CN116897202A

CN116897202A - TIGIT engineered cells and compositions thereof

Info

Publication number: CN116897202A
Application number: CN202280017000.5A
Authority: CN
Inventors: 李宗海; 石志敏
Original assignee: Keji Biomedical Shanghai Co ltd
Current assignee: Keji Biomedical Shanghai Co ltd
Priority date: 2021-02-24
Filing date: 2022-02-24
Publication date: 2023-10-17
Also published as: WO2022179567A1

Abstract

For the development of engineered immune cells for immunotherapy that have a greater persistence in the host organism and/or a high survival rate of transplantation, and methods for their preparation. A genetically engineered cell expressing a first protein that recognizes TIGIT, and a method for increasing persistence and/or transplant survival of a first immune cell in the presence of a host second immune cell.

Description

TIGIT engineered cells and compositions thereof

Related patent application

This patent application claims priority from the chinese patent application with application number 202110205868.5 filed 24 at 2 months 2021.

Technical Field

The application relates to a cell with a graft rejection resisting function, and also relates to a method for resisting graft immune rejection, in particular to a method for resisting NK cell immune rejection.

Background

Due to the immunological differences between the donor and the recipient, the donor may also be recognized and challenged by immune cells in the recipient as an exogenous donor transplant, thereby inhibiting or eliminating the exogenous transplant and producing a Host Versus Graft Reaction (HVGR). By knocking out MHC molecules in the graft cells, rejection of the host T cells to the graft can be effectively resisted, but rejection of other immune cells in the host can be caused. As in allogeneic cell transplantation, depletion of MHC class I molecules from allogeneic cells results in rejection of NK cells in the host, enhancing 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 of great importance for developing allogeneic cell transplantation therapy.

Detailed Description

The application aims to provide a cell for resisting transplant immune rejection and a method for resisting rejection.

In a first aspect of the application, there is provided a genetically engineered cell, wherein the cell expresses a first protein capable of recognizing TIGIT.

In a specific embodiment, the first protein comprises 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 comprises 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 comprising:

(i) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, cd3δ;

(ii) An antibody that recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, and cd3δ;

(iii) An antibody that recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and cd3δ; and/or

(iv) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and cd3δ.

In a specific embodiment, the cell comprises: knock-out of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules.

In a specific embodiment, the cellular TIGIT gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from any one of the sequences set forth in SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or a combination thereof.

In a specific embodiment, the cell is selected from the group consisting of a T cell, NK cell, cytotoxic T cell, NKT cell, DNT cell, NK92 cell, macrophage, CIK cell, and stem cell derived immune effector cell, or a combination 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 cell comprises a knockout of a gene encoding a TCR protein and/or an endogenous TCR molecule is expressed or not expressed, and/or a knockout of a gene encoding an MHC protein and/or an endogenous MHC is expressed or not expressed.

In a specific embodiment, the endogenous MHC molecule B2M and the endogenous TCR are knocked out using CRISPR/Cas9 technology.

In a specific embodiment, the gRNA used to knock out B2M comprises the sequences shown in SEQ ID NOS.24, 72, 73 and/or 74 and the gRNA used to knock out TCR comprises the sequences shown in SEQ ID NOS.23, 65, 66, 67, 68, 69, 70 and/or 71.

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

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

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

(iii) SEQ ID NO: 78.

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

In a specific embodiment, the first protein comprises an antibody that recognizes TIGIT and an antibody that recognizes a tumor and/or pathogen antigen, linked in a manner comprising:

(i) Light/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT-heavy/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes tumor and/or pathogen antigen-light/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigen;

(ii) Light chain (or light chain variable region) of an antibody that recognizes tumor and/or pathogen antigen-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT-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 tumor and/or pathogen antigen; 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 tumor and/or pathogen antigens-light chain (or light chain variable region) of an antibody that recognizes tumor and/or pathogen antigens-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT,

wherein the first protein is a CAR, the CAR comprising:

(i) Antibodies recognizing TIGIT and antibodies recognizing tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, cd3δ;

(ii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ;

(iii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iv) Antibodies recognizing TIGIT and antibodies recognizing tumor and/or pathogen antigens, the transmembrane region of CD28 or 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, comprising:

(i) SEQ ID NO:13, HCDR1, SEQ ID NO:14, HCDR2, SEQ ID NO:15, HCDR3, SEQ ID NO:16, LCDR1, SEQ ID NO:17, and/or the LCDR2 set forth in SEQ ID NO:18 LCDR3; or (b)

(ii) SEQ ID NO:11 and/or the heavy chain variable region set forth in SEQ ID NO:12, a light chain variable region as described in seq id no; or (b)

(iii) SEQ ID NO: 82.

In a specific embodiment, the first protein comprises SEQ ID NO: 48. 50, 52, 90 or 91.

In a specific embodiment, the cell further expresses a second protein, chemokine receptor, cytokine, siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, a safety switch, or a combination thereof that targets recognition of a tumor antigen, and/or a pathogen antigen.

In a specific embodiment, the cell is capable of killing a host NK cell, or the cell is capable of resisting killing of the cell by an activated host NK cell, when the cell is co-cultured with the host NK cell.

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 comprises a CAR comprising:

(i) Antibodies that recognize tumors and/or pathogens, transmembrane region of CD28 or CD8, cd3δ;

(ii) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ;

(iii) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iv) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, the co-stimulatory signaling domain of CD137, and cd3δ.

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

(i) Knock-out of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules; or (b)

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

(iii) Knockout of the 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) Knocking out TIGIT molecules by CRISPR/Cas9 technology;

(ii) Knocking out TCR and/or MHC molecule B2M by CRISPR/Cas9 technology; or (b)

(iii) TIGIT, TCR and MHC molecules B2M were knocked out using CRISPR/Cas9 technology.

In a specific embodiment, the cell expressing the second protein 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 a combination thereof.

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

a) Providing a first immune cell;

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

c) A polynucleotide encoding a first protein that targets TIGIT to modify the first immune cell.

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

In a specific embodiment, step b) comprises:

(i) Knocking out TIGIT molecules by using CRISPR/Cas9 technology;

(ii) Knocking out TCR and/or MHC molecules B2M using CRISPR/Cas9 technology; or (b)

In a specific embodiment, step b) comprises:

(i) The gRNA used to knock out the TIGIT molecule is selected from the group consisting of the sequences shown in SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and/or 36;

(ii) gRNAs used to knock out TCRs include the sequences shown in SEQ ID NOs 23, 65, 66, 67, 68, 69, 70 and/or 71; and/or

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

In a specific embodiment, the first protein comprises:

(i) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ; and/or

(ii) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iii) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and cd3δ;

(iv) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, and cd3δ.

In a specific embodiment, the first protein comprises:

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

(ii) SEQ ID NO:82, CLDN18 A2;

(iii) SEQ ID NO: 48. 50 or 52 and CLDN18A2 antibody tandem sequences; or (b)

(iv) SEQ ID NO: 9. 54, 56, 58, 90 or 91.

In a specific embodiment, step d) encoding a second protein targeting a tumor antigen and/or pathogen antigen and/or viral antigen, a chemokine receptor, a cytokine, an siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, or a non-endogenous polynucleotide such as a safety switch is further included to modify the first immune cell.

In a specific embodiment, the first immune cell is selected from the group consisting of a T cell, NK cell, cytotoxic T cell, NKT cell, macrophage, CIK cell, and stem cell derived immune effector cell, or a combination 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 a third aspect of the present application there is provided an engineered cell prepared by the method of the present application described above.

According to a fourth aspect of the present application there is provided a polynucleotide which encodes a nucleic acid molecule which constructs a cell as described in the present application above or which encodes a nucleic acid molecule required for the administration of a method of the present application as described above.

According to a fifth aspect of the present application there is provided a vector comprising a polynucleotide according to the present application.

According to a sixth aspect of the application there is provided a virus comprising a vector according to the application.

According to a seventh aspect of the application there is provided a composition comprising an effective amount of a cell according to the application, a polynucleotide according to the application, a vector according to the application, a virus according to the application.

According to an eighth aspect of the present application there is provided a method of treating an inflammatory disorder, viral infection and/or tumour comprising administering to a subject in need thereof a cell as described herein or a composition as described herein.

The application also relates to:

1. a genetically engineered cell, wherein the cell expresses a first protein that recognizes TIGIT;

preferably, the TIGIT amino acid sequence is as set forth in SEQ ID NO:10 is shown in the figure;

preferably, the first protein comprises an antibody or functional fragment thereof capable of recognizing TIGIT;

preferably, the first protein comprises SEQ ID NO:1 and/or the heavy chain variable region set forth in SEQ ID NO:2, and a light chain variable region as described in seq id no;

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

preferably, the antibody recognizing TIGIT or a functional fragment thereof and the antibody recognizing a tumor antigen or a pathogen antigen are linked by a linker peptide.

2. The cell of claim 1, wherein the cell is an immune effector cell or an engineered cell having immune effector cell function.

3. The cell of claim 1 or 2, wherein 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 cell is a human primary T cell;

more preferably, the cells are allogeneic T cells.

4. The cell of any one of claims 1-3, wherein the first protein is further linked to a cell activation signal,

preferably, the first protein comprises a TCR intracellular domain from the stimulatory domain of the intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha or TCR beta.

5. The cell of any one of claims 1-4, wherein MHC and/or endogenous TCR expression, activity and/or signaling in the cell is reduced; preferably, the MHC is an MHC class I molecule; more preferably, the MHC class I molecule is HLA; more preferably, the HLA is HLA-I; more preferably, the HLA-I is selected from one or more of HLA-A, HLA-B, HLA-C, B M; most preferably, the HLA-I comprises HLA-A and/or B2M; preferably, the endogenous TCR comprises one or both of the α and β chains of the TCR;

Preferably, the reducing or inhibiting is by using TAL nucleases, meganucleases, zinc finger nucleases, cas9 and Argonaute;

preferably, the engineered T 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 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 comprises SEQ ID NO:23 and/or SEQ ID NO:24, a sequence shown in seq id no;

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

preferably, the decrease in MHC and/or endogenous TCR expression, activity and/or signaling in the engineered cell is greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to the expression, activity and/or signaling of MHC and/or endogenous TCR in an un-engineered T cell;

preferably, the expression of MHC and/or endogenous TCR expressed in the engineered cell is not detected using immunoblot assay and/or in a flow-through assay.

6. The cell of any one of claims 1-5, wherein the first protein is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), a T cell fusion protein (TFP), or a combination thereof;

preferably, the first protein comprises an extracellular domain, a transmembrane region and an intracellular signaling domain.

7. The cell of any one of claims 1-6, wherein the first protein comprises:

(i) An antibody or functional fragment thereof that specifically recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, and cd3δ; and/or

(ii) An antibody or functional fragment thereof that specifically recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and cd3δ; and/or

(iii) An antibody or functional fragment thereof that specifically recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and cd3δ;

(iv) An antibody or functional fragment thereof that specifically recognizes TIGIT, a transmembrane region of CD28 or CD8, and cd3δ;

preferably, the antibody specifically recognizing TIGIT comprises SEQ ID NO:1 and/or the heavy chain variable region set forth in SEQ ID NO:2, and a light chain variable region as described in seq id no;

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

8. The cell of any one of claims 1-7, wherein the cell further expresses a second protein that targets a tumor antigen, and/or a pathogen antigen, and/or a viral antigen, a chemokine receptor, a cytokine, an siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, a safety switch, or a combination thereof;

preferably, the second protein comprises: chimeric Antigen Receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), aTCR-T, or a combination thereof;

preferably, the second protein is capable of specifically recognizing claudin18.2, GPC3, BCMA or CD19.

9. The cell of any one of claims 1-8, wherein the cell further expresses a ligand or antibody fragment of an NK cell inhibitory receptor;

preferably, the cell also expresses an NKG2A binding molecule;

preferably, the NKG2A binding molecule is a cell membrane binding protein, a secreted protein;

preferably, the NKG2A binding molecule comprises an extracellular domain, a transmembrane region; or comprises an extracellular domain, a transmembrane region and an intracellular region;

preferably, the NKG2A binding molecule is an NKG2A antibody or antibody fragment that binds to the cell membrane.

10. The cell of any one of claims 1-9, wherein the cell has reduced or inhibited endogenous TIGIT expression, activity, and/or signaling;

preferably, the immune cell comprises an inhibitory nucleic acid molecule or gRNA that targets a 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 cell is reduced by greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to the expression, activity, and/or signaling of TIGIT in an un-engineered cell;

preferably, the expression of TIGIT expressed in the cells is not detectable using immunoblot assays and/or in flow assays.

11. The cell of any one of claims 1-10, wherein the cell kills a host NK cell when the cell is co-cultured with the host NK cell.

12. The cell of any one of claims 1-11, wherein the cell is resistant to killing of the cell by a host NK cell when the cell is co-cultured with the host NK cell;

preferably, the cell is resistant to killing of the cell by a cytokine-activated NK cell in the host NK cell;

preferably, the cell is resistant to killing of the cell by a host NK cell expressing NKG 2A;

preferably, the cells are significantly resistant to killing of the cells by host NK cells that underexpress NKG 2A.

13. The cell of any one of claims 1-12, wherein the cell is administered in combination with an agent that enhances its 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.

14. A method of increasing persistence and/or transplant survival of allogeneic immune cells in the presence of host immune cells, comprising:

a) Providing allogeneic cells;

b) Modifying the cell by reduced or inhibited expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in a response to self and non-self antigen recognition;

c) A polynucleotide encoding a first protein that targets TIGIT to modify the cell.

15. The method according to claim 14, wherein the polypeptide in step b) is selected from MHC and/or endogenous TCR, the MHC being an MHC class I molecule; more preferably, the MHC class I molecule is HLA; more preferably, the HLA is HLA-I; more preferably, the HLA-I is selected from one or more of HLA-A, HLA-B, HLA-C, B M; preferably, the endogenous TCR comprises one or both of the α and β chains of the TCR; more preferably, the HLA-I comprises HLA-A and/or B2M;

preferably, step B) modifies the cell by reducing or inhibiting B2M and TCR expression, activity and/or signaling;

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 comprises SEQ ID NO:23 and/or 24;

preferably, the decrease in MHC and/or endogenous TCR expression, activity and/or signaling in the engineered cell is greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95% or 100% as compared to the expression, activity and/or signaling of MHC and/or endogenous TCR in the non-genetically engineered cell;

preferably, the expression of MHC expressed in the cell is not detected using immunoblot assays and/or in flow assays.

16. The method of claim 14 or 15, wherein the first protein is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), a T cell fusion protein (TFP), or a combination thereof;

preferably, the first protein comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain;

preferably, the cell mediates inhibition or killing of immune effector cells of the host by intracellular signaling domain;

preferably, the first protein comprises:

17. The method of any one of claims 14-16, further comprising step d) modifying the cell with a polynucleotide encoding a second protein that targets a tumor antigen, and/or a pathogen antigen, and/or a viral antigen, a chemokine receptor, a cytokine, an siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, or a safety switch, or the like;

18. The method of any one of claims 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, further comprising the step e) modifying said cell with a non-endogenous polynucleotide encoding said immune cell NKG2A binding molecule;

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

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

preferably, the cell comprises an inhibitory nucleic acid molecule or gRNA that targets a 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 expression, activity, and/or signaling of TIGIT in the engineered immune cell is reduced by greater than or greater than about 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to the expression, activity, and/or signaling of TIGIT in an un-engineered cell;

preferably, the expression of TIGIT expressed in the immune cells is not detectable using an immunoblot assay and/or in a flow-through assay.

20. The method of any one of claims 14-19, 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 cell is a human primary T cell;

more preferably, the cells are allogeneic T cells.

21. The method of any one of claims 14-20, wherein the cells produced by the method are capable of killing a host NK cell when the cells are co-cultured with the host NK cell.

22. The method of any one of claims 14-21, wherein the cells produced by the method are resistant to killing of the cells by host NK cells when the cells are co-cultured with the cells;

23. The method of any one of claims 14-22, wherein the cells prepared by the method are administered in combination with an agent that enhances their function, preferably in combination with a chemotherapeutic agent;

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

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

25. A bispecific antibody construct comprising a first binding domain that binds to human or cynomolgus 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 claim 25, wherein the second binding domain binds to human CD3 epsilon and CD3 epsilon in common marmoset, cotton top marmoset, or cynomolgus monkey;

preferably, the antibody construct is selected from the following forms: (scFv) 2, scFv-single domain mabs, bifunctional antibodies and oligomers of these forms.

27. A polynucleotide encoding the antibody construct of claim 25 or 26 or encoding the construct of the cell of any one of claims 1-13 or encoding the construct required for administration of the method of any one of claims 14-23.

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

29. A virus that infects the vector of claim 28.

30. A composition comprising an effective amount of an engineered cell of any one of claims 1-13 or 24,

preferably, further comprising a pharmaceutically acceptable carrier;

preferably, the carrier is an aqueous saline solution, dextrose solution, or 5% human serum albumin.

Preferably, a cryoprotectant is also included.

31. A kit comprising an engineered cell of any one of claims 1-13 or 24 or a composition of claim 30 and an additional agent for treating a disease.

32. A method of treating a disease comprising administering to a subject in need thereof an engineered cell of any one of claims 1-13 or 24 or a composition of claim 30 or a kit of claim 31,

preferably, the engineered cell is produced by a method according to any one of claims 14-23 prior to administration of the engineered cell;

preferably, it further comprises administering an additional agent;

preferably, the disease is selected from inflammatory disorders, infections and tumors;

preferably, the subject is a human;

preferably, wherein the engineered cells are autologous or allogeneic T cells to the subject.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the effect of cytokines on TIGIT+NK cell ratios;

FIG. 2 shows the percentage of tigit+ NK cells in activated NK cells;

FIG. 3 shows a TIGIT-Z CAR vector map;

FIG. 4 shows TIGIT-Z CAR-T cell positive rate;

FIG. 5 shows the resistance function of TIGIT-UCAR-T cells against NK cells;

figure 6 shows a vector diagram of CAR1, CAR2, CAR3 of dual targeting TIGIT & CLDN18A2 and CAR targeting CLDN18A2 only;

FIG. 7 shows the positive rates of UCAR-T1, UCAR-T2, UCAR-T3 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 for dual targeting TIGIT & CLDN18A 2;

FIG. 8 shows the killing effect of dual targeting TIGIT & CLDN18A2 UCAR-T1, UCAR-T2, UCAR-T3 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 on target cells;

FIG. 9 shows the positive rate and TIGIT expression of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 for dual targeting TIGIT & CLDN18A 2;

FIG. 10 shows the variation of T cell and NK cell ratios after U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 of dual targeting TIGIT & CLDN18A2 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 were co-cultured with PBMC cells;

FIG. 11A shows the therapeutic effect of U-UTD, UCAR-T1, UCAR-T2, UCAR-T3 and CLDN18A2-UCAR-T cells targeting only CLDN18A2 on HGC-27-CLDN18A2 transplants; FIG. 11B shows peripheral blood CD4+, CD8+ T cell survival for each of the above groups;

FIG. 12 shows the knockout efficiency of different gRNAs for TIGIT;

FIG. 13 shows CAR positive rates of CLDN18A2-UCAR-T, CLDN A2-UCAR-T-TIGITKO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells;

FIG. 14A shows the CAR positive rate and TIGIT positive cell fraction of CLDN18A2-CAR-T, CLDN A2-CAR-T-TIGIT KO cells; FIG. 14B shows the in vitro killing effect of CLDN18A2-CAR-T, CLDN A2-CAR-T-TIGIT KO cells on target cells;

FIG. 15 shows the therapeutic effect of U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells on HGC-27-CLDN18A2 transplantable tumor;

FIG. 16 shows vector diagrams of TIGIT-BBZ and TIGIT-28Z targeting TIGIT;

FIG. 17 shows the ratio of T cells to NK cells after co-culturing TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR cells with NK cells, respectively.

Detailed Description

The applicant has conducted extensive and intensive studies and unexpectedly found that expression of a chimeric antigen receptor targeting TIGIT on the surface of immune effector cells or artificially modified cells having the function of immune effector cells can destroy most NK cells in a host, thereby largely avoiding attack of the host NK cells on allogeneic immune effector cells or artificially modified cells having the function of immune effector cells, prolonging survival time of allogeneic T cells in the host, and improving antitumor effect. The present application has been completed on the basis of this finding.

Terminology

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art 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, with suitable methods and materials being 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, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise specified.

The practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are well explained in the literature. See, e.g., current Protocols in Molecular Biology (FrederickM.AUSUBEL, 2000,WileyandsonInc,Library of Congress,USA); molecular Cloning: A Laboratory Manual, third Edition, (sambrook et al,2001,Cold Spring Harbor,NewYork: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); perbal, A Practical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m.simon, eds. -in-coef, 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 a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is understood that each intervening value, to the extent that it is between the upper and lower limit of that range and any other stated or intervening value in that range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where a range includes one or both of the limits, the claimed subject matter also includes ranges excluding either or both of those limits. This applies regardless of the width of the range.

The term used herein refers to about the usual error range for each value as 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, a description of "about X" includes a description of "X". For example, "about" or "comprising" may mean within 1 or more than 1 according to the actual standard deviation in the field. Or "about" or "comprising" may mean a range of up to 10% (i.e., ±10%). For example, about 5uM may include any number between 4.5uM and 5.5 uM.

Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range recited herein is to be understood as including any integer within the range, and where appropriate, fractions thereof (e.g., tenths and hundredths of integers) of numerical values.

For a better understanding of the application, the 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 amino acid sequence of human TIGIT is shown in SEQ ID NO: shown at 10. It consists of an extracellular immunoglobulin variable region (IgV) domain transmembrane region and an intracellular domain with a classical Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and an Immunoglobulin Tyrosine Tail (ITT) motif. TIGIT is expressed in lymphocytes, particularly 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 in the treatment of tumor patients, some embodiments of the application employ different combinations of IL-2, IL-15 and IL-12 cytokines to treat NK cells to detect changes in TIGIT + NK cell ratio.

The term "CLDN18A2" refers to the Claudin 18 (Claudin 18, CLD 18) molecule (Genbank accession number: splice variant 1 (CLD 18 A1): np_057453, NM016369, and splice variant 2 (CLD 18 A2): nm_001002026, np_ 001002026) being an intrinsic transmembrane protein having a molecular weight of about 27,9/27,72 kd. Dense proteins are integral membrane proteins located in the tight junction of the epithelium and endothelium. A network of interconnected chains of particles within the tissue membrane tightly connected between adjacent cells. In tight junctions, the occluding (occludin) and claudin are the most predominant transmembrane protein components. Because of their strong intercellular adhesion properties, they create a primary barrier that prevents and controls paracellular transport of solutes and limits the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Proteins that form tight junctions are critically involved in tissue epithelial tissue architecture.

The term "transplant rejection" refers to the recognition of foreign grafts by the host's immune system as a "heteroelement" and the initiation of an immunological response to the attack, destruction, and clearance of the graft after the host has undergone allogeneic tissue, organ, or cell graft transplantation. The present application provides a cell and a method for resisting transplant rejection.

The term "graft" refers to a biological material or formulation that originates from an individual other than the host for implantation into the host. The graft may be derived from any animal source, such as mammalian sources, preferably from humans. In some embodiments, the graft may be derived from a host, such as cells derived from the host, cultured in vitro, or otherwise engineered to be re-implanted into the host. In some embodiments, the graft may be an isolated or isolated cell from an individual, such as an individual, or a cell from another individual, cultured in vitro or engineered to be implanted into a host. In some embodiments, the graft may be an organ-implanted human from a xenogeneic individual, such as from another species (e.g., mouse, pig, monkey).

The term "cell" refers to a cell of human or non-human origin that is animal.

The term "host" refers to a recipient that receives a graft transplant, and in some embodiments, may be an individual, such as a human, that receives an exogenous cell implant.

The term "individual" refers to any animal, such as a mammal or a pouched animal. Individuals of the application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaque), mice, pigs, horses, donkeys, cattle, sheep, rats, and any variety of poultry.

The term "immune effector cells" refers to cells involved in an immune response, such as T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, and the like, that produce an immune effect. In some embodiments, the immune effector cells are T cells, NK cells, NKT cells. In some embodiments, the T cells may be autologous T cells, xenogeneic T cells, allogeneic T cells. In some embodiments, the NK cells can be autologous NK cells or allogeneic NK cells.

The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that have no immune effect that have acquired immune effector cell function after being artificially modified or stimulated with a stimulus. For example, 293T cells are artificially modified to have the function of immune effector cells; such as stem cells, are induced in vitro to differentiate into immune effector cells.

The "T cells" as described herein may be PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue and natural T cells obtained from infection sites, ascites, pleural effusions, spleen tissue, tumor tissue, cell populations obtained by sorting or the like having specific phenotypic characteristics, or mixed cell populations of different phenotypic characteristics, e.g. "T cells" may be cells comprising at least one T cell subset: 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 T cell of a particular subtype, such as γδ T cells. 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 (tm) isolation and/or apheresis. T cells may be any type of T cell, and may be at any stage of development, including but not limited to cd4+/cd8+ double positive T cells, cd4+ helper T cells, such as Th1 and Th2 cells, cd8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, astronomical T cells, and the like. The 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. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells collected by apheresis may be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps. The T cells may be derived from healthy donors, or from individuals diagnosed with cancer.

The terms "activate" and "activating" are used interchangeably and may refer to the process by which a cell transitions from a resting state to an active state. The process may include a response to a phenotypic or genetic change in the antigen, migration, and/or functionally active state. For example, the term "activation" may refer to the process of stepwise activation of T cells. The activation process is co-regulated by the first stimulus signal and the co-stimulus signal. Activation of T cells is a dynamically changing process, the duration of which and the degree of activation are both affected by external stimuli. "T cell activation" or "T cell activation" refers to the state of a T cell that is stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function. Using CD3/CD28 magnetic beads, either in vitro or in vivo antigen stimulation will have an effect on the degree and duration of T cell activation. In one embodiment, the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.

The term "peripheral blood mononuclear cells" (peripheral blood mononuclear cell, PBMC) refers to cells having a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.

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

The term "engineering" refers to a comprehensive scientific technique that applies principles and methods of cell biology and molecular biology to alter, by some engineering means, genetic material within a cell or to obtain a cellular product at the cellular global or organelle level, as desired. In one embodiment, the engineering refers to one or more alterations of a nucleic acid, such as a nucleic acid within the genome of an organism. In one embodiment, the engineering refers to alterations, additions and/or deletions of genes. In one embodiment, the engineered cell or the engineered cell may also refer to a cell having added, deleted, and/or altered genes.

The terms "genetic modification", "genetically engineered" or "modified" refer to a method of modifying a cell, including but not limited to, the modification of a gene in a coding or non-coding region or an expression regulatory region thereof by means of gene editing; or by endonuclease and/or antisense RNA techniques; or increasing the introduction of exogenous proteins and/or complexes, small molecule inhibitors to alter the protein expression levels of the gene to create a gene defect. In some embodiments, the modified cells are stem cells (e.g., hematopoietic Stem Cells (HSCs) or progenitor cells, embryonic stem cells (ESs), induced Pluripotent Stem (iPS) cells), lymphocytes (e.g., T cells), which may be obtained from a subject or donor. The cells may be modified to express an exogenous construct, such as a Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR), which may be integrated into the cell genome.

The term "gene silencing" refers to a phenomenon in which a gene is not expressed or is underexpressed for various reasons. Gene silencing may be at the level of transcription due to DNA methylation, heterochromatin, positional effects, etc.; post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of the target RNA at the post-transcriptional level of the gene, including antisense RNA, co-inhibition, gene suppression, RNA interference, microrna-mediated translational inhibition, and the like; protein expression or low expression, including PROTAC, LYTAC, abTAC, ATTEC, AUTAC and membrane protein intracellular retention techniques, can also be rendered undetectable by increasing protein degradation.

By "TCR silencing" is meant that the endogenous TCR is not expressed or is underexpressed.

By "MHC silencing" is meant that endogenous MHC is not expressed or is underexpressed.

By "low expression" is herein meant that the level of protein and/or RNA expressed by the target gene in the engineered cell is lower than the level of expression prior to the cell engineering process. In particular embodiments, low expression of B2M or TCR or TIGIT refers to a decrease in expression of B2M or TCR or TIGIT in a cell of 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 may be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting (Western Blotting) or flow cytometry using antibodies specific for B2M or TCR or TIGIT.

The term "MHC", which is a histocompatibility complex, is a generic term for all gene groups encoding biocompatible complex antigens, which are expressed in all tissues of higher vertebrates, called HLA antigens in human cells, play an important role in the transplantation reaction, and are mediated by T cells that react to the histocompatibility antigens on the surface of the implanted tissues. MHC antigens are classified into NHC class I antigens and MHC class II antigens.

The term "human leukocyte antigen" (Human leukocyte antigen, HLA) is a gene encoding the major histocompatibility complex of humans, located on chromosome 6 (6p21.31), and is closely related to the function of the human immune system. HLA includes class I, class II and class III gene portions. Antigens expressed by HLA class I and class II genes are located on cell membranes, are MHC-I (HLA-A, HLA-B, HLA-C site encoding) and MHC-II (HLA-D region encoding), are distributed on almost all cell surfaces of the body, are heterodimers composed of heavy chains (alpha chains) and beta 2 microglobulin (B2M), and class II is glycoprotein localized on the surfaces of macrophages and B lymphocytes.

The term "B2M" is a 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, as opposed to other MHC genes located as a cluster of genes on chromosome 6. Studies have shown that when mutations occur in the B2M gene, hematopoietic grafts from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice, suggesting that defective expression of MHC I molecules renders the cells susceptible to rejection by the host immune system (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. Classical TCR receptors, consisting of two peptide chains, a and β, each of which can be divided into a variable region (V region), a constant region (C region), a transmembrane region, a cytoplasmic region, etc., where antigen specificity resides, V regions (vα, vβ) each having three hypervariable regions CDR1, CDR2, CDR3, in one aspect, T cells expressing classical TCRs can induce T cell TCR specificity for a target antigen by, for example, antigen stimulation of T cells. TCRs fall into two categories: TCR1 and TCR2; TCR1 consists of two chains, γ and δ, and TCR2 consists of two chains, α and β. The term "TRAC" refers to the constant region of the TCR alpha chain.

The term "gene editing" refers to genetic engineering techniques that use site-specific nucleases to insert, knock out, modify or replace DNA at specific locations in the genome of an organism, altering the DNA sequence. This technique is sometimes referred to as "gene clipping" or "genome engineering". Gene editing can be used to achieve accurate, efficient gene knockouts or gene knockins.

Nuclease-directed genomic targeted modification techniques typically consist of a DNA recognition domain and a non-specific endonuclease domain, with the recognition of the target site by the DNA recognition domain, localization of the nuclease to the genomic region to be edited, and subsequent cleavage of the DNA duplex by the non-specific endonuclease, causing DNA fragmentation self-repair mechanisms, thereby initiating mutation of the gene sequence and promoting homologous recombination. The endonuclease may be a Meganuclease (Meganuclease), a zinc finger nuclease, a CRISPR/Cas9 nuclease, an MBBBD-nuclease, or a TALEN-nuclease. In preferred embodiments, the endonuclease is a CRISPR/Cas9 nuclease, TALEN-nuclease. Gene knockout techniques using nucleases include CRISPR/Cas9 techniques, ZFN techniques, TALE techniques, and TALE-CRISPR/Cas9 techniques, base Editor techniques, guided editing techniques, and/or homing endonuclease techniques.

The term "artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN)" technology, which is a first generation nuclease site-directed modification technology, utilizes a zinc finger motif (motif) capable of specifically recognizing a triplet DNA fragment instead of a base as a basic unit for recognizing a specific DNA sequence. The most classical zinc finger nucleases fuse a non-specific endonuclease fokl to a zinc finger containing domain, naturally with the aim of cleaving a specific sequence.

The term "Transcription Activator Like Effector (TALE)" has DNA binding specificity, has a module capable of specifically recognizing a base, and is simple and convenient to operate. The TALE-DNA binding domain consists of tandem repeat units, most of which contain 3 4 amino acids, with amino acids 12 and 13 of the unit designed as variable regions (repeat variable residues, RVD). The RVD of TALE recognizes 4 bases of DNA sequence with high specificity, and amino acid 13 is directly combined with DNA base specificity. Can construct specific TALEDN recognition binding domain at any site according to DNA sequence, and can be widely used for gene sequence mutation modification, gene targeting and the like. The DNA target sequence was set, the TALE-DNA binding domain was assembled, and the non-specific DNA cleavage domain of Fok I endonuclease was fused, and assembled into TALE nuclease (tanscription activator-like effector nucleases, TALENs). TALENs target binding to DNA, creating DNA double-strand breaks (DSBs).

CRISPER/Cas9 is a third generation gene editing technology. One embodiment of the application employs CRISPR/Cas9 technology to prepare UCAR-T cells. The term "CRISPR (Clustered regularly interspaced short palindromicrepeats)" refers to short palindromic repeats at regular clustered intervals. The term "Cas9 (CRISPRassociated nuclease)" is a CRISPR-associated nuclease, a RNA-guided technique for editing a targeted gene with a Cas9 nuclease. The Cas9 enzyme may be a wild-type Cas9 or a engineered Cas9. The "CRISPER/Cas9 system" is collectively referred to as transcripts and other elements involved in the expression of or directing the activity of Cas9 enzyme genes, including sequences encoding Cas9 genes, tracr (transactivation CRISPR) sequences (e.g., tracrRNA or active moiety tracrRNA), tracr mate sequences (covering "orthographic repeats" and partial orthographic repeats of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems, i.e., grnas), or other sequences and transcripts from CRISPR loci. CRISPR systems are characterized by elements that promote the formation of CRISPR complexes (also referred to as the forebay in the context of endogenous CRISPR systems) at the site of a target sequence. In the context of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. Complete complementarity is not necessary provided that sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR complex. After CRISPR complex is formed, specific loci of a genome can be cut under the action of cas9 enzyme, and gene mutation is introduced; expression of the gene may also be regulated, such as by activation or inhibition. A target sequence may 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.

In general, a guide sequence (gRNA) is any ribonucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct the sequence-specific binding of a CRISPR complex to the target sequence. The application relates to a sequence of gRNA, which can be a targeted DNA sequence or a complete Cas9 guide sequence formed by ribonucleotides corresponding to the DNA and crRNA, tracrRNA. gRNA is used to guide, bind or recognize Cas enzymes. 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%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. The optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needman-Wunsch algorithm, the Basil Yu Baluo Siderurg-Wheater transform (Burley-Wheeler Transform) algorithm (e.g., the Burley-Wheater alignment tool (Burrows Wheeler Aligner)), clustalW, clustal X, BLAT, novoalign (Novocraft technologies), ELAND (Ill.) by Ill. The term "sgRNA" refers to short gRNA.

In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains other than the CRISPR enzyme). The CRISPR enzyme fusion protein may comprise any other protein, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter sequences, and one or more protein domains having the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus Hemagglutinin (HA) tags, myc tags, VSV-G tags, and thioredoxin (Trx) tags.

In gene editing, the given gRNA, tracr mate sequence, and tracr sequence may be administered alone or in one complete RNA sequence.

Cas9 protein and gRNA binding can realize DNA cleavage at specific sites, the CRISPR/Cas system recognition sequence from Streptococcus pyogenes is 23bp and can target 20bp, and the last 3 rd NGG sequence of the recognition site is called PAM (protospacer adjacent motif) sequence.

CRISPR/Cas transgenes can be delivered by vectors (e.g., AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electrotransport.

The application simultaneously knocks out genes TRAC and B2M to inactivate functions of TCR and MHC molecules, thus obtaining general T cells or general CAR-T cells. In one embodiment, the exons of the corresponding coding genes in the constant region of one or both of the alpha and beta strands of B2M, TCR are knocked out using the CRISPER/Cas technique, respectively. In one embodiment, the gRNA used to knock out the endogenous TCR is selected from the sequences set forth in SEQ ID NO. 23, 65, 66, 67, 68, 69, 70 or 71. In one embodiment, the endogenous B2M gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the sequences set forth in SEQ ID NO. 24, 72, 73 or 74.

In one embodiment, the cellular TIGIT gene is knocked out. Illustratively, the TIGIT gene is knocked out using CRISPR/Cas9 technology using a gRNA selected from the sequences shown in SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.

By "inhibiting" or "suppressing" expression of B2M or TCR or TIGIT is meant that expression of B2M or TCR or TIGIT in the cell 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 may be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting (Western Blotting) or flow cytometry using antibodies specific 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 interfering agents include, but are not limited to, nucleic acid molecules of RNA molecules homologous to the target gene or fragment thereof, short interfering RNAs (sirnas), shrnas or mirnas, and small molecules that interfere with or inhibit expression of the target gene by RNA interference (RNAi).

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

The term "transfection" refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of 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 (biolistics).

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

The term "sequence" as used herein when used in reference to a nucleotide sequence may include DNA or RNA, and may be single-stranded or double-stranded.

The term sequence "identity" as used herein determines the percent identity by comparing two optimally matched sequences over a comparison window (e.g., at least 20 positions), wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps), such as 20% or less (e.g., 5 to 15%, or 10 to 12%) of the gap(s) for the optimally matched two sequences compared to the reference sequence (which does not comprise additions or deletions). The percentage is typically calculated by determining the number of positions at which the same nucleobase or amino acid residue occurs in both sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to produce the percentage of sequence identity.

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

The term "vector" as used herein is a composition comprising an isolated nucleic acid and useful for delivering 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, such as polylysine compounds, liposomes, and the like, that facilitate transfer of nucleic acids into cells may also be included.

The term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously by the organism itself, or that is not expressed at levels sufficient to achieve overexpression.

The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., derived from the organism itself.

The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments thereof that specifically bind an antigen or an antigenic determinant so long as they exhibit the desired antigen-binding activity. The present application provides T cells expressing a first protein comprising an antibody that recognizes TIGIT. In particular embodiments, the application also provides T cells expressing a first protein comprising a tandem antibody that recognizes TIGIT and a tumor antigen. In particular embodiments, the application also provides T cells expressing a first protein comprising a tandem antibody that recognizes TIGIT and a pathogen antigen.

"antibody fragment" refers to a fragment of an antibody that specifically binds an antigen or epitope, non-limiting antibody fragments include Fab, F (ab') 2, fv, CDR, scFv.

The term "variable region or variable domain" refers to the domain of an antibody heavy or light chain that is involved in antigen binding of an antibody. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain comprising four conserved FR and three CDRs. In a specific embodiment, the application provides a T cell expressing a first protein comprising a tandem antibody that recognizes TIGIT and a tumor antigen, or TIGIT and a pathogen antigen, in tandem form comprising:

(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 a tumor and/or pathogen-light chain (or light chain variable region) of an antibody that recognizes a tumor and/or pathogen-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 regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops") and/or contain residues that contact an antigen ("antigen contacts"). Typically, an antibody comprises six CDRs: three of VH (HCDR 1, HCDR2, HCDR 3) and three of VL (LCDR 1, LCDR2, LCDR 3). Illustratively, an antibody or functional fragment thereof that recognizes TIGIT of the present application contains a heavy chain variable region comprising 3 CDRs, heavy chain CDR1 shown in SEQ ID No. 3, heavy chain CDR2 shown in SEQ ID No. 4, heavy chain CDR3 shown in SEQ ID No. 5. The antibody or a functional fragment thereof recognizing TIGIT of the present application contains a light chain variable region comprising 3 CDRs, a light chain CDR1 shown in SEQ ID NO. 6, a light chain CDR2 shown in SEQ ID NO. 7, and a light chain CDR3 shown in SEQ ID NO. 8. The antibody or the functional fragment thereof for recognizing TIGIT contains a heavy chain variable region shown in SEQ ID NO. 1 and/or a light chain variable region shown in SEQ ID NO. 2. Illustratively, an antibody or functional fragment thereof of the application that recognizes CLDN18A2 contains a heavy chain variable region comprising 3 CDRs, heavy chain CDR1 as shown in SEQ ID No. 13, heavy chain CDR2 as shown in SEQ ID No. 14, heavy chain CDR3 as shown in SEQ ID No. 15. The antibody or functional fragment thereof recognizing CLDN18A2 contains a light chain variable region comprising 3 CDRs, light chain CDR1 shown in SEQ ID NO. 16, light chain CDR2 shown in SEQ ID NO. 17, light chain CDR3 shown in SEQ ID NO. 18. The antibody or the functional fragment thereof for recognizing the CLDN18A2 contains a heavy chain variable region shown in SEQ ID NO. 11 and/or a light chain variable region shown in SEQ ID NO. 12.

The term "chimeric receptor", i.e., a fusion molecule comprising a gene recombinant technology linked to DNA fragments of different origins or corresponding cdnas of proteins, includes an extracellular domain, a transmembrane region, and an intracellular domain. Chimeric receptors include, but are not limited to: chimeric Antigen Receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC).

The term "chimeric T cell receptor" consists of a TCR subunit associated with an antigen binding domain (e.g., an antibody domain), wherein the TCR subunit comprises at least a portion of a TCR extracellular domain, a transmembrane region (or referred to as a transmembrane domain), a stimulatory domain of a TCR intracellular signaling domain; the TCR subunit and the antibody domain are operably linked. In specific embodiments, the extracellular, transmembrane, intracellular signaling domain of the TCR subunit is derived from CD3 epsilon, CD3 gamma, CD3z, the alpha chain of the TCR, or the beta chain of the TCR, and the chimeric T cell receptor is capable of forming a complex with TCR/CD3 expressed on a T cell.

The term "T cell antigen coupler (T cell antigen coupler, TAC)", includes three functional domains: (1) An antigen binding domain comprising a single chain antibody, a designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting group; (2) An extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; (3) Transmembrane domain and intracellular region of CD4 co-receptor, wherein the intracellular region is linked to the protein kinase LCK, which catalyzes the phosphorylation of the Immunoreceptor Tyrosine Activation Motif (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 application provides a T cell capable of resisting killing of autologous or allogeneic NK cells. In particular, the application provides a CAR-T cell expressing a cell comprising a recognized TIGIT. The application provides T cells expressing a CAR comprising a recognized TIGIT and having an endogenous TIGIT knockout. The application provides universal T cells expressing CARs comprising recognized TIGIT, and endogenous TCR and B2M knockouts. The application provides universal T cells expressing CARs comprising recognized TIGIT, and endogenous TIGIT, TCR, and B2M knockouts.

The application also provides a T cell which can resist the killing of autologous or allogeneic NK cells and can obviously kill tumor cells. In particular, the application provides T cells expressing a CAR comprising a dual target that recognizes TIGIT and a tumor antigen. The application provides T cells expressing a CAR comprising a dual target that recognizes TIGIT and a pathogen antigen, and knocks out endogenous TIGIT. The application provides universal T cells expressing a CAR comprising a dual target that recognizes TIGIT and a pathogen antigen, and knocks out endogenous TCRs and B2M. The application provides universal T cells expressing a CAR comprising a dual target that recognizes TIGIT and a pathogen antigen, and knocks out endogenous TIGIT, TCR, and B2M.

The manner of linking antibodies recognizing TIGIT and tumor antigens in the antigen binding region of the above-described dual-target-recognizing CAR 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 tumor antigen-light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes tumor antigen;

(ii) Light chain (or light chain variable region) of an antibody that recognizes a tumor antigen-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT-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 a tumor antigen; 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 a tumor antigen-light chain (or light chain variable region) of an antibody that recognizes a tumor antigen-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT.

The application also provides a T cell which can resist the killing of autologous or allogeneic NK cells and can obviously kill tumor cells. In particular, the application provides T cells expressing a CAR that comprises a TIGIT-recognizing CAR and a CAR that recognizes a tumor antigen. The application provides T cells expressing a CAR comprising a recognized TIGIT and a CAR that recognizes a tumor antigen, and knocked out endogenous TIGIT. The application provides universal T cells expressing a CAR comprising a CAR that recognizes TIGIT and a CAR that recognizes a tumor antigen, and knocked out endogenous TCRs and B2M. The application provides universal T cells expressing a CAR comprising a recognized TIGIT and a CAR that recognizes a tumor antigen, and knocked out endogenous TIGIT, TCR, and B2M.

The application also provides a combination of T cells provided by the application that are resistant to killing by autologous or allogeneic NK cells with T cells expressing a second protein (e.g. CAR) that recognizes a tumor antigen. The T cells provided by the application, which can resist killing by autologous or allogeneic NK cells, can promote the survival of the T cells expressing a second protein (such as CAR) for recognizing tumor antigens in the presence of autologous or allogeneic immune cells. In particular embodiments, the endogenous TIGIT of the T cell expressing the second protein (e.g., CAR) that recognizes the tumor antigen is knocked out. In particular embodiments, the T cells expressing a second protein (e.g., CAR) that recognizes a tumor antigen are universal T cells in which the endogenous TCR and B2M are knocked out. In particular embodiments, the T cells expressing a second protein (e.g., CAR) that recognizes a tumor antigen are universal T cells with endogenous TIGIT, TCR, and B2M knockouts.

The term "chimeric antigen receptor" (CAR) includes an extracellular domain, a transmembrane region, and an intracellular signaling domain. Intracellular signaling domains include functional signaling domains of stimulatory molecules and/or co-stimulatory molecules (referred to as intracellular signaling domains and co-stimulatory signaling domains, respectively), in one aspect, stimulatory molecules are delta chains that bind to the T cell receptor complex; in one aspect, the cytoplasmic signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD 137), CD27, and/or CD28. In certain embodiments, the sets of polypeptides are linked to each other. Illustratively, TIGIT-targeted CARs comprise SEQ ID NO:9, the CLDN18 A2-targeted CAR comprises the sequence set forth in SEQ ID NO: 21. 75, 76 or 77. The CAR targeting TIGIT and CLDN18A2 simultaneously comprises SEQ ID NO: 48. 50 or 52; or comprises SEQ ID NO: 54. 56 or 58. In a specific embodiment, an engineered T cell that targets TIGIT and CLDN18A2 simultaneously comprises SEQ ID NO: 54. 56 or 58; or comprises SEQ ID NO: 53. 55 or 57.

The term "primary signal domain" modulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signal domain is triggered by binding of, for example, 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 signal domain acting in a stimulatory manner may comprise an immunoreceptor tyrosine activation motif or a signaling motif of ITAM. Examples of primary signal domains comprising ITAM that are particularly useful in the present application include, but are not limited to, sequences derived from tcrζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d, in particular inventive CARs, the intracellular signaling domain in any one or more of the inventive CARs comprises an intracellular signaling sequence, such as the primary signaling domain of CD3 ζ.

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by either producing a second messenger or by acting as an effector in response to such a messenger. The intracellular signaling domain may comprise all intracellular portions of the molecule, or all native intracellular signaling domains, or functional fragments or derivatives thereof.

The term "co-stimulatory molecule" refers to a signal that, in combination with a cell stimulatory signaling molecule, e.g., TCR/CD3, results in up-or down-regulation of T cell proliferation and/or a key molecule. Is a cognate binding partner on T cells that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, including but not limited to cell proliferation. Costimulatory molecules are cell surface molecules or ligands thereof that are non-antigen receptors required for an effective immune response. Co-stimulatory 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 (CD 11a/CD 18) and 4-1BB (CD 137).

The intracellular signaling domain (or referred to as a structural region) may be selected from any one of the co-stimulatory domains of table 1. In some embodiments, the domain may be modified such that the identity to the reference domain may be 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 Co-stimulatory domains

In some cases, the intracellular signaling domain may be designed to comprise several possible co-stimulatory signaling domains, may comprise a single co-stimulatory domain, such as the delta chain (first generation CAR), or it may further comprise one co-stimulatory domain, such as the delta chain with CD28 or 4-1BB (second generation CAR), or it may further comprise two co-stimulatory domains, such as the delta chain with CD28/OX40 or CD28/4-1BB (third generation CAR). The signaling pathways used by these costimulatory molecules are all synergistic with the primary T cell receptor activation signal. The signals provided by these costimulatory signaling regions may cooperate with a major-effect activation signal derived from one or more ITAM motifs (e.g., the CD3zeta signaling domain) and may fulfill the requirements of T cell activation. Exemplary, signaling domains of TIGIT-targeted, CLDN18 A2-targeted, and/or CLDN18 A2-targeted CARs include cd3δ. In a specific embodiment, the CD3δ is a human CD3δ molecule comprising the sequence set forth in SEQ ID NO. 46. Exemplary, signaling domains of TIGIT-targeted, CLDN18 A2-targeted, and/or dual-targeted TIGIT and CLDN18A2 CARs include the CD28 intracellular domain. In a specific embodiment, the CD28 intracellular domain comprises the sequence shown as SEQ ID NO. 44.

The term "CD3 ζ" (also referred to as CD3 Zeta) "is defined as the protein provided by genbank accession No. BAG36664.1, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc. "CD 3zeta domain" is defined as the amino acid residue from the cytoplasmic domain of the zeta chain that is sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 164 of genbank accession No. BAG36664.1, functional orthologs thereof-equivalent residues from non-human species such as mice, rodents, monkeys, apes, and the like. CD3 ζ is also known as the T cell receptor T3 delta chain or CD247. This domain is part of the T cell receptor-CD 3 complex and plays an important role in combining antigen recognition of several intracellular signaling pathways with activation of the primary effect of T cells. As used herein, cd3δ refers primarily to human cd3δ and isoforms thereof, as known from Swissprot entry P20963, including proteins having substantially the same sequence. As part of the chimeric antigen receptor, the whole T cell receptor T3 delta chain is not required, and any derivative thereof comprising the signal domain of the T cell receptor T3 delta chain is suitable, including any functional equivalent thereof. In the present application, "CD3 delta" is used interchangeably with "CD3Z" and "CD 3Z".

In one aspect of the application, a CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain that contains a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at an amino acid of the CAR protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane. In one embodiment, the leader sequence comprises 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. Typically, the extracellular domain of the CAR is derived from a mouse or humanized or human monoclonal antibody.

Chimeric antigen receptors can be designed with a variety of antigen binding regions, including single chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors. In some embodiments, the extracellular domain may comprise scFv, fab, or natural ligand, and 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 may bind to an 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; a bifunctional antibody, a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. The extracellular domain, e.g., scFv, fab, or natural ligand, may be part of a CAR that determines antigen specificity. The extracellular domain may bind any complementary target. The extracellular domain may be derived from antibodies of known variable region sequences. The extracellular domain may be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, the extracellular domain may be obtained from whole exo-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 the light chain CDRs and the heavy chain CDRs. The application provides a CAR-T cell expressing a cell comprising a recognized TIGIT. In particular embodiments, the application also provides a CAR-T cell expressing a double target comprising a recognition TIGIT and a tumor antigen. In particular embodiments, the application also provides a CAR-T cell expressing a double target comprising a recognition TIGIT and a pathogen antigen.

In some embodiments, the extracellular domain of the CAR includes an antigen binding region and a linker fragment (also referred to as a hinge, spacer, or linker). The linker fragment may be considered to be part of a CAR for providing flexibility to the extracellular domain. In some cases, the difference in the linker fragments may result in a CAR on the cell surface that is not activated by the target antigen, or is activated to a very low degree, or the T cells may undergo significant depletion after activation and loss of function, possibly due to the difference in the degree of complementarity of the spatial structure formed by the antibody or antibody fragment of the extracellular domain to that of the target antigen. For example, whether the length of the linker fragment derived from an immunoglobulin needs to be optimized depends on the location where the extracellular domain targets the target epitope.

The composition and length of the linked fragments of the CAR polypeptide are both adjustable. The spatial conformation of the immunological synaptic binding between T cells and target cells determines the distance that the membrane distal epitope on the target molecule cannot functionally bridge to the CAR, nor does a CAR using a short linker fragment allow the synaptic distance to reach an approximation of signal transduction. Likewise, the membrane proximal CAR target epitope only observed signal output in the context of long linker CARs. The linker fragment may be regulated depending on the extracellular domain used. The connecting segments may be of any length. Illustratively, in one embodiment of the application, the CAR comprises a hinge domain, which is a CD 8. Alpha. Hinge, preferably, the CD 8. Alpha. Hinge domain comprises the amino acid of SEQ ID NO. 40.

The Transmembrane (TM) domain of the CAR (or referred to as a structural region) may anchor the CAR to the plasma membrane of the cell. The natural transmembrane portion of CD28 is available for CAR. In other cases, the natural transmembrane portion of CD8 a may also be used in the CAR. "CD8" may be the same reference number as NCBI: NP-001759 or fragments thereof having stimulatory activity have a protein of at least 85, 90, 95, 96, 97, 98, 99 or 100% identity. A "CD8 nucleic acid molecule" may be a polynucleotide encoding a CD8 polypeptide, in some cases the transmembrane domain may be the natural transmembrane portion of CD28, and "CD28" may refer to the sequence that is associated with NCBI reference number: NP-006130 or fragments thereof having stimulatory activity have a protein that is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical. A "CD28 nucleic acid molecule" may be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion may comprise a CD8 a region. Illustratively, in one embodiment of the application, the TM is a CD28 transmembrane region. In a preferred embodiment, it is the human CD28 transmembrane region. Preferably, the CD28 transmembrane region comprises the amino acid of SEQ ID NO. 42.

With respect to pharmaceutical compositions, a pharmaceutically acceptable carrier may be one of the carriers conventionally used and is limited only by chemical-physical considerations, such as solubility and non-reactivity with the active agent, as well as the route of administration. Pharmaceutically acceptable carriers described herein, such as adjuvants, adjuvants and diluents, are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is a carrier which is harmless under the conditions of use and has no toxic or side effects. The pharmaceutical compositions of the present application are in a variety of suitable dosage forms. Methods of preparing administrable (e.g., parenteral) compositions are known or apparent to those skilled in the art.

The engineered cells of the application can be administered to a subject in any suitable manner. Preferably, the CAR material of the application is administered by injection (e.g., subcutaneously, intravenously, intratumorally, intraarterially, intramuscularly, intradermally, intraperitoneally, or intrathecally). Preferably, the CAR material of the application is administered intravenously. Suitable pharmaceutically acceptable carriers for the injectable CAR materials of the application can include any isotonic carrier, for example, physiological saline (about 0.90% w/v NaCl in water, about 300mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), ambient temperature, or an electrolyte solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

An "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to prevent or treat a disease (cancer) in an individual. The effective dose for therapeutic or prophylactic use will depend on the disease being treatedStage and severity, age, weight and general health of the subject, and judgment of the prescribing physician. The size of the dosage 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 a particular active substance, and the desired physiological effect. One or more rounds, or multiple administrations of the CAR materials of the present application may be required, as determined by the prescribing physician or skilled artisan. By way of example and not limitation, when the CAR material of the application is a host cell, an exemplary dose of host cell may be at least one million cells (1 x 10 ⁶ Individual cells/dose).

Embodiments of the application also include removing lymphocytes from a mammal prior to administration of the CAR material of the application, including, but not limited to, non-myeloablative lymphocyte depleting chemotherapy, systemic irradiation, and the like.

The term "treatment" refers to interventions that attempt to alter the course of a disease, both prophylactic and clinical, as well. Therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of progression of a disease, improving or alleviating a condition, alleviating or improving prognosis, and the like. In specific embodiments, the engineered T-cells provided herein are capable of inhibiting tumor cell proliferation, and/or inhibiting tumor cell proliferation in vivo, tumor volume increase.

The term "prevention" refers to intervention prior to attempting to develop a disease such as rejection by cell transplantation.

The application provides a cell, nucleic acid, expression vector, host cell, or composition of the application for use in the treatment or prevention of a tumor.

The provided engineered T cells of the application are useful for the treatment, prevention or amelioration of autoimmune diseases or inflammatory diseases, particularly inflammatory diseases associated with autoimmune diseases, such as arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis (arthritis chronicaprogrediente) and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spinal arthropathy (including ankylosing spondylitis), reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, start-stop point inflammation, hypersensitivity reactions (including airway hypersensitivity and skin hypersensitivity reactions) and allergies. The engineered T cells provided herein are useful for the treatment and prevention of conditions including autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure erythrocyte anemia, and idiopathic thrombocytopenia), systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory muscle diseases (dermatomyositis), periodontitis, polychondritis, scleroderma, wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, stefan Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel diseases (including, for example, ulcerative colitis, crohn's disease, and irritable bowel syndrome), endocrine ocular diseases, graves ' disease, sarcoidosis, multiple sclerosis, systemic sclerosis, fibrotic diseases, primary biliary cirrhosis, juvenile diabetes (type I diabetes), uveitis, keratoconjunctivitis sicca, and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, peri-prosthetic osteolysis, glomerulonephritis (with and without nephrotic syndrome), for example including idiopathic nephrotic syndrome or minuscular nephropathy), multiple myeloma, other types of tumors, inflammatory diseases of the skin and 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 autoimmune thyroid diseases including hashimoto's thyroiditis, medium and small vascular primary vasculitis, macrovasculitis including giant cell arteritis, suppurative sweat gland, neuromyelitis optica, sjogren's syndrome, behcet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle diseases, autoimmune peripheral neuropathy, immune kidney, liver and thyroid diseases, inflammatory and atherosclerosis, auto-inflammatory fever syndrome, immune hematologic disorders, and bullous diseases of the skin and mucous membranes.

The engineered T-cells provided by the application can be used for treating, preventing or improving asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema and other obstructive or inflammatory diseases of the airways.

The engineered T cells of the application may be administered as the sole active ingredient or in combination (e.g., as an adjuvant or in combination) with other drugs such as immunosuppressants or immunomodulators or other anti-inflammatory or other cytotoxic or anticancer agents, e.g., to treat or prevent diseases associated with immune disorders. For example, the antibodies of the application may be used in combination with the following drugs: DMARDs such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors such as cyclosporin a or FK 506; modulators of lymphocyte recirculation, such as FTY720 and FTY720 analogs; mTOR inhibitors, such as rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, CCI779, ABT578, AP23573, or TAFA-93; ascomycins having immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or an immunosuppressive homolog, analog or derivative thereof; immunosuppressive monoclonal antibodies, e.g., directed against leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. Monoclonal antibodies to CD45, CD58, CD80, CD86 or ligands thereof; other immunomodulatory compounds.

The tumor of the present application may be any tumor including acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal or middle ear cancer, oral cancer, vulvar cancer, chronic Lymphocytic Leukemia (CLL), chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid, head and neck cancer (such as head and neck squamous cell carcinoma), hodgkin lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (such as non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin lymphoma, B-chronic lymphocytic leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), pre-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, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer. Preferably, the tumor is characterized by BCMA expression, and more preferably is multiple myeloma characterized by BCMA expression.

"tumor antigen" refers to an antigen that is either newly emerged or overexpressed during the development, progression, or development of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present application is cancer.

The tumor antigen of the application can be a solid tumor antigen or a blood tumor antigen.

Tumor antigens of the application include, but are not limited to: thyroid Stimulating Hormone Receptor (TSHR); CD171; CS-1; c-type lectin-like molecule-1; ganglioside GD3; a Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD 276), B7H6; KIT (CD 117); interleukin 13 receptor subunit alpha (IL-13 ra); interleukin 11 receptor alpha (IL-11 ra); prostate Stem Cell Antigen (PSCA); prostate Specific Membrane Antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; tyrosinase; mesothelin; epCAM; protease serine 21 (PRSS 21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR 2); lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1 (MUC 1), MUC6; the epidermal growth factor receptor family and mutants thereof (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural Cell Adhesion Molecules (NCAM); carbonic Anhydrase IX (CAIX); LMP2; ephrin-type a receptor 2 (EphA 2); fucosyl GM1; sialic acid based lewis adhesion molecules (sLe); ganglioside GM3; TGS5; high Molecular Weight Melanoma Associated Antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD 2); a folate receptor; tumor vascular endothelial marker 1 (TEM 1/CD 248); tumor vascular endothelial marker 7-associated (TEM 7R); 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; embryo type AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligands; PSC1; ROR1; sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variants of tumor necrosis zone; g protein coupled receptor class C group 5-member D (GPRC 5D); x chromosome open reading frame 61 (CXORF 61); CD97; CD179a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1 (PLAC 1); a hexose moiety of globohglycyceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK 2); hepatitis a virus cell receptor 1 (HAVCR 1); adrenergic receptor beta 3 (ADRB 3); pannexin 3 (PANX 3); g protein-coupled receptor 20 (GPR 20); lymphocyte antigen 6 complex gene locus K9 (LY 6K); olfactory receptor 51E2 (OR 51E 2); tcrγ alternate reading frame protein (TARP); a wilms tumor protein (WT 1); ETS translocation variable gene 6 (ETV 6-AML); sperm protein 17 (SPA 17); x antigen family member 1A (XAGE 1); angiogenin binds to cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-associated antigen 1; a p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; a melanoma inhibitory agent of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS 2) ETS fusion gene); n-acetylglucosaminyl transferase V (NA 17); pairing box protein Pax-3 (Pax 3); androgen receptor; cyclin B1; a V-myc avian myeloblastosis virus oncogene neuroblastosis derived homolog (MYCN); ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP 1B 1); CCCTC binding factor (zinc finger protein) like (BORIS); squamous cell carcinoma antigen 3 (SART 3) recognized by T cells; pairing box protein Pax-5 (Pax 5); the proaacrosin binding protein sp32 (OYTES 1); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX 2); CD79a; CD79b; CD72; leukocyte associated immunoglobulin-like receptor 1 (LAIR 1); an Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA 2); CD300 molecular-like family member f (CD 300 LF); c lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2 (BST 2); containing EGF-like module mucin-like hormone receptor-like 2 (EMR 2); lymphocyte antigen 75 (LY 75); glypican-3 (GPC 3); fc receptor like 5 (FCRL 5); immunoglobulin lambda-like polypeptide 1 (IGLL 1). Preferably, the tumor antigen is CLDN18A2, GPC3, BCMA or CD19.

The pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigen is selected from: giant cell virus antigen, epstein-barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.

Provided herein is a method of genetically engineering TIGIT-CARs expressed by autologous or allogeneic cells, such as T cells, for use against NK cell killing, thereby providing a means of increasing the persistence and/or the survival rate of transplantation of autologous or allogeneic first immune cells in the presence of host second immune cells. For clarity, the "host" is the recipient of the "first immune cell", e.g., subject, patient, etc.; the "first immune cell" is any one of the other immune cells in the host other than the "first immune cell" after being engineered and transplanted into the host, and is called a "second immune cell". The "first immune cell" and the "second immune cell" may be cells derived from the same individual or may be allogeneic cells. In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR. In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR and the endogenous TIGIT is knocked out using gene editing techniques. 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 techniques. 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 techniques.

In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR, which cell is also genetically engineered to express at least one non-TIGIT-targeted chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or a combination thereof). In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or combination thereof) that is not targeted to TIGIT, and the endogenous TIGIT is knocked out using gene editing techniques. In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one non-TIGIT-targeted chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or combination thereof), and endogenous B2M and TCR are knocked out using gene editing techniques. In some embodiments, the first immune cell is genetically engineered to express a TIGIT-CAR, the cell is also genetically engineered to express at least one non-TIGIT-targeted chimeric receptor (CAR, modified TCR, TFP, TAC, aTCR, or combination thereof), and endogenous tigis, B2M, and TCRs are knocked out using gene editing techniques.

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

The application includes, for example, chinese patent application publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN 108884459A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176A, CN109880803A, CN 110055275A, CN110123837A, CN 110438082A, CN 110468105a international patent application publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018/018958A1, WO2014180306A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A1, WO2018108106A1, WO 2018/219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/141270, WO 2019/149979, WO 2019/147 A1, WO 2019/210863, WO2019/219029, and methods of making those CAR-T cells.

When constructing CAR-T cells, chimeric antigen receptor targeting NK cell surface inhibitory receptor TIGIT was simultaneously expressed on both TRAC and B2M gene double knockout T cells. The strategy can avoid the attack of host NK cells on T cells with TRAC and B2M genes knocked out, and prolong the survival time of the general T cells in the host.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Examples

Example 1 cytokine activation leads to an increase in tigit+nk cell ratio

PBMC were isolated from donor peripheral blood by density gradient centrifugation using Ficoll-Paque (GE bioscience) and NK cells were isolated using the NK cell isolation kit (purchased from Methaemal-Twai). 36% NK cell surface expressed TIGIT was detected by flow cytometry using an APC-TIGIT (Invitrogen) antibody (see FIG. 1A).

TABLE 2

After in vitro incubation for 24 hours with IL-2 (250U/ml), IL-15 (150U/ml), IL-12 (10 ng/ml) added separately or in combination to the above isolated NK cell culture medium according to Table 2, the percentage of TIGIT+NK cells was detected using the APC-TIGIT (Invitrogen) antibody.

The flow cytometry detection results are shown in FIG. 1, and the ratios of TIGIT+NK cells under the action of the single cytokines IL-12 and IL-15 are respectively 35.5% (see FIG. 1B) and 37.5% (see FIG. 1C); the individual cytokines IL-2 or combinations of two or three significantly increased the proportion of TIGIT+NK cells, corresponding to the ratio of TIGIT+NK cells, such as shown in FIGS. 1D-1H, of 69.7% (IL-2), 68.9% (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), respectively.

PBMC were isolated from peripheral blood of 5 donors by density gradient centrifugation using Ficoll-Paque (GE bioscience) and NK cells were isolated using NK cell isolation kit (purchased from Methaemal and Twai). IL-2 (500U/ml) and IL-15 (150U/ml) were added in combination to NK cell culture medium for 10 days, and the percentage of TIGIT+NK cells was detected by flow cytometry using an APC-TIGIT (Invitrogen) antibody.

The results of flow cytometry assays are shown in FIG. 2, where the percentage of TIGIT+NK cells in NK cells of 5 donors after cytokine activation was 86.1%, 76.2%, 74.1%, 38.3% and 55%, respectively. The proportion of tigit+nk cells in 3 out of 5 cases was over 70%.

The result suggests that: TIGIT proteins are expressed on resting NK cells and activated NK cells.

EXAMPLE 2 preparation of CAR-T cells

tigit-CAR-T cell preparation

Engineered T cells comprising TIGIT-targeted CARs were constructed and their resistance to NK cells was observed.

Expression vectors were constructed using methods conventional in the art of molecular biology (FIG. 3). A vector TIGIT-CAR comprising a chimeric antigen receptor of CD8 alpha signal peptide (SEQ ID NO: 38), anti-TIGIT single chain antibody (VH has the amino acid sequence shown in SEQ ID NO:1, VL has the amino acid sequence shown in SEQ ID NO: 2), CD8 alpha hinge region (amino acid sequence shown in SEQ ID NO: 40), CD28 transmembrane region (amino acid sequence shown in SEQ ID NO: 42), and T cell activator CD3 delta (amino acid sequence shown in SEQ ID NO: 46) was designed and constructed, and the lentivirus formed by packaging thereof was named PRRL-TIGIT.

And (3) performing density gradient centrifugation by using Ficoll-Paque (GE bioscience), separating mononuclear cells (PBMC) from human peripheral blood, and adding anti-CD 3/CD28 magnetic beads for in vitro activation to obtain T cells. And infecting the T cells with the lentivirus PRRL-TIGIT, and culturing and amplifying the T cells 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, the cell density was adjusted to 2X 10≡7/mL. TRAC and B2M double gene knockout was performed on TIGIT CAR-T cells. According to the instructions for the reagents (GeneArt) ^TM Precision gRNA Synthesis Kit, thermo Tisher) in vitro to synthesize sgRNA sequences targeting TRAC and B2M. Cas 9 enzyme (purchased from NEB) and sgRNA were prepared at 1:4, incubating at room temperature for 10 minutes to obtain an RNP complex, wherein the TRAC-gRNA (gRNA for targeted TRAC knockout) has a nucleic acid sequence shown in SEQ ID NO:23, the nucleic acid sequence of the B2M-gRNA (for targeted knockout of the B2M gRNA) is set forth in SEQ ID NO: shown at 24. 1 x 10≡6 TIGIT CAR-T cells were mixed with RNP complex (final concentration of Cas 9 enzyme 2. Mu.M), and RNP complex containing TRAC-gRNA and B2M-gRNA was introduced into TIGIT CAR-T cells using a maxcyte electrotransport. On day 4 after electrotransformation, gene knockdown was detected using flow cytometry.

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

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

In vitro amplifying UTD cells of single TRAC, TIGIT CAR-T cells of double knockout B2M and TRAC, UTD cells of double knockout B2M and TRAC, regulating the cell density to 1 x 10≡7/mL on 4 th day after electrotransformation, marking the cells by using an anti-PE-HLA-ABC antibody (Invitrogen) and a PE-B2M antibody (Invitrogen), sorting the marked cells by using anti-PE magnetic beads through a sorting column, and collecting TRAC negative cells, TRAC and B2M double negative cells (sorting kit is purchased from Methaemal day, so as to obtain more than 99% TRAC-deleted/UTD cells, TRAC and B2M double-deleted TIGIT CAR-T cells (TIGIT-UCAR-T cells), and TRAC and B2M double-deleted UTD cells (U-UTD cells).

The CAR-T cells are marked by using a Biotin marked goat anti-human Fab antibody (Jackson), then the CAR-T cells are marked by using a PE-strepitavidin secondary antibody, and the CAR expression condition of the TIGIT-UCAR-T cells is detected by using a flow cytometry, wherein the experimental result is shown in figure 4, and the positive rate can reach more than 80%.

The scFv against TIGIT is fully human and can be labeled with a universal anti-human Fab antibody. To sort TIGIT-UCAR-T cells, we used Biotin-labeled goat anti-human Fab antibodies (Jackson) to label CAR positive T cells. And then the PE-strepitavidin secondary antibody is used for marking, and after the marked cells are separated by a separation column by using PE-resistant magnetic beads, TIGIT-UCAR-T cells are collected. The positive proportion of the TIGIT-UCAR-T cells after screening reaches more than 95 percent.

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

PBMC are separated from peripheral blood of a donor by density gradient centrifugation using Ficoll-Paque (GE bioscience), NK cells in the PBMC cells are separated by an NK cell separation kit (purchased from Meitian and Twain), and the cell density is adjusted to 1 x 10 x 6/ml or 2 x 10 x 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 1X 10≡6/ml. According to the number proportion of NK cells to T cells of 1:1 or 2:1 were inoculated into 24-well plates and incubated in an incubator for 0hr,24hr and 48hr, respectively. NK cells were labeled with APC-CD56 (Invitrogen) antibody in the NK cell+TRAC-/-UTD group, and with APC-HLA-ABC antibody (Invitrogen) in the other mixed culture groups, the ratio and number of the T cells and the NK cells were measured at different time points of co-incubation, respectively.

As shown in FIG. 5, the ratio of U-UTD cells gradually decreased with time and the ratio of TIGIT-UCAR-T cells gradually increased when the cells were co-cultured with NK cells. TIGIT-UCAR-T cells have higher survival rates and survival numbers than U-UTD cells at NK: T ratios of 2:1 and 1:1.

The above results indicate that TIGIT-targeted CAR-T cells are effective against NK cell killing.

Example 4 preparation of double target UCAR-T cells targeting TIGIT and tumor antigen

To see if TIGIT-targeted CAR-T cells could enhance their anti-tumor activity in the presence of NK cells, we exemplified the construction of TIGIT-and CLDN18 A2-targeted CAR-T cells simultaneously, observing their effect on anti-tumor activity (targeting tumor antigens, e.g., CLDN18 A2) and on NK cell clearance (targeting non-tumor antigens, e.g., TIGIT).

According to the differences of the antibody tandem mode and the hinge domain connection mode of the double-targeting antigen (see table 3), three different forms of TIGIT and CLDN18A2 double-target CAR vectors are constructed.

TABLE 3 Table 3

Exemplary, single chain antibodies to CLDN18A2 (VH sequence shown in SEQ ID NO:11, VL sequence shown in SEQ ID NO: 12, HCDR1 sequence shown in SEQ ID NO:13, HCDR2 sequence shown in SEQ ID NO:14, HCDR3 sequence shown in SEQ ID NO:15, LCDR1 sequence shown in SEQ ID NO:16, LCDR2 sequence shown in SEQ ID NO:17, LCDR3 sequence shown in SEQ ID NO: 18), single chain antibodies to TIGIT (VH sequence shown in SEQ ID NO:1, VL sequence shown in SEQ ID NO:2, HCDR1 sequence shown in SEQ ID NO:3, HCDR2 sequence shown in SEQ ID NO:4, HCDR3 sequence shown in SEQ ID NO:5, LCDR1 sequence shown in SEQ ID NO:6, LCDR2 sequence shown in SEQ ID NO:7, LCDR3 sequence shown in SEQ ID NO: 8), G4S (SEQ ID NO: 60), (G4S) 3 (SEQ ID NO: 62), and linker1 (SEQ ID NO: 64).

Tandem fragment 1 (SEQ ID NO: 48), tandem fragment 2 (SEQ ID NO: 50), tandem fragment 3 (SEQ ID NO: 52) (see Table 3) were constructed with CD 8. Alpha. 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 CD3 delta (SEQ ID NO: 46) respectively as CAR1 fragment (SEQ ID NO: 54), CAR2 fragment (SEQ ID NO: 56), CAR3 fragment (SEQ ID NO: 58), and then inserted into vectors PRRLsin, PRRLsin-CAR1, PRRLsin-CAR2, lsin-CAR3 (FIG. 6), respectively, and packaged to form lentiviruses PRRL-CAR1, PRRL-CAR2, PRRL-CAR3, respectively.

Construction of a chimeric antigen receptor expressing CLDN18A2 lentiviral plasmid PRRLsin-CLDN18A2-CAR (fig. 6), comprising a single chain antibody to CLDN18A2 (SEQ ID NO:11, 12), a CD 8a signal peptide (SEQ ID NO: 38), a CD8 hinge region (SEQ ID NO: 40), a CD28 transmembrane region (SEQ ID NO: 42) and an intracellular domain (SEQ ID NO: 44), T cell activating factor cd3δ (SEQ ID NO: 46); packaging to form lentivirus PRRL-CLDN18A2-CAR.

CLDN18A2-CAR-T targeting CLDN18A2 was prepared as described in reference to example 2; CAR-T1 (CAR 1-expressing), CAR-T2 (CAR 2-expressing), CAR-T3 (CAR 3-expressing) cells that dual target TIGIT and CLDN18 A2. TRAC and B2M double knockdown cells were prepared as described in example 2: U-UTD (T cells with only TRAC and B2M knocked out) as a negative control, clDN18A2-UCAR-T targeting clDN18A2 only, UCAR-T1 (CAR 1-expressing), UCAR-T2 (CAR 2-expressing), UCAR-T3 (CAR 3-expressing) cells dual targeting TIGIT and clDN18A 2.

Taking 1×10 ⁶ Each UCAR-T cell described above was stained with PE-labeled anti-CLDN 18A2scFv antibody. Each UCAR-T cell positive rate was measured using flow cytometry (FIG. 7). The average fluorescence intensities of CLDN18A2-CAR, CAR1, CAR2, CAR3 on T cells were 21520, 13355, 15973 and 5338, respectively.

Example 5 detection of target cell killing by double target UCAR-T cells targeting TIGIT and tumor antigen

Human CLDN18A2 (SEQ ID NO:19, 20) is transfected with pancreatic cancer cells BXPC-3 (American ATCC) and gastric cancer cells HGC-27 (department of Chinese sciences cell bank) which are not expressed by endogenous CLDN18A2 respectively through lentiviruses by using conventional molecular biology technology, positive monoclonal is selected through a limiting dilution method, and BXPC-3-A2 and HGC-27-A2 stable transgenic cell lines which express the CLDN18A2 are constructed.

75. Mu.L of 2X 10 cells were inoculated in 96-well plates ⁵ BXPC-3-A2, HGC-27-A2 cells (target cells); adding effector cells U-UTD, CLDN18A2-CAR-T, CLDN A2-UCAR-T, UCAR-T1, UCAR-T2 and UCAR-T3 cells according to the effective target ratio of 3:1, 1:1 or 1:3 respectively, and setting an effector cell spontaneous LDH control hole, a target cell maximum LDH control hole, a volume correction control hole and a culture medium background control, wherein the number of the effector cells spontaneous LDH control holes, the target cell maximum LDH control holes and the culture medium background control are 4 multiple holes respectively; LDH release was detected 18h later using Cytotoxicity Detection Kit (LDH, roche) and killing of target cells by each group of cells was calculated (fig. 8).

The results show that three different forms of TIGIT & CLDN18A2 dual-target UCAR-T cells (UCAR-T1, UCAR-T2, UCAR-T3) did not significantly differ from CLDN18A2-UCAR-T in killing the target cells, and the dual-target CAR-T cells did not reduce killing of the target cells.

EXAMPLE 6 resistance of double-target UCAR-T cells targeting TIGIT and tumor antigen to PBMC cells

Double-target UCAR-T cells targeting TIGIT and tumor antigens are co-cultured with allogeneic PBMC cells, and whether the cells can resist killing of NK cells in the PBMC cells is observed.

TIGIT and CLDN18A2 dual-target UCAR-T cells were prepared as in example 4 and tested for CAR-T cell positive rate and TIGIT expression (fig. 9). Some U-UTD, CLDN18A2-UCAR-T cells express TIGIT protein on their surface, UCAR-T1, UCAR-T2, UCAR-T3 cells do not detect TIGIT protein. Taking 1×10 ⁶ U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells and 5X 10, respectively ⁶ PBMC cells were co-cultured and the proportion of U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells and the proportion of NK cells were examined at 0h, 24h and 48h of co-culture using PE-anti-HLA-ABC antibody (labeled U-UTD and UCAR-T cells) and APC-anti-CD 56 antibody (labeled NK cells in PBMC cells) (FIG. 10).

The results show that the proportion of the three forms of TIGIT and CLDN18A2 double-target UCAR-T cells and PBMC cells after being co-cultured for 48 hours is higher than that of U-UTD and CLDN18A 2-UCAR-T; after co-culture, NK cells in UCAR-T1, UCAR-T2 and UCAR-T3 groups are lower than those in U-UTD and CLDN18A2-UCAR-T groups. The results show that the TIGIT and CLDN18A2 double-target UCAR-T cells can effectively resist NK cell killing in PBMC cells, and the UCAR-T2 has the best effect of resisting NK cells.

EXAMPLE 7 therapeutic Effect of double target UCAR-T cells targeting TIGIT and tumor antigen on transplantation tumor

The target TIGIT and the tumor antigen targeted double-target UCAR-T can resist the foreign NK cells so as to keep higher survival rate. Further we examined the anti-tumor effect of the double-target UCAR-T cells in vivo.

Subcutaneous inoculation of HGC-27-A2 transplants (designated D0) on NPG in immunodeficient mice, each mouse was inoculated with 5X 10 ⁶ HGC-27-A2 cells, tumor volume was measured on day 15 and grouped, transplanted tumor volume was about 90mm3, 5 groups (U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3), 5 per group; tail vein injection is 1×10 respectively ⁶ U-UTD, CLDN18A2-UCAR-T, UCAR-T1, UCAR-T2, UCAR-T3 cells. After injection, body weight was measured 2 times per week (including the day of group dosing and euthanasia), tumor length and diameter were recorded by vernier caliper measurement, tumor volume was calculated, tumor growth curves were plotted from tumor volume, and differences in tumor growth curves between groups were compared (tumor volume: v=1/2×length×diameter) ² ). (FIG. 11A).

The results show that UCAR-T2 and UCAR-T1 treated groups have significant differences in the therapeutic effects of the transplanted tumors compared to U-UTD.

14 days after UCAR-T cell treatment, mouse peripheral blood was taken and the density of human CD4+ and CD8+ T cells in the mouse peripheral blood was examined.

The results are shown in FIG. 11B, where the CLDN18A2-UCAR-T cell treated group had a higher density of human CD4+, CD8+ T cells than the TIGIT & CLDN18A2 double-target UCAR-T cell treated group. The result is probably that the partial UCAR-T cells themselves express TIGIT protein, so that the double-target UCAR-T cells kill themselves, thereby reducing the number of human CD4+ and CD8+ T cells to a certain extent, and reducing the anti-tumor effect

EXAMPLE 8 TIGIT gRNA screening

Since some T cells also express TIGIT, to avoid the killing of CAR-T cells by CAR-T cells themselves, the TIGIT gene of CAR-T cells is further knocked out on the basis of the TRAC/B2M double knockout. For the TIGIT gene, we designed 12 pairs of gRNA1-12 (SEQ ID NOS: 25-36) targeting the TIGIT gene. And according to the instructions for the reagents (GeneArt ^TM Precision gRNA Synthesis Kit, thermo Tisher) in vitro to synthesize sgRNA sequences targeting TIGIT.

The T cells are activated by using CD3-CD28 antibody coated magnetic beads, and the T cells on the 5 th day of activation are electrotransformed by using a Maxcyte electrotransformation instrument. The electrotransformation system was 0.5. Mu.MCas 9 +2. Mu.M TIGIT sgRNA. On day 4 post-electrotransformation, each gRNA knockout efficiency was tested using the APC-TIGIT antibody. Specific experimental procedures are described with reference to example 2.

The results are shown in FIG. 12: the knockdown efficiencies of gRNA6 and gRNA10 were highest, 76% and 72%, respectively. Whereas gRNAs 11, 5, 7, 8 also enabled TIGIT knockouts.

In order to avoid the damage of TIGIT & CLDN18A2 double-target UCAR-T to self cells, the TIGIT gene of UCAR-T cells needs to be knocked out again on the basis of TRAC/B2M knocking out. On day 0, T cells were activated with CD3-CD8 antibody coated magnetic beads and after 48 hours lentiviruses PRRL-CLDN18A2-CAR, PRRL-CAR1, PRRL-CAR2, PRRL-CAR3 were infected respectively. After 24 hours, the solution was centrifuged. Continuing to culture until the 6 th day, adopting a Maxcyte electrotransfer instrument to double-knock out TRAC & B2M or triple-knock out TRAC & B2M & TIGIT genes, wherein an electrotransfer system comprises 1 mu M Cas9 (Kai organism) +2 mu M TRAC sgRNA+2mu M B M sgRNA, and 2.5 mu M Cas9 (Kai organism) +2 mu M TRAC sgRNA+2mu M B M sgRNA+6mu M TIGIT sgRNA; wherein the nucleotide sequence of the TIGIT sgRNA is shown in SEQ ID NO:30, the TRAC-gRNA has the nucleic acid sequence shown in SEQ ID NO:23, the nucleic acid sequence of the B2M-gRNA is shown as SEQ ID NO: shown at 24. Thus, TRAC and B2M double-gene knockout CLDN18A2-UCAR-T cells, 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 were prepared. Specific experimental procedures are described with reference to example 2.

The above CAR-T cells were stained with PE-labeled anti-CLDN 18A2scFv antibodies and each CAR-T cell positive rate was detected using flow cytometry. The CAR-T cell positive rate is shown in FIG. 13. Wherein the knockout efficiency of UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO and UCAR-T3-TIGIT KO cell TIGIT genes is respectively 84.3%, 85.2% and 84.7%.

Example 9 Effect of TIGIT gene knockout on CAR-T cell in vitro killing Activity

Knocking out the endogenous TIGIT gene may affect the killing of the target cells by the CAR-T cells. To assess the effect of knockout TIGIT genes on CAR-T in vitro killing activity we compared the in vitro killing activity of CLDN18A2-CAR-T, CLDN A2-CAR-T-TIGIT KO cells on target cells.

The T cells were activated by coating magnetic beads with CD3-CD28 antibody, and after 48 hours, PRRL-CLDN18A2-CAR lentivirus infection was performed on the activated T cells. Moi=10. Centrifuge after 24 hours after infection. Continuing to culture until the 6 th day, electrotransferring the CLDN18A2-CAR-T cells by using a Maxcyte electrotransfer instrument, wherein the electrotransfer system is 1.5 mu MCas9 +6mu M TIGIT sgRNA, and the nucleic acid sequence of the TIGIT sgRNA is shown as SEQ ID NO:30, CLDN18A2-CAR-T-TIGIT KO cells were obtained. After 72 hours of electric conversion, 1X 10 is taken ⁶ Cells, CAR-T cell positive rate and TIGIT positive cell fraction were detected using PE-anti-CLDN 18A2scFv antibody and APC-TIGIT antibody, respectively (fig. 14A). Specific experimental procedures are described with reference to example 2.

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

75. Mu.L of 2X 10 cells were inoculated in 96-well plates ⁵ BXPC-3-A2, HGC-27-A2 cells (target cells); adding effector cells UTD, CLDN18A2-CAR-T, CLDN A2-CAR-T-TIGIT KO cells according to the effective target ratio of 3:1, 1:1 or 1:3 respectively, and setting an effector cell spontaneous LDH control hole, a target cell maximum LDH control hole, a volume correction control hole and a culture medium background control, wherein 4 compound holes are respectively arranged; detection of LDH Release after 18h using Cytotoxicity Detection Kit (LDH, roche)And killing of target cells by each group of cells was calculated (fig. 14B).

The results show that knockout of the endogenous TIGIT gene does not affect the killing of the target cells by the CAR-T cells.

Example 10 therapeutic Effect of double target UCAR-T-TIGIT KO cells targeting TIGIT and tumor antigen on transplantation tumor

To investigate whether endogenous TIGIT expression affects the therapeutic effect of dual-target UCAR-T in example 7 on tumors, we performed the knockout of TIGIT gene in UCAR-T cells with reference to example 9 to observe the therapeutic effect of dual-target UCAR-T on transplanted tumors in case of TIGIT knockout.

On day 0, NPG mice were inoculated subcutaneously with HGC-27-A2 transplants, each with 5X 10 ⁶ HGC-27-A2 cells. Measuring tumor volume size and grouping on day 20, transplanting tumor volume about 200mm3, grouping 6 groups (U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO), 5 per group; tail vein administration 2×10 ⁶ U-UTD-TIGIT KO, CLDN18A2-UCAR-T, CLDN A2-UCAR-T-TIGIT KO, UCAR-T1-TIGIT KO, UCAR-T2-TIGIT KO, UCAR-T3-TIGIT KO cells. After injection, body weight was measured 2 times per week (including the day of group dosing and euthanasia), tumor length and diameter were recorded by vernier caliper measurement, tumor volume was calculated, tumor growth curves were plotted from tumor volume, and differences in tumor growth curves between groups were compared (tumor volume: v=1/2×length×diameter) ² ). (FIG. 15).

The results show that compared with the control group, the UCAR-T3-TIGIT KO with the endogenous TIGIT knocked-out has a significantly improved treatment effect on the transplanted tumor.

Because the TIGIT gene is knocked out, the UCAR-T cells are prevented from killing the UCAR-T cells, and the anti-tumor effect is obviously improved. Furthermore, UCAR-T3-TIGIT KO had significantly better therapeutic effects on HGC-27-A2 transplants than the control group, as compared to the control group in example 7.

Example 11 treatment effect of double target U-CAR T-TIGIT KO cells targeting TIGIT and tumor antigen on transplantation tumor in the Presence of NK cells

In order to investigate the in vivo anti-tumor effect of TIGIT and tumor antigen double-target U-CAR T cells in the presence of NK cells, we compared the therapeutic effect of CLDN18A2-UCAR T-TIGIT KO+NK, UCAR-T1-TIGIT KO+NK, UCAR-T2-TIGIT KO+NK and UCAR-T3-TIGIT KO on HGC-27-A2 transplants.

Day 0 NPG mice were inoculated subcutaneously with HGC-27-A2, 5X 10 each ⁶ And (3) cells. On day 13, HGC-27-A2 transplantable tumor volumes were measured at about 100mm ³ And 6 groups, wherein groups 1,3,4,5,6 are given 5X 10 ⁶ PBMC cells. Day 14, group 1 tail vein injection 5 x 10 ⁵ U-UTD-TIGIT KO cells, group 2 tail vein injection 5X 10 ⁵ CLDN18A2-UCAR T-TIGIT KO, group 3 tail vein injection 5×10 ⁵ CLDN18A2-UCAR T-TIGIT KO, group 4 tail vein injection 5×10 ⁵ U-CAR-T1-TIGIT KO cells, group 5 administration of 5X 10 ⁵ UCAR-T2-TIGIT KO cells, group 6 tail vein administration 5X 10 ⁵ UCAR-T3-TIGIT KO cells. After injection, body weight was measured 2 times per week (including the day of group dosing and euthanasia), tumor length and diameter were recorded by vernier caliper measurement, tumor volume was calculated, tumor growth curves were plotted from tumor volume, and differences in tumor growth curves between groups were compared (tumor volume: v=1/2×length×diameter) ² )。

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

The experimental data show that the dual-target CAR-T cell anti-tumor effect of targeting TIGIT and tumor antigen is better than that of the CAR-T cell of targeting tumor antigen alone under the condition of NK cell existence.

EXAMPLE 12 resistance of TIGIT-targeted CAR-T cells to NK cells

1. Preparation of TIGIT-BBZ-CAR T and TIGIT-28Z-CAR T cells reference example 2A CAR vector of TIGIT-BBZ and TIGIT-28Z was constructed. Fragments shown in Table 4 were inserted separately for construction of TIGIT-targeted CAR-T cells.

TABLE 4 chimeric antigen receptor

Wherein the single chain antibody against TIGIT (VH has an amino acid sequence shown in SEQ ID NO:1, VL has an amino acid sequence shown in SEQ ID NO: 2), the CD8 alpha hinge region (amino acid sequence shown in SEQ ID NO: 40), the CD8 transmembrane region (amino acid sequence shown in SEQ ID NO: 86) or the CD28 transmembrane region (amino acid sequence shown in SEQ ID NO: 42), the CD28 intracellular domain (amino acid sequence shown in SEQ ID NO: 44) or the CD137 intracellular domain (amino acid sequence shown in SEQ ID NO: 88), the T cell activating factor CD3 delta (amino acid sequence shown in SEQ ID NO: 46)

The nucleic acid fragments of TIGIT-BBZ and TIGIT-28Z in Table 4 are respectively inserted into lentiviral vectors to construct lentiviral plasmids PRRL-TIGIT-BBZ and PRRL-TIGIT-28Z (see figure 16), and the lentiviral vectors are respectively transferred into transfected 293T cells, and packed to obtain lentiviral TIGIT-BBZ and TIGIT-28Z, and the T cells are respectively infected to obtain TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells of targeted TIGIT.

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

TRAC and B2M double negative TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells were prepared by knocking out TRAC and B2M in TIGIT-BBZ-CAR T cells and TIGIT-28Z-CAR T cells, respectively, according to reference example 2. TRAC-/-UTD was prepared as a positive control by TRAC single gene knockout on UTD cells (T cells not transfected with virus). TRAC and B2M double-deleted UTD cells (U-UTD cells) served as negative controls.

The cell concentration is adjusted to 1 x 10 x 6/ml. According to the number proportion of NK cells to T cells of 1:1 were inoculated into 24-well plates and incubated in an incubator for 0hr,24hr and 48hr, respectively. NK cells were labeled with APC-CD56 (Invitrogen) antibody in the NK cell+TRAC-/-UTD group, and with APC-HLA-ABC antibody (Invitrogen) in the other mixed culture groups, the ratio of T cells and NK cells was varied at different time points of co-incubation, respectively.

As a result, as shown in FIG. 17, the ratio of TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells was gradually increased and the ratio of NK cells was gradually decreased as the co-culture time was prolonged, when the cells were co-cultured with NK cells. This suggests that TIGIT-targeted CAR-T cells are able to effectively resist NK cell killing.

Preparation of TIGIT-BBZ-UCAR T-TIGIT KO cells with TIGIT gene knocked out, TIGIT-28Z-UCAR T-TIGIT KO cells

TIGIT-BBZ-UCAR T cells and TIGIT-28Z-UCAR T cells were deleted, respectively, by the procedure of example 9, to prepare TIGIT-BBZ-UCAR T-TIGIT KO cells and TIGIT-28Z-UCAR T-TIGIT KO cells.

Example 13 TIGIT-targeted CAR-T cells enhance the resistance of tumor antigen-targeted CAR-T cells to NK cells and their antitumor activity

Taking CAR T cells targeting tumor antigen CLDN18A2 as an example, we detected in vitro whether TIGIT-targeting CAR-T cells could enhance survival and anti-tumor activity of tumor antigen-targeting CAR-T cells in the presence of NK cells.

Adjusting 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 1X 10 ⁶ /ml. Co-culture was performed according to the following ratios, 500. Mu.l of each cell was added. The co-cultivation time is 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

Detecting the ratio and number change of HGC-27-A2 tumor cells and CLDN18A2-UCAR T-TIGIT KO cells of the co-culture system. The results show that: the CLDN18A2-UCAR T-TIGIT KO cells ratio and cell number were higher in co-culture systems (2) and (3) compared to control (1), while the HGC-27-A2 cell ratio and cell number were significantly decreased.

The experimental result shows that the CAR-T cells targeting the TIGIT can obviously improve the survival of the CAR-T cells targeting the tumor antigen in the presence of NK cells, thereby improving the anti-tumor activity of the CAR-T cells.

EXAMPLE 14 TIGIT-targeting CAR-T cells enhance the resistance of tumor antigen-targeting CAR-T cells to NK cells and their in vivo anti-tumor Activity

In order to confirm that the combined targeting TIGIT-UCAR T-TIGIT KO can enhance the anti-tumor effect of the universal CAR T cells in the presence of NK cells, the therapeutic effects of CLDN18A2-UCAR T-TIGIT KO, CLDN18A2-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 on HGC-27-A2 transplantation tumors were compared with the example of CLDN18A2-UCAR T-TIGIT KO.

Day 0 NPG mice were inoculated subcutaneously with HGC-27-A2, 5X 10 each ⁶ And (3) cells. On day 13, HGC-27-A2 transplantable tumor volumes were measured at about 100mm ³ And 5 groups, wherein groups 1, 3, 4, 5 are given 5X 10 respectively ⁶ PBMC cells. Day 14, group 1 tail vein injection 5 x 10 ⁵ U-UTD-TIGIT KO cells, group 2 tail vein injection 5X 10 ⁵ CLDN18A2-UCAR T-TIGIT KO, group 3 tail vein injection 5×10 ⁵ CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ U-UTD-TIGIT KO cells, group 4 was given 5X 10 ⁵ CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ TIGIT-BBZ-UCAR-TIGIT-KO cells, 5 x 10 by tail vein administration of group 5 ⁵ CLDN18A2-UCAR T-TIGIT KO+5×10 ⁵ TIGIT-28Z-UCAR-TIGIT-KO cells. After injection, body weight was measured 2 times per week (including the day of group dosing and euthanasia), tumor length and diameter were recorded by vernier caliper measurement, tumor volume was calculated, tumor growth curves were plotted from tumor volume, and differences in tumor growth curves between groups were compared (tumor volume: v=1/2×length×diameter) ² )。

The results show that: the treatment effect of the 5 th group on HGC-27-A2 transplanted tumor is obviously better than that of the 3 rd group, which shows that the CAR-T cells targeting TIGIT can enhance the in vivo anti-tumor function of the CAR-T cells targeting tumor antigens in the presence of NK cells.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence:

Claims

a genetically engineered cell, wherein the cell expresses a first protein that recognizes TIGIT.
The cell of claim 1, wherein the first protein comprises 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 comprises a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.
A cell according to any one of claims 1 to 3, wherein the first protein is a CAR comprising:

(i) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, cd3δ;

(ii) An antibody that recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, and cd3δ;

(iii) An antibody that recognizes TIGIT, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and cd3δ; and/or

(iv) Antibodies that recognize TIGIT, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and cd3δ.
The cell of any one of claims 1-4, wherein the cell comprises: knock-out of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules.
The cell of claim 5, wherein the CRISPR/Cas9 technique is used to knock out the TIGIT gene of the cell and the gRNA used is selected from any one of the sequences set forth in SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or a combination thereof.
The cell of any one of claims 1-6, wherein the cell is selected from the group consisting of a T cell, an NK cell, a cytotoxic T cell, an NKT cell, a DNT cell, an NK92 cell, a macrophage, a CIK cell, and a stem cell derived immune effector cell, or a combination thereof.
The cell of any one of claims 1-7, wherein the cell is an autologous or allogeneic T cell, a primary T cell, or an autologous T cell derived from a human.
The cell of any one of claim 1 to 8, wherein the cell comprises,

knock-out of the gene encoding the TCR protein and/or low or no expression of endogenous TCR molecules, and/or

Knockout of genes encoding MHC proteins and/or endogenous MHC low expression or non-expression.
The cell of claim 9, wherein the endogenous MHC molecule B2M and the endogenous TCR are knocked out using CRISPR/Cas9 technology.
A cell according to claim 10, wherein the gRNA used to knock out B2M comprises the sequences set forth in SEQ ID NOS.24, 72, 73 and/or 74 and the gRNA used to knock out the TCR comprises the sequences set forth in SEQ ID NOS.23, 65, 66, 67, 68, 69, 70 and/or 71.
The cell of any one of claims 2-11, wherein the antibody that recognizes TIGIT comprises:

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

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

(iii) SEQ ID NO: 78.
The cell of any one of claims 1-12, wherein the first protein further recognizes a tumor and/or a pathogen; preferably, the tumor expression comprises BCMA, CD19, GPC3, CLDN18A2, EGFR, or a combination thereof.
The cell of claim 13, wherein the first protein comprises an antibody that recognizes TIGIT and an antibody that recognizes a tumor and/or pathogen antigen, linked in a manner comprising:

(i) Light/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes TIGIT-heavy/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes tumor and/or pathogen antigen-light/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes tumor and/or pathogen antigen;

(ii) Light chain (or light chain variable region) of an antibody that recognizes tumor and/or pathogen antigen-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT-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 tumor and/or pathogen antigen; 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 tumor and/or pathogen antigens-light chain (or light chain variable region) of an antibody that recognizes tumor and/or pathogen antigens-heavy chain (or heavy chain variable region) of an antibody that recognizes TIGIT,

wherein the first protein is a CAR, the CAR comprising:

(i) Antibodies recognizing TIGIT and antibodies recognizing tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, cd3δ;

(ii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ;

(iii) Antibodies that recognize TIGIT and antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iv) Antibodies recognizing TIGIT and antibodies recognizing tumor and/or pathogen antigens, the transmembrane region of CD28 or 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, comprising:

(i) SEQ ID NO:13, HCDR1, SEQ ID NO:14, HCDR2, SEQ ID NO:15, HCDR3, SEQ ID NO:16, LCDR1, SEQ ID NO:17, and/or the LCDR2 set forth in SEQ ID NO:18 LCDR3; or (b)

(ii) SEQ ID NO:11 and/or the heavy chain variable region set forth in SEQ ID NO:12, a light chain variable region as described in seq id no; or (b)

(iii) SEQ ID NO: 82.
The cell of any one of claims 13-15, wherein the first protein comprises SEQ ID NO: 48. 50, 52, 90 or 91.
The cell of any one of claims 1-16, wherein the cell further expresses a second protein that targets recognition of a tumor antigen, and/or a pathogen antigen, a chemokine receptor, a cytokine, an siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, a safety switch, or a combination thereof.
The cell of any one of claims 1-17, wherein the cell is capable of killing a host NK cell, or the cell is capable of resisting killing of the cell by an activated host NK cell when the cell is co-cultured with the host NK cell.
The cell of any one of claims 1-18, wherein the cell is administered in combination with an agent that enhances its 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.
The cell of claim 19, wherein the second protein comprises a CAR comprising:

(i) Antibodies that recognize tumors and/or pathogens, transmembrane region of CD28 or CD8, cd3δ;

(ii) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ;

(iii) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iv) Antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD28, the co-stimulatory signaling domain of CD137, and cd3δ.
The cell of claim 19 or 20, wherein the cell expressing the second protein comprises:

(i) Knock-out of the gene encoding TIGIT protein and/or low or no expression of endogenous TIGIT molecules; or (b)

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

(iii) Knockout of the 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 expresses the second protein:

(i) Knocking out TIGIT molecules by CRISPR/Cas9 technology;

(ii) Knocking out TCR and/or MHC molecule B2M by CRISPR/Cas9 technology; or (b)

(iii) TIGIT, TCR and MHC molecules B2M were knocked out using CRISPR/Cas9 technology.
The cell of any one of claims 19-22, wherein the cell expressing the second protein 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 a combination 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.
A method of increasing persistence and/or transplant survival of a first immune cell in the presence of a host second immune cell comprising:

a) Providing a first immune cell;

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

c) A polynucleotide encoding a first protein that targets TIGIT to modify the first immune cell.
The method of claim 25, wherein the polypeptide in step b) is selected from MHC, TCR, and/or TIGIT.
The method of claim 26, wherein step b) comprises:

(i) Knocking out TIGIT molecules by using CRISPR/Cas9 technology;

(ii) Knocking out TCR and/or MHC molecules B2M using CRISPR/Cas9 technology; or (b)

(iii) TIGIT, TCR and MHC molecules B2M were knocked out using CRISPR/Cas9 technology.
The method of claim 27, wherein step b) comprises:

(i) The gRNA used to knock out the TIGIT molecule is selected from the group consisting of the sequences shown in SEQ ID NOs 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and/or 36;

(ii) gRNAs used to knock out TCRs include the sequences shown in SEQ ID NOs 23, 65, 66, 67, 68, 69, 70 and/or 71; and/or

(iii) The gRNA used to knock out MHC molecule B2M includes the sequences shown in SEQ ID NO 24, 72, 73 and/or 74.
The method of any one of claims 25-28, wherein the first protein comprises 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 comprises:

(i) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD28, and cd3δ; and/or

(ii) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signal domain of CD137, and cd3δ; and/or

(iii) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and cd3δ;

(iv) Antibodies that recognize TIGIT, optionally antibodies that recognize tumors and/or pathogens, the transmembrane region of CD28 or CD8, and cd3δ.
The method of claim 25, wherein the first protein comprises:

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

(ii) SEQ ID NO:82, CLDN18 A2;

(iii) SEQ ID NO: 48. 50 or 52 and CLDN18A2 antibody tandem sequences; or (b)

(iv) SEQ ID NO: 9. 54, 56, 58, 90 or 91.
The method of any one of claims 25-31, further comprising step d) modifying the first immune cell with a non-endogenous polynucleotide encoding a second protein that targets a tumor antigen and/or a pathogen antigen and/or a viral antigen, a chemokine receptor, a cytokine, an siRNA that reduces expression of PD-1, a protein that blocks binding of PD-L1 to PD-1, or a safety switch.
The method of any one of claims 25-32, wherein the first immune cell is selected from the group consisting of a T cell, NK cell, cytotoxic T cell, NKT cell, macrophage, CIK cell, and stem cell derived immune effector cell, or a combination thereof.
The method of any one of claims 25-33, wherein the first immune cell is an autologous or allogeneic T cell, a primary T cell, or a human derived autologous T cell.
An engineered cell prepared by the method of any one of claims 25-34.
A polynucleotide encoding a nucleic acid molecule that constructs the cell of any one of claims 1-24, 35 or encoding a nucleic acid molecule required for administration of the method of any one of 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 of any one of claims 1-24, 35, the polynucleotide of claim 36, the vector of claim 37, the virus of claim 38.
A method of treating an inflammatory disorder, a viral infection, and/or a tumor comprising administering to a subject in need thereof the cell of any one of claims 1-24 or 35 or the composition of claim 39.