) Use CRISPR/Cas9 technology to knock out TIGIT molecules; (ii) Use CRISPR/Cas9 technology to knock out TCR and/or MHC molecule B2M; or (iii) Use CRISPR/Cas9 technology to knock out TIGIT, TCR and MHC molecule B2M. The method of claim 27, wherein step b) includes: (i) The gRNA used to knock 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; (ii) The gRNA used to knock out TCR includes the sequences shown in SEQ ID NO: 23, 65, 66, 67, 68, 69, 70 and/or 71; and/or (iii) The gRNA used to knock out the MHC molecule B2M includes the sequence shown in SEQ ID NO: 24, 72, 73 and/or 74. The method of any one of claims 25-28, wherein the first protein includes a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof . The method of any one of claims 25-29, wherein the first protein includes: (i) an antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28 and CD3δ; and/or (ii) an antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD137 and CD3δ; and/or (iii) an antibody that recognizes TIGIT, optionally an antibody that recognizes tumors and/or pathogens, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ; (iv) An antibody that recognizes TIGIT, optionally an antibody that recognizes a tumor and/or pathogen, CD28 or the transmembrane region of CD8, and CD3δ. The method of claim 25, wherein the first protein includes: (i) scFv of TIGIT shown in SEQ ID NO: 78; (ii) scFv of CLDN18A2 shown in SEQ ID NO: 82; (iii) The tandem sequence of the TIGIT antibody and CLDN18A2 antibody shown in SEQ ID NO: 48, 50 or 52; or (iv) The first protein shown in SEQ ID NO: 9, 54, 56, 58, 90 or 91. The method according to any one of claims 25 to 31, further comprising step d) encoding a second protein, a chemokine, or a chemokine receptor that targets tumor antigens and/or pathogen antigens and/or viral antigens. The first immune cell is modified with non-endogenous polynucleotides such as antibodies, cytokines, siRNA that reduces PD-1 expression, proteins that block the binding of PD-L1 to PD-1, or safety switches. The method of any one of claims 25-32, wherein 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 cells. Immune effector cells or combinations thereof. The method according to any one of claims 25 to 33, wherein the first immune cell is an autologous or allogeneic T cell, a primary T cell or an autologous T cell derived from a human. An engineered cell prepared by the method as described in any one of claims 25-34. A polynucleotide encoding a nucleic acid molecule for constructing a cell according to any one of claims 1-24 and 35 or encoding a nucleic acid molecule required for administering any one of the methods described in claims 25-34. A vector comprising the polynucleotide of claim 36. A virus comprising the vector of claim 37. A composition comprising an effective amount of the cell according to any one of claims 1-24 and 35, the polynucleotide according to claim 36, the vector according to claim 37, and the vector according to claim 38. Virus. A method of treating inflammatory conditions, viral infections and/or tumors, comprising administering to a subject in need thereof a cell according to any one of claims 1-24 or 35 or a combination according to claim 39 things.