WO2024193714A1 - Cell immunotherapy composition and method - Google Patents

Abstract

Provided are a fusion protein having a function of preventing transplantation rejection and an engineering cell expressing the fusion protein. The present invention further relates to a method against transplantation immunological rejection, in particular to a method against NK cell immunological rejection.

Description

Compositions and methods for cellular immunotherapy

References to sequence listings

This specification is submitted together with the sequence listing, the file name of the sequence listing is 350A001WO02.XML, the size is 49,152 bytes, and it was created on March 25, 2024; the full text of which is incorporated herein by reference.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. CN202310294098.5 filed on March 23, 2023, which is fully incorporated herein by reference.

Technical Field

The present invention relates to a fusion protein, an engineered cell expressing the fusion protein, and a method for applying the fusion protein and the engineered cell thereof to immune cell therapy.

Background Art

Due to the immunogenetic differences between the donor and the recipient, when performing exogenous donor transplantation, as an exogenous graft, the donor may also be recognized and attacked by the immune cells in the recipient, thereby inhibiting or eliminating the exogenous graft and generating a host-versus-graft reaction (HVGR). By knocking out the MHC molecules in the graft cells, the host T cells’ rejection of the graft can be effectively resisted, but it may cause rejection reactions by other immune cells in the host. For example, in allogeneic cell transplantation, when the MHC-I class molecules of allogeneic cells are missing, it will lead to NK cell rejection in the host, enhancing the clearance of allogeneic cells. Therefore, how to effectively prevent the immune rejection of host NK cells is crucial to the development of allogeneic cell transplantation therapy.

Summary of the invention

The first aspect of the present invention provides a fusion protein, which includes, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular region includes a domain that binds to Fas ligand (FasL).

In one example, the domain that binds to FasL includes the extracellular segment of Fas or a fragment or variant thereof that can bind to FasL, an anti-FasL antibody or a fragment thereof, or a synthetic binding domain.

In one embodiment, the domain that binds to FasL comprises an anti-FasL antibody or a fragment thereof.

In one embodiment, an anti-FasL antibody or a fragment thereof comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 and VL1 respectively have (1) the amino acid sequences shown in SEQ ID NOs:16 and 17; (2) the amino acid sequences shown in SEQ ID NOs:18 and 17; (3) the amino acid sequences shown in SEQ ID NOs:20 and 21; (4) the amino acid sequences shown in SEQ ID NOs:22 and 21; (5) the amino acid sequences shown in SEQ ID NOs:24 and 25; (6) the amino acid sequences shown in SEQ ID NOs:26 and 27; or (7) the amino acid sequences shown in SEQ ID NOs:28 and 27.

In one embodiment, the VH1 and VL1 have the amino acid sequences shown in (1) SEQ ID NOs: 18 and 17, respectively.

In one example, the anti-FasL antibody is selected from: whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody or IgG4 antibody.

In one example, the anti-FasL antibody is a scFv. In one example, the anti-FasL antibody is a single domain antibody. In one example, the anti-FasL antibody is a DDPP antibody.

In one example, the FasL-binding domain includes the extracellular segment of Fas or a variant thereof that can bind to FasL.

In one embodiment, the extracellular segment of Fas comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 29.

In one embodiment, the FasL binding domain comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 19, 23, or 42.

The fusion protein provided by the present invention includes an intracellular signal transduction domain. In one example, the intracellular signal transduction domain includes an immunoreceptor tyrosine activation motif or an ITAM signal transduction motif.

In one example, the intracellular signal transduction domain includes an intracellular signal transduction domain selected from: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof.

In one embodiment, the intracellular signal transduction domain includes the CD3ζ intracellular signal transduction domain (SEQ ID NO: 7).

In one example, the intracellular signal transduction domain included in the fusion protein provided by the present invention further includes a co-stimulatory domain.

In one example, the co-stimulatory domain is selected from the intracellular signaling domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof.

In one embodiment, the co-stimulatory structure is selected from the intracellular signal transduction domain of CD137 (4-1BB) (SEQ ID NO: 6) or the intracellular signal transduction domain of CD28 (SEQ ID NO: 5).

The fusion protein provided by the present invention includes a transmembrane domain. In one example, the transmembrane domain is selected from: CD2, CD3ε, CD3δ, CD3ζ, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 The transmembrane domain of (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or Zap70.

In one embodiment, the transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 2).

In one embodiment, the transmembrane domain is the CD8 transmembrane domain (SEQ ID NO: 3).

In one embodiment, the fusion protein provided by the present invention includes a CD28 transmembrane domain (SEQ ID NO: 2), a CD3ζ intracellular signaling domain (SEQ ID NO: 7), and a CD28 intracellular signaling domain (SEQ ID NO: 5).

In one example, in the fusion protein provided by the present invention, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide.

In one example, the connecting polypeptide is selected from: CD28 hinge region, CD8 hinge region, IgG spacer region or fragments thereof, or a combination thereof.

In one example, the connecting polypeptide is a CD8 hinge region (SEQ ID NO: 10). In one example, the connecting polypeptide is an IgG1 spacer region (SEQ ID NO: 8). In one example, the connecting polypeptide is an IgG4 spacer region (SEQ ID NO: 9).

In one example, the extracellular domain of the fusion protein further includes a domain that binds to an immune cell marker other than FasL.

In one example, the fusion protein further comprises a chimeric receptor 1, wherein the chimeric receptor 1 binds to at least one or more immune cell markers different from FasL.

In one example, the immune cell marker is a T cell marker and/or a NK cell marker.

In one embodiment, the immune cell marker is NK inhibitory receptor (NKIR).

In one example, the immune cell marker is selected from: NKG2/CD94, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, SIGLEC family members, Ly49 family members, or a combination thereof. In one example, the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

In one example, the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD2 44. CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

In one example, the immune cell marker is CD38. In one example, the immune cell marker is NKG2A.

In one example, the extracellular domain of the fusion protein further includes a domain that binds to an immune cell marker other than FasL, and the domain includes a ligand, a synthetic binding domain, an antibody or a fragment thereof that binds to the immune cell marker.

In one example, the extracellular domain comprises an antibody or a fragment thereof that binds to the immune cell marker.

In one example, the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one example, the anti-FasL antibody or fragment thereof comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment thereof binding to the immune cell marker comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).

In one embodiment, the extracellular domain of the fusion protein includes, from N-terminus to C-terminus, (1) VH1-VL1-VH2-VL2, (2) VL1-VH1-VL2-VH2, (3) VH1-VL1-VL2-VH2, (4) VL1-VH1-VH2-VL2, (5) VH1-VH2-VL2-VL1, (6) VL1- VH2-VL2-VH1, (7)VH1-VL2-VH2-VL1, (8)VL1-VL2-VH2-VH1, (9)VH2-VH1-VL1-VL2, (10)VL2-VH1-VL1-VH2, (11)VH2-VL1-VH1-VL2, or (12)VL2-VL1-V H1-VH2.

In one example, the extracellular domain includes, from N-terminus to C-terminus, VL1-VH2-VL2-VH1.

In one example, the extracellular domain includes, from N-terminus to C-terminus, VH1-VL2-VH2-VL1.

In one example, the extracellular domain includes, from N-terminus to C-terminus, VL2-VH1-VL1-VH2.

In one example, the extracellular domain includes, from N-terminus to C-terminus, VH2-VL1-VH1-VL2.

In one embodiment, the fusion protein wherein the extracellular domain comprises an antibody or a fragment thereof that binds to CD38, and the VH2 and VL2 respectively have (1) the amino acid sequences shown in SEQ ID NOs:34 and 35; (2) the amino acid sequences shown in SEQ ID NOs:36 and 37; (3) the amino acid sequences shown in SEQ ID NOs:38 and 39; or (4) the amino acid sequences shown in SEQ ID NOs:40 and 41.

In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively.

In one embodiment, the extracellular domain of the fusion protein includes an antibody or a fragment thereof that binds to NKG2A, and the VH2 and VL2 have (1) the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively; or (2) the amino acid sequences shown in SEQ ID NOs: 32 and 33.

In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively.

In one example, the extracellular domain of the fusion protein further includes a domain that binds to a target antigen on a pathological cell.

In one example, the extracellular domain comprises a ligand, a synthetic binding domain, an antibody or a fragment thereof that binds to the target antigen.

In one embodiment, the pathological cells are selected from malignant cells or infected cells.

In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases.

In one example, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis/dermatomyositis.

In one embodiment, the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.

The second aspect of the present invention provides an engineered cell, which comprises the fusion protein as described in the first aspect.

In one example, the engineered cell further comprises a chimeric receptor 1, wherein the chimeric receptor 1 binds to at least one or more immune cell markers other than FasL.

In one example, the immune cell marker is selected from: T cell markers and/or NK cell markers. In one example, the immune cell marker is NK inhibitory receptor (NKIR).

In one example, the immune cell marker is selected from: NKG2/CD94, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, SIGLEC family members, Ly49 family members, or a combination thereof. In one example, the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

In one example, the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD2 44. CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

In one embodiment, the chimeric receptor 1 also binds to a target antigen on a pathological cell.

In one example, the engineered cells also express chimeric receptor 2 that binds to a target antigen on a pathological cell.

In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases.

In one example, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis/dermatomyositis.

In one example, the engineered cells further include: low expression or no expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS and/or CD58.

In one example, the engineered cell further comprises: no expression of endogenous TCR and B2M.

In one example, the engineered cell further comprises: no expression of endogenous TCR/B2M/FAS.

In one example, the engineered cell further comprises: no expression of endogenous TCR/B2M/CD58.

In one example, the engineered cells are immune cells, neurons, epithelial cells, endothelial cells, stem cells or a combination thereof.

In one example, the immune cells are autologous or allogeneic cells selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells or a combination thereof.

In one example, the engineered cells are allogeneic T cells.

In one example, the engineered cells have a prolonged survival time or enhanced proliferation capacity in the presence of host immune cells.

In one example, the engineered cells have an inhibitory or killing function on the host's immune cells.

In one example, the engineered cells have an inhibitory or killing function on the host's T cells and NK cells.

In one example, the engineered cell can enhance the survival, proliferation, and killing of pathological cells by another engineered cell that is previously, simultaneously, or later introduced into a subject.

The third aspect of the present invention provides a polynucleotide, which is a nucleic acid molecule encoding the fusion protein constructed in the first aspect.

The fourth aspect of the present invention provides a vector comprising the polynucleotide as described in the third aspect.

The fifth aspect of the present invention provides a virus comprising the vector as described in the fourth aspect.

The sixth aspect of the present invention provides a pharmaceutical composition, which comprises an effective amount of the fusion protein described in the first aspect, the engineered cell described in the second aspect, the polynucleotide of the third aspect, the vector described in the fourth aspect, or the virus described in the fifth aspect, and a pharmaceutically acceptable carrier.

The seventh aspect of the present invention provides the use of the fusion protein described in the first aspect or the engineered cell described in the second aspect.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to prepare a drug for preventing or resisting transplant rejection.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to prevent or resist transplant immune rejection.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to prepare a drug for treating autoimmune diseases or inflammatory diseases.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to treat, prevent or improve an autoimmune disease or inflammatory disease in a subject in need thereof.

In one embodiment, the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis/dermatomyositis.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to prepare a drug for treating tumors.

In one example, the fusion protein described in the first aspect and the engineered cell described in the second aspect are used to treat, prevent or improve tumors in subjects in need thereof.

In one embodiment, the tumor is a solid tumor or a hematological tumor.

In one embodiment, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B show the expression of FasL in primary NK (Figure 1A), activated NK (aNK, Figure 1B).

Figures 2A-2B show the schematic structural diagrams of FasL-CAR (Figure 2A) and dual-target CAR (Figure 2B).

FIG3 shows the CAR positivity rate and cell viability of FasL-CAR-T.

Figure 4 shows the aNK killing ability, cytokine secretion and self-survival of FasL-CAR-T cells.

FIG5 shows the ability of FasL-CAR-T cells to kill primary NK cells and their own survival.

Figures 6A-6B show the expression of FasL and NKG2A on primary NK (Figure 6A) and aNK cells (Figure 6B).

Figure 7 shows the killing of aNK by dual-target CAR-T cells, cytokine secretion and their own survival.

FIG8 shows dual-target CAR-T cells killing primary NK cells.

FIG. 9 shows dual-target CAR-T cells killing primary NK.

FIG. 10 shows that endogenous FAS knockout improves CAR-T cell survival.

FIG. 11 shows that endogenous CD58 knockout improves CAR-T cell survival.

Figures 12A-12B show CAR expression on FAS-CAR-T cells (Figure 12A) and cell proliferation (Figure 12B).

FIG. 13 shows that FAS-CAR-T cells kill NK.

FIG. 14 shows the survival of FAS-CAR-T cells themselves.

Figures 15A-15B show the expression of CD38 on primary NK cells (Figure 15A) and aNK cells (Figure 15B).

FIG. 16 shows a diagram of the working state of T cells expressing FasL-CAR targeting NK cells.

DETAILED DESCRIPTION

The present invention relates to a cell which binds to Fas ligand (FasL) and has the function of resisting transplant rejection, and also relates to a method for resisting transplant immune rejection.

Unless specifically defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, wherein suitable methods and materials are described herein. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification, including definitions, shall prevail. In addition, unless otherwise provided, the materials, methods and embodiments of the present invention are illustrative only and are not intended to be limiting. According to the present disclosure, it should be understood by those skilled in the art that many changes or modifications can be made in the disclosed specific embodiments, and the same or similar results are still obtained without departing from the spirit and scope of the present invention. The present invention is not limited in scope to the specific embodiments described herein (which are only intended to be illustrative of various aspects of the present invention), and functionally equivalent methods and components are within the scope of the present invention. The present invention includes modifications and modifications to the subject matter of the present invention for various uses and conditions.

Unless otherwise indicated, the practice of the present invention will employ 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 fully explained in the literature.

In the present application, the description in range format is only for convenience and brevity and should not be regarded as an unchangeable limitation on the scope of the present application. Therefore, the description of the range should be considered to specifically disclose all possible sub-ranges and individual numerical values within the range.

definition

The term "about" refers to the usual error range of each value that is readily known to those skilled in the art. References to "about" values or parameters herein include embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X". In this article, "about" can be an acceptable error range in the technical field. For example, it can refer to a value or parameter within the range of ±10% of an "about" value or parameter, for example, about 5uM can include any number between 4.5uM and 5.5uM.

Fas (NCBI Entrez Gene: 355) is a TNF-receptor superfamily protein that participates in the proliferation of T cells and fibroblasts through the cell programmed necrosis signaling pathway. As used herein, "Fas" refers to the Fas gene or the protein encoded and any variants, derivatives or isoforms. The Fas polypeptide is a type I transmembrane protein, consisting of an extracellular domain (ECD), a transmembrane domain (TM) and an intracellular domain (ICD). The extracellular domain contains three cysteine-rich regions (CRD1, CDR2, CDR3), in which the CDR region binds to its ligand FasL; the intracellular domain ICD region contains: a calcium inducing domain (CID) and a death domain (DD). Human Fas has multiple isoforms produced by alternative splicing. For example, isoform 1 has 335 amino acids (Uniprot: P25445-1, SEQ ID NO: 42), which includes an extracellular domain (amino acids 26-173), a transmembrane domain (amino acids 174-190) and a cytoplasmic domain (amino acids 191-335). Its extracellular domain can specifically bind to FasL (Fas ligand). In one embodiment, the Fas-fragment includes at least 90% identity of the sequences shown in SEQ ID NO: 19, 23, 29, 42.

FasL (Fas ligand) (Gene ID: 356) is a member of the tumor necrosis superfamily and activates the apoptosis signaling pathway of target cells by binding to Fas. FasL is a type II transmembrane protein, including an intracellular proline-rich domain (PRD) at the N-terminus, a transmembrane domain (TM), a stalk region (SR) and a TNF homology domain (THD) at the C-terminus. Human FasL has at least two isoforms produced by alternative splicing, the first of which is selected as the canonical sequence, including 281 amino acids (Uniprot: P48023-1, SEQ ID NO: 1). In one embodiment, FasL includes at least 90% identity to the sequence shown in SEQ ID NO: 1.

Term "CD58": CD58 (Gene ID: 965), CD58 is expressed on the surface of a variety of cells, including hematopoietic cells and non-hematopoietic cells. CD58 binds to CD2 on T cells, which is very important for the recognition between T cells and antigen-presenting cells. When CD58 on target cells interacts with CD2 on T cells, it can promote the activation and expansion of T cells and NK cells, providing their cell adhesion ability.

The term "CD38" refers to CD38 (Gene ID: 952). The functions of CD38 include that it can act as a receptor to bind to CD31 on T cells, thereby activating T cells to produce various cytokines. CD38 can also act as a cyclic ADP nuclease, which can catalyze NAD+ to form ADP ribose and cyclic ADP ribose. It is expressed on the surface of many immune cells, including CD4 T, CD8 T, B lymphocytes and NK cells. CD38 plays a role in cell adhesion, signal transduction and calcium signaling.

Term "NKG2A": NKG2A (Gene ID: 3821), NKG2A is a type II transmembrane protein with a C-type lectin domain. NKG2A and CD94 form dimers on NK cells and act as inhibitory receptors of NK, and its ligand is HLA-E. NKG2A is generally expressed on NK cells and a small number of CD8 T cells.

The term "host-versus-graft reaction (HVGR)" generally refers to the following: due to the immunogenetic differences between the donor and the recipient (or host), during exogenous donor transplantation, the donor as an exogenous transplant will be recognized and attacked by the host's immune cells (such as NK cells), thereby inhibiting or eliminating the donor.

The term "graft-versus-host disease (GVHD)" generally refers to the following: due to the diversity of TCRs of exogenous transplanted donor T lymphocytes and incompatibility with the host's HLA molecules, donor T lymphocytes will recognize antigens on the host's normal tissues, proliferate and release a series of cytokines to attack host cells.

The term "antibody" is used in the broadest sense herein and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), domain antibodies, and antibody fragments thereof that can specifically bind to an antigen or antigenic determinant, as long as they exhibit the desired antigen binding activity. The term "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region; (v) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; (vi) single-chain Fv molecule antigen binding sites; (vii) bispecific single-chain Fv dimers; (viii) "dibodies" or "tribodies", multivalent or multispecific fragments constructed by gene fusion; and (ix) scFv genetically fused to the same or different antibodies. For example, the antibody is selected from: whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, scFv-Fc antibody or IgG4 antibody. In one example, the antibodies of the present invention include typical antibodies, scFv and combinations thereof, wherein, for example, DDpp is covalently linked (e.g., by a peptide bond or by a chemical linker) to the N-terminus of the heavy chain and/or light chain of a typical complete (full-length) antibody, or inserted into the H chain and/or L chain of a full-length antibody. For example, a DDpp antibody that recognizes FasL is prepared with reference to the preparation method of CN111727250A.

The terms "recognize", "bind", and "target" are used interchangeably and refer to selective binding to a target antigen. For example, binding to a target cell refers to binding to a target antigen (eg, a target molecule) on a target cell.

The term "scFv" refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., connected by a synthetic linker, such as a short flexible polypeptide linker), and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL. The antigen binding function of an antibody can be performed by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". The term "antigen binding unit" also includes any molecular structure containing a polypeptide chain having a specific shape suitable for and recognizing an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

The term "DDpp" refers to a target binding D domain (DD) polypeptide based on a non-traditional antibody structural scaffold. D domain polypeptides (DDpp) are characterized by high target binding affinity and a non-antibody structural scaffold. DDpp can be monovalent or multivalent. In some embodiments, DDpp is monospecific or multispecific. In other embodiments, monospecific and multivalent. In other embodiments, DDpp is multispecific and multivalent. For more information, see CN111727250A.

The term "variable region or variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in antibody antigen binding. The heavy chain variable domain (VH) and light chain variable domain (VL) of a natural antibody generally have similar structures, wherein each domain comprises four conserved FRs and three CDRs. A single VH or VL domain can confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains using a VH or VL domain from an antibody that binds to the antigen, respectively.

The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each region of an antibody variable domain whose sequence is hypervariable, and/or forms structurally defined loops ("hypervariable loops"), and/or contains residues that contact the antigen ("antigen contacts"). Typically, an antibody comprises six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).

The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

"Framework (FR)" refers to variable domain residues other than hypervariable region (CDR) residues. The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. In VH (or VL), CDR and FR sequences usually appear in the following order:

FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

The term "natural antibody" refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by a disulfide bond. From N-terminal to C-terminal, each heavy chain has a variable region (VH), which is also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), which is also referred to as a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) domain. The light chain of an antibody can be assigned to one of two types based on the amino acid sequence of its constant domain, referred to as κ (κ) and λ (λ).

The terms "whole antibody", "full length antibody" and "intact antibody" are used interchangeably and refer to a full-length antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein or including an intact full-length antibody with an antigen binding region.

The term "single domain antibody (sdAb)" may also be referred to as "VHH", or "VHH polypeptide", and comprises a variable VHH domain responsible for antigen recognition. Antigen binding of the VHH domain is mediated by three CDRs, flanked by four relatively constant framework regions (FRs). It refers to a type of antibody that lacks the antibody light chain and has only the heavy chain variable region, and is also called a nanobody because of its small molecular weight. In certain embodiments, the VHH may be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and/or FR4, or lacks one or two of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

The term "single domain antibody" refers to an antibody comprising all or part of the heavy chain variable domain, or all or part of the light chain variable domain. The single domain antibody may be a human single domain antibody.

The terms "monoclonal antibody", "monoclonal antibody" refer to an antibody obtained from a population of substantially homologous antibodies, that is, the antibody molecules comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or generated during the preparation of the monoclonal antibody preparation, which variants are usually present in small amounts. In contrast to polyclonal antibody preparations (which typically include different antibodies directed against different determinants (epitopes)), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the term "monoclonal" indicates that the nature of the antibody is obtained from a substantially homologous antibody population, and is not considered to require that the antibody be prepared by any particular method. For example, it can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.

The term "fully human antibody (or fully human antibody)" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from an antibody of non-human origin using a human antibody library or other human antibody encoding sequence. The definition of a fully human antibody explicitly excludes humanized antibodies containing non-human antigen binding residues. Fully human antibodies can be generated by phage display technology. Fully human antibodies can be produced by engineered strains and/or engineered cells.

The term "fusion polypeptide" or "fusion protein" refers to a fusion molecule formed by connecting DNA fragments from different sources, or cDNA or peptide segments corresponding to proteins. For example, the fusion protein includes any protein or protein domain that binds to FasL, including natural proteins or domains, and can also be an artificially synthesized protein or domain. For example, the fusion protein includes a secreted protein that binds to FasL. For example, the fusion protein includes a bispecific molecule or a multispecific molecule that binds to FasL. For example, the fusion protein includes a membrane protein that binds to FasL. For example, the fusion protein includes a synthetic binding domain that binds to FasL. For example, the fusion protein includes a D domain polypeptide (DDpp, CN111727250A). For example, the fusion protein includes an antibody or a fragment thereof that binds to FasL. For example, the fusion protein includes an extracellular domain, a transmembrane domain, and an intracellular domain. For example, the fusion protein includes, but is not limited to, a chimeric antigen receptor (CAR), a recombinant TCR receptor. For example, the extracellular domain of the fusion protein includes a binding domain that binds to FasL. For example, the binding domain includes a FasL antibody. For example, the binding domain includes a Fas polypeptide or a fragment thereof or a variant thereof. For example, the binding domain includes a synthetic binding domain that binds FasL. For example, the binding domain includes an artificially synthesized D domain polypeptide that binds FasL. For example, the extracellular domain includes the entire extracellular domain of Fas, or includes at least 175, 173, 166 or 162 amino acids at the N-terminus of Fas. For example, the extracellular domain also includes a connecting polypeptide. For example, the connecting polypeptide includes a multimerization domain. For example, the connecting polypeptide includes the self-assembly domain (AS) of FasL. For example, the connecting polypeptide includes a hinge region/spacer region. For example, the connecting polypeptide includes a CD8 hinge region, a CD28 hinge region or an IgG spacer region. For example, the connecting polypeptide includes an IgG1, 2, 3 or 4 spacer region or a fragment thereof. For example, the transmembrane domain is selected from the group consisting of CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD -L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, or the transmembrane domain of Zap70. For example, the intracellular domain includes an intracellular signaling domain of CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or a combination thereof.

The term "variant" refers to a different protein or polypeptide with respect to a protein or polypeptide having a specific sequence feature ("control protein" or "reference polypeptide"), having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions and/or additions compared to the control protein or reference polypeptide. The change in the amino acid sequence can be an amino acid substitution. The change in the amino acid sequence can be a conservative amino acid substitution. The functional fragment or functional variant of a protein or polypeptide maintains the basic structure and functional properties of the control protein or polypeptide.

The term "cell marker" is also called a cell surface molecule or a cell surface protein, and preferably is a molecule present on the surface of an immune cell membrane. For example, "markers of T cells and/or NK cells" refer to markers present in T cells or NK cells, respectively, or in both T cells and NK cells, including but not limited to: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD160, CD161CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, SLAMF7, and TIGIT. For example, NK cell markers are selected from: NKG2 receptor family, killer immunoglobulin-like receptor (KIR) family, natural cytotoxicity receptor (NCR), and/or other NK cell-specifically expressed antigens. The NKG2 receptor family includes NKG2A, NKG2D, and NKG2C. The KIR family includes KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, and KIR3DS1. NCR includes NKP30, NKP44, NKP46, and NKp80. Other NK cell-specifically expressed antigens include CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, and IL-15R.

The term "NK inhibitory receptor (NK inhibitory receptor, NKIR)" refers to a class of receptors on NK cells that can transduce killing inhibitory signals and inhibit the killing function of NK cells. Including HLA-specific and non-HLA-specific inhibitory receptors. Among them, HLA-specific inhibitory receptors include CD94, NKG2A and KIR. Non-HLA-specific NK inhibitory receptors include PD-1, Siglec7, LAIR1 and CD300A. NKIR includes, but is not limited to, immunoreceptor tyrosine-based inhibitory motifs (ITIM). For example, NKIR includes: NKG2/CD94 components, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, immune checkpoint inhibitors, SIGLEC family members, Ly49 family members, or combinations thereof. For example, the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94. For example, the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3. For example, the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8. For example, the NKR-P1 family member is selected from NKR-P1B and NKR-P1D. For example, immune checkpoint inhibitors include: (a) one or more antagonists of checkpoint molecules, which include PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, M ICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A/HLA-E or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab and pembrolizumab, or one or more of venetoclax, azacitidine, pomalidomide. For example, the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3. For example, the SIGLEC family member is selected from SIGLEC7 and SIGLEC9. For example, the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

The term "pathological cells" generally refers to any type of cell present in a patient that is believed to contribute to a worsening health condition, or to a malignant or infected cell that needs to be reduced or eliminated to obtain patient remission. The present invention relates to methods of novel adoptive immunotherapy strategies for treating diseases associated with the development of pathological cells, such as cancer (tumors), infections, and autoimmune diseases.

The term "tumor antigen" refers to an antigen that emerges or is overexpressed during the development and progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present invention refers to cancer. Hyperproliferative disorders are called cancer or tumors. Tumor antigens include solid tumor antigens and blood tumor antigens. The tumor described in the present invention is a solid tumor or a blood tumor.

The term "chimeric receptor" refers to a fusion molecule formed by connecting DNA fragments or corresponding cDNAs of proteins from different sources using genetic recombination technology, including an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs) and recombinant TCR receptors.

The term "chimeric antigen receptor" (CAR) includes at least one extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a co-stimulatory molecule. For example, the stimulatory molecule is from a ζ chain (such as CD3Z) that binds to a T cell receptor complex. For example, the intracellular signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and/or CD28. For example, polypeptide groups are connected to each other. For example, the intracellular signaling domain (or structural region) can be selected from the intracellular co-stimulatory domain of any one or more of the following polypeptides: CD27, CD28, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CD83.

The term "recombinant T cell receptor (recombinant TCR)" includes chimeric receptors derived from one or more TCR subunits. For example, a recombinant TCR includes an extracellular domain, a transmembrane domain, and a TCR intracellular domain of at least part of a TCR subunit, and the TCR subunit portion is effectively connected to an antigen binding domain. For example, the TCR subunits in the recombinant TCR are derived from CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ and/or TCRδ subunits. For example, the recombinant TCR can be integrated into a TCR/CD3 complex expressed on a T cell. For example, a recombinant TCR includes constant regions and intracellular domains of TCRα and TCRβ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, a recombinant TCR includes constant regions and intracellular domains of TCRγ and TCRδ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, the recombinant TCR comprises a CD3ζ, CD3ε, CD3γ or CD3δ subunit, and the extracellular domain of the subunit is operably linked to an antigen binding domain.

The term "primary signal domain" or "primary signal domain" regulates the initial activation of the TCR complex in a stimulating manner. On the one hand, the primary signal domain is triggered by the combination of, for example, the TCR/CD3 complex with the MHC molecule loaded with the peptide, thereby mediating T cell responses (including but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain that acts in a stimulating manner may include a signal transduction motif of an immunoreceptor tyrosine activation motif or an ITAM. For example, a fragment of the primary signal domain containing ITAM includes, but is not limited to, an intracellular signal transduction domain derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD79a, CD79b, CD278 and CD66d.

The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of a cell via a defined signaling pathway by producing a second messenger or by acting as an effector in response to such a messenger. An intracellular signaling domain may include the entire intracellular portion of a molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

The term "costimulatory signaling domain" or "costimulatory molecule": generally refers to the intracellular domain of a costimulatory molecule that is capable of binding to a cell stimulatory signaling molecule, such as TCR/CD3, in combination with a signal that results in T cell proliferation and/or upregulation or downregulation of key molecules. Costimulatory molecules are typically cognate binding partners on T cells that specifically bind to costimulatory ligands and mediate co-stimulatory responses of T cells, including but not limited to proliferation. Costimulatory molecules are non-antigen receptor cell surface molecules or their ligands that are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).

The term "CD3ζ (also known as CD3 Zeta)" includes the protein provided by GenBank accession number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. "CD3ζ" is used interchangeably with "CD3z" and "CD3Z" in this application.

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. The classical TCR receptor is composed of two peptide chains, α and β, each of which can be divided into a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region. Its antigen specificity exists in the V region, and the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3. In one aspect, T cells expressing classical TCR can induce T cell TCR specific responses to target antigens by using methods such as antigen stimulation on T cells.

In one example, the fusion protein is a chimeric receptor. For example, the fusion protein is a CAR or a recombinant TCR. In one example, the fusion protein is used in combination with a chimeric receptor that binds to a pathological cell. In one example, the fusion protein is used in combination with a chimeric receptor that binds to an NK cell marker. In one example, the fusion protein is used in combination with a chimeric receptor that binds to a pathological cell and a chimeric receptor that binds to an NK cell marker. In one example, a chimeric receptor that binds to a pathological cell and an NK cell marker is used in combination with a fusion protein.

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

The term "host" or "subject" refers to a recipient of a transplant, for example, an individual, such as a human, into which exogenous cells are implanted. For example, a "subject" may be a clinical patient, a clinical trial volunteer, an experimental animal, or the like. The subject may be suspected of having a disease characterized by cell proliferation or may be diagnosed with a disease characterized by cell proliferation. For example, the subject may be suffering from or may be suffering from an immune disease such as an autoimmune disease, or may be suffering from a tumor, or may be suffering from an inflammatory disease.

The term "engineering" or "engineering" refers to the application of the principles and methods of cell biology and molecular biology, through some engineering means, at the level of the whole cell or the organelle level, to change the genetic material in the cell or obtain cell products according to people's wishes. For example, "engineering" refers to one or more changes in nucleic acids (such as nucleic acids in the genome of an organism). "Engineering" can refer to the change, addition and/or deletion of genes. "Engineered cells" can also refer to cells with added, deleted and/or changed genes.

In one example, the engineering cell is an immune cell, a neuron, an epithelial cell, an endothelial cell or a stem cell. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells). For example, the engineering cell is an immune cell. For example, the engineering cell is a primary cell. For example, the engineering cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, a NKT cell or a combination thereof. The engineering cell can be a cell of autologous or allogeneic origin. For example, the engineering cell is derived from human PBMC cells.

The term "immune cell" refers to a cell that participates in an immune response and produces an immune effect, such as a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a dendritic cell, a CIK cell, a macrophage, a mast cell, etc. For example, the immune cell is a T cell, an NK cell, or a NKT cell. For example, the T cell can be an autologous T cell, a heterologous T cell, or an allogeneic T cell. For example, the NK cell can be an autologous NK cell or an allogeneic NK cell. For example, the immune cell is obtained by sorting a donor's peripheral blood mononuclear cells (PBMC).

The term "T cell" may be a natural T cell obtained from PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and from infection sites, ascites, pleural effusion, spleen tissue, tumor tissue, or a cell population with specific phenotypic characteristics obtained through sorting, etc., or a mixed cell population with different phenotypic characteristics, such as "T cells" may be cells comprising at least one T cell subpopulation: memory 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, "T cells" may be T cells of a certain specific subtype, such as αβT cells and γδT cells. In some cases, T cells may be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as FicollTM separation and/or apheresis. For example, T cells are derived from induced pluripotent stem cells. For example, cells from circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. For example, the cells collected by apheresis can be washed to remove plasma molecules and the cells can be placed in a suitable buffer or culture medium for subsequent processing steps. The T cells can be derived from healthy donors, or from individuals diagnosed with cancer. T cells can be autologous T cells or allogeneic T cells. T cells can be primary T cells. In one example, "T cells" can also be T cells carrying a fusion protein that binds FasL. For example, CAR T cells, recombinant TCR-T cells.

The term "cell composition" generally refers to a combination of at least two types of cells, wherein the first type of cells can at least bind to FasL; the second type of cells bind to NK cell markers and/or target antigens on pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases). For example, each type of cell can be present in different containers, and can also be formulated into a desired preparation with a suitable adjuvant simultaneously or separately when necessary; each type of cell can be from different sources (e.g., prepared, produced or sold by different manufacturers; for example, naturally occurring T cells isolated from donors and T cells derived from stem cells, respectively); each type of cell can be prepared into an independent preparation (solid, liquid, gel, etc.); each type of cell can be present in a mixed form. The cell composition can also include an effective amount of antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells, and can also include a pharmaceutically acceptable carrier.

The term "MHC" stands for histocompatibility complex. In human cells, MHC is called HLA antigen and plays an important role in transplantation reactions, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.

The term "human leukocyte antigen" (HLA) is the encoding gene of the human major histocompatibility complex, which is closely related to the human immune system function. HLA includes class I, class II and class III gene parts. HLA class I is a heterodimer composed of a heavy chain (α chain) and a light chain β2 microglobulin (B2M). The term "B2M" is β-2 microglobulin, also known as B2M, which is the light chain of MHC class I molecules. HLA-II class genes include the HLA-D family, mainly HLA-DP, HLA-DQ and HLA-DR, etc., which are mainly distributed on the surface of professional antigen presenting cells such as B lymphocytes, macrophages and dendritic cells.

The term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function it has when overexpressed.

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

The terms "activation" and "activation" are used interchangeably and refer to the process by which cells change from a quiescent state to an active state. The process may include responses to phenotypic or genetic changes in antigens, migration and/or functional activity states. For example, the term "activation" may refer to the process by which T cells are gradually activated. The activation process is regulated by the first stimulation signal and the co-stimulation signal. The activation of T cells is a dynamically changing process, and its duration and degree of activation are affected by external stimulation. "T cell activation" or "T cell activation" refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production and/or detectable effector function. Using CD3/CD28 magnetic beads, in vitro antigen stimulation or in vivo antigen stimulation will affect the degree and duration of T cell activation. For example, the engineered T cells are co-incubated with tumor cells containing specific target antigens or activated after viral infection.

The term "Genome editing" refers to a genetic engineering technology that uses site-specific nucleases to insert, knock out, modify or replace DNA at a specific location in the genome of an organism to change the DNA sequence. Gene editing can be used to achieve precise and efficient gene knockout or gene knock-in. Gene knockout technology using nucleases includes CRISPR/Cas technology, ZFN technology, TALEN technology and TALEN-CRISPR/Cas technology, single base editing (Base Editor) technology, guide editing (Prime Editor) technology, and homing nuclease (Meganuclease) technology. The guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence, and the gRNA can guide the sequence-specific binding of the CRISPR complex to the target sequence. Whenever the sequence of gRNA is involved in this application, it can be a targeted DNA sequence, or it can be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA and crRNA and TracrRNA. gRNA is used to guide, bind or recognize the Cas enzyme. For example, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the 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. For example, endogenous TCR/B2M/FAS knockout or endogenous TCR/B2M knockout engineered cells are constructed using CRISPR technology. The gRNA sequences targeting TCR, B2M, FAS, and CD58 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

"Low expression" as described in this application means that the protein and/or RNA level expressed by the target gene in the engineered cell is lower than the expression level before the cell engineering treatment. For example, low expression of B2M, TCR, FAS, NKG2A or CD58 refers to a decrease in the expression of B2M, TCR, FAS, NKG2A or NKG2D ligands in the cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of a specific protein in the cell can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting or flow cytometry using specific antibodies. CRISPR library screening technology has been widely used in the screening of various biological problems, including screening for tumor resistance regulatory genes, screening for cell therapy product function enhancement genes, etc. This application uses large-scale library screening to identify the FAS-FADD-BID-BAK1 signaling axis, which may play an important role in resisting allogeneic immune cell applications.

The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

The terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid strand. For example, a nucleic acid sequence encodes an amino acid sequence. When referring to a nucleotide sequence, the "sequence" may include DNA or RNA, and may be single-stranded or double-stranded.

The term "subject" refers to any animal, such as a mammal or marsupial. The subject of the present invention includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

The term "peripheral blood mononuclear cell" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc. Density-based cell separation methods, for example, by lysing red blood cells or without lysing red blood cells and obtaining PBMCs by Percoll Ficoll gradient centrifugation of peripheral blood or a single sample or leukapheresis sample preparation.

The term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat an individual disease (cancer). The effective dose for therapeutic or preventive use depends on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose also depends on the selected active substance, 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 specific active substance, and the desired physiological effect. According to the judgment of the prescribing physician or a person skilled in the art, one or more rounds, or multiple administrations of the engineered cells of the present application may be required.

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

The term "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. It includes, but is not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. For example, it includes autonomously replicating plasmids or viruses. It also includes non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.

The term "modulate" refers to a positive or negative change. Examples of modulation include 1%, 2%, 10%, 25%, 50%, 75% or 100% change. In a specific embodiment, it refers to a negative change.

The term "treatment" refers to intervention measures that attempt to change the course of a disease, which can be either preventive or intervention in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or alleviating the condition, and alleviating or improving prognosis.

The term "prevention" refers to interventions that attempt to occur before disease develops (eg, rejection of a cell transplant).

The term "transplant immune rejection" refers to the process in which, after a host receives an allogeneic tissue, organ, or cell transplant, the foreign transplant is recognized by the host's immune system as a "foreign component" and an immunological response is initiated to attack, destroy, and remove the transplant. The present invention provides a cell and method for resisting transplant immune rejection.

The term "graft" refers to a biological material or preparation derived from an individual other than a host, which is used to be implanted into a host. The graft may be from any animal source, such as a mammalian source, preferably from a human. The graft may be from a host, such as cells from a host that are cultured in vitro or transformed and then implanted into the host. The graft may be from other individuals of the same species, such as cells from other people that are cultured in vitro or transformed and then implanted into the host. The graft may be from a xenogeneic individual, such as an organ from another species (such as a mouse, pig, or monkey) that is implanted into a human. Xenotransplantation includes, but is not limited to, vascularized xenotransplantation, partially vascularized xenotransplantation, non-vascularized xenotransplantation, xenodressing, xenobandage, and xenostructures.

The term "autologous" refers to originating from the same organism. For example, a sample can be removed from a subject (e.g., a cell), processed, and returned to the subject (e.g., a patient) at a later time. Autologous procedures are distinguished from allogeneic procedures in which the donor and recipient are different subjects.

The term "autologous transplantation" includes any procedure involving the transplantation, implantation or infusion of cells, tissues or organs into a recipient, wherein the subject and the donor are the same individual. Transplantation of cells, organs and/or tissues described herein can be used for autologous transplantation into humans. Autologous transplantation includes, but is not limited to, vascularized autologous transplantation, partially vascularized autologous transplantation, non-vascularized autologous transplantation, autologous dressings, autologous bandages and autologous structures.

The term "allogeneic transplantation" includes any procedure involving the transplantation, implantation or infusion of cells, tissues or organs into a subject, wherein the subject and the donor are different individuals of the same species. Transplantation of cells, organs and/or tissues described herein can be used for allogeneic transplantation into humans. Allogeneic transplantation includes, but is not limited to, vascularized allogeneic transplantation, partially vascularized allogeneic transplantation, non-vascularized allogeneic transplantation, allodressing, allobandage and allogeneic structures.

Fusion Protein

The present invention is based on the screening of cell libraries for resistant NK cells. Studies have found that among the genes related to resistant NK cells, the signal axis FAS-FADD-BID-BAK1 may play an important role.

The present invention provides a fusion protein targeting FasL, which has an anti-transplant rejection function. Engineered cells (such as T cells) expressing the fusion protein can resist transplant rejection, especially transplant rejection mediated by NK cells of allogeneic hosts. CART cells expressing the fusion protein can effectively kill NK cells of allogeneic hosts.

In one example, the fusion protein is a membrane protein targeting FasL. In one example, the fusion protein includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus, and the extracellular region includes a domain that binds to FasL. The domain that binds to FasL includes an extracellular segment of Fas or a fragment or variant thereof that can bind to FasL, an anti-FasL antibody or a fragment thereof, or a synthetic binding domain. Among them, the domain targeting FasL includes an antigen binding unit that binds to FasL.

In one example, the fusion protein includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus, wherein the extracellular region includes a domain that binds to FasL; the domain that binds to FasL includes an anti-FasL antibody or a fragment thereof. Any antibody described in the present invention or known in the art that has a high affinity for FasL can be used as the FasL-binding domain in the fusion protein of the present invention. In one example, the anti-FasL antibody is selected from: a whole antibody, scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody, or an IgG4 antibody. In one example, the anti-FasL antibody is a scFv. In one example, the anti-FasL antibody is a single domain antibody. In one example, the anti-FasL antibody is a DDPP antibody.

In one example, the anti-FasL antibody or fragment thereof comprises a heavy chain variable region (VH1) and a light chain variable region (VL1). In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 16, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 17. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 18, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 17. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 20, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 21. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 22, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 21. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 24, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 25. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 26, and VL CDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 27. In one embodiment, the VH1 includes VH CDR1, VH CDR2, and VH CDR3, and the VL1 includes VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are derived from VH having an amino acid sequence as shown in SEQ ID NO:28, and VL CDR1, VL CDR2, and VL CDR3 are derived from VL having an amino acid sequence as shown in SEQ ID NO:27.

In one embodiment, the anti-FasL antibody or fragment thereof includes VH1 and VL1. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 16 and 17, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 18 and 17, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 20 and 21, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 22 and 21, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 24 and 25, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 26 and 27, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 28 and 27, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto.

It is well known in the art that some amino acid substitutions can occur in the VH and VL region sequences while the CDR sequence remains unchanged and the affinity for the target is retained; and some amino acid substitutions can occur in the CDR sequence while the amino acids in contact with the target remain unchanged and the affinity for the target is retained.

In one example, the anti-FasL antibody or its fragment is a scFv (see Figure 2A). The VH (heavy chain variable region) and VL (light chain variable region) in the scFv can be connected by a connecting peptide chain, and the positions can be interchanged. In one example, the anti-FasL antibody includes, from the N-terminus to the C-terminus, VH, a connecting peptide chain, and VL. In one example, the anti-FasL antibody includes, from the N-terminus to the C-terminus, VL, a connecting peptide chain, and VH. Any connecting peptide chain that connects VH and VL together to form a functionally complete single-chain antibody is applicable. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO: 47), (GGGGS) 3 (SEQ ID NO: 48), or (GGGS) 4 (SEQ ID NO: 49).

In one embodiment, the FasL-binding domain of the extracellular region of the fusion protein includes the extracellular segment of Fas or a variant thereof that can bind to FasL. In one embodiment, the FasL-binding domain includes the extracellular segment of Fas (e.g., SEQ ID NO: 29). In one embodiment, the FasL-binding domain includes a variant of the extracellular segment of Fas that can bind to FasL, including at least 90%, at least 95%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO: 29.

In one embodiment, the FasL-binding domain comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 19. In one embodiment, the FasL-binding domain comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 23. In one embodiment, the FasL-binding domain comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 42.

The fusion protein of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus. When the intracellular signal transduction domain contains one or more signal transduction domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM), a kinase domain, a co-stimulatory domain, etc., it can directly promote cell responses. In other embodiments, the intracellular signal transduction domain will indirectly promote cell responses by associating with one or more other proteins, which in turn directly promote cell responses. In some embodiments, the intracellular signal transduction domain or its functional fragment can be from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2 or any combination thereof. The fusion protein provided by the present invention includes an intracellular signal transduction domain. In one example, the intracellular signal transduction domain includes a signal transduction motif of an immunoreceptor tyrosine activation motif or an ITAM.

In one example, the intracellular signal transduction domain includes an intracellular signal transduction domain selected from: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof. In one example, the intracellular signal transduction domain includes a CD3ζ intracellular signal transduction domain (SEQ ID NO: 7).

In one example, the intracellular signal transduction domain included in the fusion protein provided by the present invention further includes a co-stimulatory domain. In one example, the co-stimulatory domain is selected from the intracellular signal transduction domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof. In one example, the co-stimulatory structure is the intracellular signal transduction domain of CD137 (4-1BB) (SEQ ID NO: 6). In one example, the co-stimulatory structure is the intracellular signal transduction domain of CD28 (SEQ ID NO: 5).

The fusion protein of the present invention includes a transmembrane domain. In one example, the transmembrane domain is selected from: CD2, CD3ε, CD3δ, CD3ζ, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70 transmembrane domain. In one example, the transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 2). In one example, the transmembrane domain is a variant of the transmembrane domain of CD28 (SEQ ID NO: 2) with no more than three amino acid mutations. In one example, the transmembrane domain is the CD8 transmembrane domain (SEQ ID NO: 3). In one embodiment, the transmembrane domain is a variant of the CD8 transmembrane domain (SEQ ID NO: 3) with no more than three amino acid mutations.

In one example, in the fusion protein provided by the present invention, the extracellular domain and the transmembrane domain are connected by a connecting polypeptide.

In one example, the connecting polypeptide is selected from: CD28 hinge region, CD8 hinge region, IgG spacer or fragment thereof, or a combination thereof. In one example, the connecting polypeptide can be IgG1 spacer or fragment thereof, IgG2 spacer or fragment thereof, IgG3 spacer or fragment thereof, or IgG4 spacer or fragment thereof.

In one example, the connecting polypeptide is a CD8 hinge region (SEQ ID NO: 10). In one example, the connecting polypeptide is an IgG1 spacer region (SEQ ID NO: 8). In one example, the connecting polypeptide is an IgG4 spacer region (SEQ ID NO: 44). In one example, the connecting polypeptide is an IgG4 spacer region fragment (SEQ ID NO: 9).

In one example, the fusion protein includes a signal peptide. The signal peptide is a peptide sequence that is usually present at the N-terminus of newly synthesized proteins to guide them into the secretory pathway. The signal peptide is covalently linked to the N-terminus of the extracellular antigen binding domain of the CAR as a fusion protein. As is well known in the art, any suitable signal peptide can be applied to CAR to provide cell surface expression in immune cells (see Gierasch Biochem. 28: 923-930 (1989); von Heijne, J. Mol. Biol. 184 (1): 99–105 (1985)).

In one embodiment, the fusion protein provided by the present invention includes (1) a Fas extracellular region that binds to FasL (e.g., SEQ ID NO: 29) or a domain of an anti-FasL antibody (VH/VL selected from SEQ ID NOs: 16-18, 20-22, and 24-28), (2) a CD8 hinge region (SEQ ID NO: 10), an IgG1 spacer region (SEQ ID NO: 8) or an IgG4 spacer region fragment (SEQ ID NO: 9), (3) a CD28 transmembrane domain (SEQ ID NO: 2), (4) a CD28 intracellular signaling domain (SEQ ID NO: 5), and (5) a CD3ζ intracellular signaling domain (SEQ ID NO: 7). The extracellular region may include any of the FasL-binding domains mentioned above, such as the extracellular segment of Fas or an antibody that binds to FasL.

In one embodiment, the extracellular domain of the fusion protein provided by the present invention includes the extracellular segment of Fas (e.g., SEQ ID NO: 29). The fusion protein provided by the present invention includes, for example, Fas-CAR1, Fas-CAR2, Fas-CAR3, Fas-CAR4, Fas-CAR5, Fas-CAR6, and Fas-CAR7 in Table 1, and variants thereof.

For example, the fusion protein provided by the present invention includes, from N-terminus to C-terminus, Fas4 (1-173) (SEQ ID NO: 29), CD28 transmembrane domain (SEQ ID NO: 2), CD28 intracellular signaling domain (SEQ ID NO: 5) and CD3ζ intracellular signaling domain (SEQ ID NO: 7). In one example, the fusion protein provided by the present invention is Fas-CAR4 (Table 1), or a variant thereof.

In one example, the extracellular domain of the fusion protein provided by the present invention includes the domain of the anti-FasL antibody (VH/VL is selected from SEQ ID NOs: 16-18, 20-22, and 24-28). For example, the fusion protein provided by the present invention includes 1-28Z, 2-28Z, 3-28Z, 4-28Z, 5-28Z, 11-28Z, 12-28Z, and 10-28Z in Table 1, and variants thereof. The fusion protein variants in these examples include, but are not limited to, for example, a fusion protein obtained by swapping the positions of VH and VL in the scFv structure of the anti-FasL antibody part; a fusion protein obtained by replacing the connecting peptide chain with other connecting peptide chains; a fusion protein in which the anti-FasL antibody part is replaced with other antibody structures (such as Fab, single domain antibody, etc.); and a fusion protein in which the sequences of the VH and VL of the anti-FasL antibody part can undergo certain changes but do not affect the binding of the anti-FasL antibody to FasL; and so on. Those skilled in the art can obtain the above possible variants through common knowledge in the art and routine experiments.

In one example, the fusion protein includes a domain that binds to an immune cell marker different from FasL, and the domain can together with the domain that binds to FasL constitute the extracellular region of the fusion protein. In one example, the extracellular domain of the fusion protein also includes a domain that binds to an immune cell marker different from FasL.

In one example, the domain that binds to an immune cell marker includes a ligand, a synthetic binding domain, an antibody or a fragment thereof that binds to the immune cell marker. In one example, the extracellular domain includes an antibody or a fragment thereof that binds to the immune cell marker. In one example, the antibody or fragment that binds to the immune cell marker is selected from: a whole antibody, scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody. In one example, the antibody or fragment that binds to the immune cell marker is a scFv. In one example, the antibody or fragment that binds to the immune cell marker is a single domain antibody. In one example, the antibody or fragment that binds to the immune cell marker is a Fab fragment.

In one example, the antibody or fragment thereof that binds to the immune cell marker includes a heavy chain variable region (VH2) and a light chain variable region (VL2). In one example, the extracellular domain of the fusion protein includes an anti-FasL antibody or fragment thereof, and an antibody or fragment thereof that binds to the immune cell marker; wherein the anti-FasL antibody or fragment thereof includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the anti-immune cell marker antibody or fragment thereof includes a heavy chain variable region (VH2) and a light chain variable region (VL2). Different variable regions are connected by a connecting peptide chain. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO: 47), (GGGGS)3 (SEQ ID NO: 48), or (GGGS)4 (SEQ ID NO: 49).

The fusion protein extracellular domain can have different conformations, such as some of those shown in Figure 2B.

In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL1-VH2-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH1-VL2-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL1-VL2-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH1-VH2-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VH2-VL2-VL1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH2-VL2-VH1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL2-VH2-VL1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VL2-VH2-VH1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH2-VH1-VL1-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL2-VH1-VL1-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH2-VL1-VH1-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL2-VL1-VH1-VH2.

In one example, the immune cell marker is a NK inhibitory receptor (NKIR). In one example, the immune cell marker is selected from: NKG2/CD94, a KIR family member, a LIR family member, a NKR-P1 family member, an immune checkpoint receptor, a SIGLEC family member, a Ly49 family member, or a combination thereof. In one example, The NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4. For example, the natural cytotoxicity receptor (NCR) family includes: NKP30, NKP44, NKP46, NKp80, etc.

In one example, the domain that binds to an immune cell marker other than FasL is an immune checkpoint inhibitor, including: (a) one or more antagonists of checkpoint molecules, which include PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, ED O, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A/HLA-E or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab and pembrolizumab, or one or more of venetoclax, azacitidine, pomalidomide. For example, the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3. For example, the SIGLEC family member is selected from SIGLEC7 and SIGLEC9. For example, the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

In one example, the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD2 44. CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

In one example, the immune cell marker is CD38. In one example, the fusion protein includes an antibody or fragment against CD38. In one example, the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one embodiment, the anti-CD38 antibody or fragment comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). In one embodiment, the VH2 and VL2 have (1) the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively; (2) the amino acid sequences shown in SEQ ID NOs: 36 and 37, respectively; (3) the amino acid sequences shown in SEQ ID NOs: 38 and 39, respectively; or (4) the amino acid sequences shown in SEQ ID NOs: 40 and 41. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 36 and 37, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 38 and 39, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 40 and 41, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the anti-CD38 antibody or fragment is an scFv comprising the above-mentioned combination of VH2 and VL2, and VH2 and VL2 are connected by a connecting peptide chain. In one example, the anti-CD38 antibody or fragment comprises, from N-terminus to C-terminus, VH2, a connecting peptide chain, and VL2. In one example, the anti-CD38 antibody or fragment comprises, from N-terminus to C-terminus, VL2, a connecting peptide chain, and VH2. In one embodiment, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO:47), (GGGGS)3 (SEQ ID NO:48), or (GGGS)4 (SEQ ID NO:49).

In one embodiment, the anti-CD38 antibody or fragment thereof includes a variant of the above-mentioned scFv. For example, scFv can be replaced with other antibody structures (such as single domain antibodies (sdAb) etc.); and the sequences of VH2 and VL2 can be changed to a certain extent without affecting the binding of the anti-CD38 antibody to CD38; etc. Those skilled in the art can obtain the above-mentioned possible variants through common knowledge in the art and routine experiments.

In one example, the immune cell marker is NKG2A. In one example, the fusion protein includes an antibody or fragment against NKG2A. In one example, the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one embodiment, the anti-NKG2A antibody or fragment includes a heavy chain variable region (VH2) and a light chain variable region (VL2). In one embodiment, the VH2 and VL2 have (1) the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively; or (2) the amino acid sequences shown in SEQ ID NOs: 32 and 33. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 32 and 33, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the anti-NKG2A antibody or fragment is a scFv comprising the above-mentioned combination of VH2 and VL2, and VH2 and VL2 are connected by a connecting peptide chain. In one example, the anti-NKG2A antibody or fragment comprises, from N-terminus to C-terminus, VH2, a connecting peptide chain, and VL2. In one example, the anti-NKG2A antibody or fragment comprises, from N-terminus to C-terminus, VL2, a connecting peptide chain, and VH2. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO: 47), (GGGGS)3 (SEQ ID NO: 48), or (GGGS)4 (SEQ ID NO: 49).

In one example, the anti-NKG2A antibody or fragment thereof includes a variant of the above-mentioned scFv. For example, scFv can be replaced with other antibody structures (such as single domain antibodies (sdAb) etc.); and the sequences of VH2 and VL2 can be changed to a certain extent without affecting the binding of the anti-NKG2A antibody to NKG2A; etc. Those skilled in the art can obtain the above-mentioned possible variants through common knowledge in the art and routine experiments.

For example, the fusion proteins comprising the domains binding to NKG2A provided by the present invention include 6-28Z, 13-28Z, 14-28Z, 15-28Z in Tables 1 and 2, and variants thereof.

In one example, the fusion protein includes a domain that binds to a target antigen on a pathological cell. The domain that binds to a target antigen on a pathological cell can constitute the extracellular region of the fusion protein together with the domain that binds to FasL.

In one example, the extracellular domain of the fusion protein also includes a domain that binds to a target antigen on a pathological cell. In one example, the domain that binds to the target antigen includes a ligand that binds to the target antigen, a synthetic binding domain, an antibody or a fragment thereof. In one example, the extracellular domain includes an antibody or a fragment thereof that binds to the target antigen. In one example, the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one example, the extracellular domain of the fusion protein includes an anti-FasL antibody or a fragment thereof, and an antibody or a fragment thereof that binds to the target antigen; wherein the anti-FasL antibody or a fragment thereof includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the anti-target antigen antibody or a fragment thereof includes a heavy chain variable region (VH2) and a light chain variable region (VL2). Different variable regions are connected by a connecting peptide chain. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO: 47), (GGGGS)3 (SEQ ID NO: 48), or (GGGS)4 (SEQ ID NO: 49).

The fusion protein extracellular domain can have different conformations, such as some of those shown in Figure 2B.

In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL1-VH2-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH1-VL2-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL1-VL2-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH1-VH2-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VH2-VL2-VL1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VH2-VL2-VH1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH1-VL2-VH2-VL1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL1-VL2-VH2-VH1. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH2-VH1-VL1-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL2-VH1-VL1-VH2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VH2-VL1-VH1-VL2. In one example, the extracellular domain thereof includes, from N-terminus to C-terminus, VL2-VL1-VH1-VH2.

In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases. In one example, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; blood tumors are selected from: leukemia, lymphoma, myeloma; autoimmune diseases are selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (systemic lupus erythematosus, SLE), rheumatoid arthritis (rheumatoid arthritis, RA), ankylosing spondylitis (AS), Sjogren syndrome (Sjogren syndrome, SS), polymyositis/dermatomyositis.

In one embodiment, the target antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit α (IL-13Rα); interleukin 11 receptor α (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); cancer embryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7 related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell BCMA; CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of tumor necrosis zone; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphocyte ALK; polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); nephroblastoma protein (WT1 ); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 ( PAX3), androgen receptor, cyclin B1, V-myc avian myelocytotoxic viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OYTES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2) ; CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is BCMA or CD19. In one embodiment, the tumor antigen is selected from: the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.

In one example, the target antigen is a pathogen. In one example, the target antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites. In one example, the target antigen is a viral antigen. The viral antigen is selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.

In one example, the fusion protein also binds to a target antigen on a pathological cell. For example, the pathological cell includes a malignant cell or an infected cell. For example, the pathological cell is a solid tumor cell, a blood tumor cell, or a pathological cell of an autoimmune disease. In one example, the fusion protein that binds to FasL includes, from the N-terminus to the C-terminus: a Fas polypeptide fragment, an intracellular signaling domain; or a Fas polypeptide extracellular domain, a connecting fragment, a transmembrane domain, an intracellular signaling domain; or a synthetic domain, an antibody or a fragment thereof that binds to FasL, a connecting fragment, a transmembrane domain, and an intracellular signaling domain.

In one example, the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, CD94, FCRH5, EGFR and its mutants, or a combination thereof.

In one example, the fusion protein includes a domain that binds to FasL, a domain that binds to an immune cell marker, and a domain that binds to a target antigen on a pathological cell. These domains can be connected in sequence by a connecting peptide chain (for example, sequentially from the N-terminus to the C-terminus), or they can be cross-connected. For example, in one example, the extracellular domain of the fusion protein includes an antibody or fragment against FasL, an antibody or fragment against an immune cell marker, and an antibody or fragment against a target antigen on a pathological cell. In one example, these antibodies or fragments include VH and VL, respectively, and these VH and VL that bind to different targets can be arranged in sequence or cross-arranged to achieve simultaneous binding to multiple targets. Those skilled in the art can find out the extracellular domain sequence that achieves simultaneous binding to multiple targets through routine experiments.

In one example, the fusion protein includes, from N-terminus to C-terminus: a FasL binding domain, a transmembrane domain and an intracellular signaling domain, and optionally, the binding domain is connected to the transmembrane domain by a connecting polypeptide. For example, the binding domain of the fusion molecule is selected from: (1) an extracellular segment of a natural receptor that binds to FasL, a synthetic binding domain, an antibody or a fragment thereof, and one or more natural ligands (receptors), synthetic binding domains, antibodies or fragments thereof of immune cell markers other than FasL; (2) a natural receptor that binds to FasL, a synthetic binding domain, an antibody or a fragment thereof, and a natural ligand (receptor), synthetic binding domain, antibody or a fragment thereof of a target antigen on a pathological cell; (3) a natural receptor that binds to FasL, a synthetic binding domain, an antibody or a fragment thereof, one or more natural ligands (receptors), synthetic binding domains, antibodies or fragments thereof of an immune cell marker other than FasL, and a natural ligand (receptor), synthetic binding domain, antibody or a fragment thereof of a target antigen on a pathological cell. For example, the arrangement order of the two binding domains that bind to FasL and immune cell markers other than FasL can be adjusted. For example, the arrangement order of the three binding domains that bind to FasL, an immune cell marker different from FasL, and a target antigen on a pathological cell can be adjusted.

The sequences provided in the present application are not limited to the BiTEs having specific amino acid sequences as shown in SEQ ID NO: 16, 17, 18, 20, 21, 22, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and/or 41, which are modified on the basis of the amino acid sequence, and/or one or more amino acid substitutions, and/or deletions and/or additions of one or more amino acids. BiTEs having amino acid sequences that are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to the amino acid sequences shown in SEQ ID NO:16, 17, 18, 20, 21, 22, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and/or 41 and have the same function are also within the scope of protection of the present application.

Table 1 Examples of fusion proteins binding to FasL

Table 2 Chimeric receptors binding to immune cell markers

In Tables 1 and 2 above: αFasL_1H: VH of anti-FasL antibody 1 (SEQ ID NO: 16); αFasL_1L: VL of anti-FasL antibody 1 (SEQ ID NO: 17); αFasL_2H: VH of anti-FasL antibody 2 (SEQ ID NO: 18); αFasL_2L: VL of anti-FasL antibody 2 (SEQ ID NO: 17); αFasL_3H: VH of anti-FasL antibody 3 (SEQ ID NO: 19); SEQ ID NO:20); αFasL_3L: VL of anti-FasL antibody 3 (SEQ ID NO:21); αFasL_4H: VH of anti-FasL antibody 4 (SEQ ID NO:22); αFasL_4L: VL of anti-FasL antibody 4 (SEQ ID NO:21); αFasL_5H: VH of anti-FasL antibody 5 (SEQ ID NO: 24); αFasL_5L: anti-FasL VL of antibody 5 (SEQ ID NO:25); αFasL_6H: VH of anti-FasL antibody 6 (SEQ ID NO:26); αFasL_6L: VL of anti-FasL antibody 6 (SEQ ID NO:27); αFasL_7H: VH of anti-FasL antibody 7 (SEQ ID NO:28); αFasL_7L: VL of anti-FasL antibody 7 (SEQ ID NO:27) SEQ ID NO:27);αNKG2A_ αNKG2A_1H: VH of anti-NKG2A antibody 1 (SEQ ID NO:30); αNKG2A_1L: VL of anti-NKG2A antibody 1 (SEQ ID NO:31); anti-αNKG2A_2H: VH of anti-NKG2A antibody 2 (SEQ ID NO:32); αNKG2A_2L: VL of anti-NKG2A antibody 2 (SEQ ID NO:33); αCD38_1H: VH of anti-CD38 antibody 1 (SEQ ID NO:34); D NO:34); αCD38_1L: VL of anti-CD38 antibody 1 (SEQ ID NO:35); αCD38_2H: VH of anti-CD38 antibody 2 (SEQ ID NO:36); CD38_2L: VL of anti-CD38 antibody 2 (SEQ ID NO:37); αCD38_3H: VH of anti-CD38 antibody 3 (SEQ ID NO:38); αCD38_3L: VL of anti-CD38 antibody 3 (SEQ ID NO:39); αCD38_4H: VH of anti-CD38 antibody 4 (SEQ ID NO:40); αCD38_4L: VL of anti-CD38 antibody 4 (SEQ ID NO:41); (L): connecting peptide chain (e.g.: SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49); (S): signal peptide (e.g.: SEQ ID NO:4); CD8(H): C D8 hinge (SEQ ID NO:10); CD28 (TM): CD28 transmembrane region (SEQ ID NO:2); CD28 (C): CD28 intracellular domain (SEQ ID NO:5); CD3ζ (C): CD3ζ intracellular domain (SEQ ID NO:7); IgG1 (H): IgG1-spacer region (SEQ ID NO:8); IgG4 (H): IgG4-spacer region (SEQ ID NO:44); sIgG4(H): IgG4-spacer-short (SEQ ID NO:9); Fas1(1-335): Fas fragment 1 (SEQ ID NO:42); Fas2(1-225): Fas fragment 2 (SEQ ID NO:19)Fas3(1-190): Fas fragment 3 (SEQ ID NO:23); Fas4(1-173): Fas fragment 4 (SEQ ID NO:29).

The present invention also provides a polynucleotide encoding the fusion protein disclosed in the present invention. The present invention also provides a vector comprising the polynucleotide disclosed in the present invention. The present invention also provides a virus comprising the vector disclosed in the present invention.

Engineered cells

The present invention provides an engineered cell, which comprises the fusion protein disclosed in the present invention.

In one example, the engineered cell further comprises a chimeric receptor 1, wherein the chimeric receptor 1 binds to at least one or more immune cell markers other than FasL.

In one example, the immune cell marker is selected from: T cell markers and/or NK cell markers. In one example, the immune cell marker is NK inhibitory receptor (NKIR).

In one example, the immune cell marker is selected from: NKG2/CD94, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, SIGLEC family members, Ly49 family members, or a combination thereof. In one example, the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

In one example, the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD2 44. CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

In one example, the engineered cell provided by the present invention comprises the fusion protein disclosed in the present invention, and a chimeric receptor 1 comprising a domain that binds to NKG2A. In one example, the chimeric receptor 1 comprises an antibody or fragment against NKG2A. In one example, the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one embodiment, the anti-NKG2A antibody or fragment includes a heavy chain variable region (VH2) and a light chain variable region (VL2). In one embodiment, the VH2 and VL2 have (1) the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively; or (2) the amino acid sequences shown in SEQ ID NOs: 32 and 33. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 32 and 33, respectively, or each has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the anti-NKG2A antibody or fragment is a scFv comprising the above-mentioned combination of VH2 and VL2, and VH2 and VL2 are connected by a connecting peptide chain. In one example, the anti-NKG2A antibody or fragment comprises, from N-terminus to C-terminus, VH2, a connecting peptide chain, and VL2. In one example, the anti-NKG2A antibody or fragment comprises, from N-terminus to C-terminus, VL2, a connecting peptide chain, and VH2. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO: 47), (GGGGS)3 (SEQ ID NO: 48), or (GGGS)4 (SEQ ID NO: 49).

In one example, the anti-NKG2A antibody or fragment thereof includes a variant of the above-mentioned scFv. For example, scFv can be replaced with other antibody structures (such as single domain antibodies (sdAb) etc.); and the sequences of VH2 and VL2 can be changed to a certain extent without affecting the binding of the anti-NKG2A antibody to NKG2A; etc. Those skilled in the art can obtain the above-mentioned possible variants through common knowledge in the art and routine experiments.

For example, the engineered cells provided by the present invention include the fusion protein disclosed in the present invention, and a chimeric receptor 1 that binds to NKG2A, such as 7-28Z in Table 2, and variants thereof.

In one example, the engineered cell provided by the present invention comprises the fusion protein disclosed in the present invention, and a chimeric receptor 1 comprising a domain that binds to CD38. In one example, the chimeric receptor 1 comprises an anti-CD38 antibody or fragment. In one example, the antibody or fragment is selected from: a whole antibody, scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, an sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.

In one embodiment, the anti-CD38 antibody or fragment comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). In one embodiment, the VH2 and VL2 have (1) the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively; (2) the amino acid sequences shown in SEQ ID NOs: 36 and 37, respectively; (3) the amino acid sequences shown in SEQ ID NOs: 38 and 39, respectively; or (4) the amino acid sequences shown in SEQ ID NOs: 40 and 41. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one embodiment, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 36 and 37, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 38 and 39, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 40 and 41, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In one example, the anti-CD38 antibody or fragment is an scFv comprising the above-mentioned combination of VH2 and VL2, and VH2 and VL2 are connected by a connecting peptide chain. In one example, the anti-CD38 antibody or fragment comprises, from N-terminus to C-terminus, VH2, a connecting peptide chain, and VL2. In one example, the anti-CD38 antibody or fragment comprises, from N-terminus to C-terminus, VL2, a connecting peptide chain, and VH2. In one embodiment, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS (SEQ ID NO:47), (GGGGS)3 (SEQ ID NO:48), or (GGGS)4 (SEQ ID NO:49).

In one embodiment, the anti-CD38 antibody or fragment thereof includes a variant of the above-mentioned scFv. For example, scFv can be replaced with other antibody structures (such as single domain antibodies (sdAb)); and the sequences of VH2 and VL2 can be changed to a certain extent without affecting the binding of the anti-CD38 antibody to CD38; etc. Those skilled in the art can obtain the above-mentioned possible variants through common knowledge in the art and routine experiments.

For example, the engineered cells provided by the present invention include the fusion protein disclosed in the present invention, and a chimeric receptor 1 including a domain that binds to CD38, such as 8-28Z in Table 2, and variants thereof.

In one example, the engineered cell includes a fusion protein that binds to FasL and a chimeric receptor 1. The chimeric receptor 1 binds to at least one or more immune cell markers different from FasL. In one example, the engineered cell includes a fusion protein that binds to FasL and a chimeric receptor 1, wherein the fusion protein that binds to FasL includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain of a domain that binds to FasL; the chimeric receptor 1 includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain of a domain that binds to an immune cell marker. In one example, the fusion protein that binds to FasL and the chimeric receptor 1 are constructed on a carrier. In one example, the fusion protein that binds to FasL and the chimeric receptor 1 are connected by a hydrolyzable connecting peptide. In one example, the hydrolyzable connecting peptide is P2A (SEQ ID NO: 43). In one example, the hydrolyzable connecting peptide is T2A (SEQ ID NO: 11). In one example, the hydrolyzable connecting peptide is E2A (SEQ ID NO: 45). In one example, the hydrolyzable connecting peptide is F2A (SEQ ID NO: 46). In one example, the fusion protein binding to FasL and the chimeric receptor 1 are constructed on different carriers respectively. In one example, the chimeric receptor 1 includes a ligand, a synthetic binding domain, an antibody or a fragment thereof that binds to the immune cell marker. In one example, the chimeric receptor 1 includes an antibody or a fragment thereof that binds to the immune cell marker. In one example, the antibody or fragment that binds to the immune cell marker is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody. In one example, the antibody or fragment that binds to the immune cell marker is a scFv. In one example, the antibody or fragment that binds to the immune cell marker is a single domain antibody. In one example, the antibody or fragment that binds to the immune cell marker is a Fab fragment.

In one embodiment, the chimeric receptor 1 also binds to a target antigen on a pathological cell.

In one example, the engineered cells also express chimeric receptor 2 that binds to a target antigen on a pathological cell.

In one example, the engineered cell includes a fusion protein that binds to FasL and a chimeric receptor 2, which includes a domain that binds to a target antigen on a pathological cell. In one example, the fusion protein that binds to FasL and the chimeric receptor 2 are constructed on a carrier. In one example, the fusion protein that binds to FasL and the chimeric receptor 2 are connected by a hydrolyzable connecting peptide. In one example, the hydrolyzable connecting peptide is P2A (SEQ ID NO: 43), T2A (SEQ ID NO: 11), E2A (SEQ ID NO: 45), or F2A (SEQ ID NO: 46). In one example, the fusion protein that binds to FasL and the chimeric receptor 2 are constructed on different carriers.

In one example, the engineered cell includes a fusion protein that binds to FasL, a chimeric receptor 1, and a chimeric receptor 2, wherein the chimeric receptor 1 includes a domain that binds to the immune cell marker, and the chimeric receptor 2 includes a domain that binds to the target antigen on the pathological cell. In one example, the fusion protein that binds to FasL, the chimeric receptor 1, and the chimeric receptor 2 are constructed on one vector. In one example, the fusion protein that binds to FasL and the chimeric receptor 1 are constructed on one vector, and the chimeric receptor 2 is constructed on a different vector. In one example, the chimeric receptor 1 and the chimeric receptor 2 are constructed on one vector, and the fusion protein that binds to FasL is constructed on a different vector. In one example, the hydrolyzable connecting peptide is P2A (SEQ ID NO: 43), T2A (SEQ ID NO: 11), E2A (SEQ ID NO: 45), or F2A (SEQ ID NO: 46). In one example, the fusion protein that binds to FasL, the chimeric receptor 1, and the chimeric receptor 2 are constructed on different vectors, respectively.

In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases.

In one example, the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis/dermatomyositis.

In one embodiment, the present invention provides an engineered cell, which includes a polynucleotide encoding the present invention. In one embodiment, the present invention provides an engineered cell, which includes a vector encoding the present invention. In one embodiment, the present invention provides an engineered cell, which includes a virus encoding the present invention.

In one example, the engineered cells are immune cells, neurons, epithelial cells, endothelial cells, stem cells, or a combination thereof. In one example, the engineered cells are immune cells. In one example, the immune cells are autologous cells. In one example, the immune cells are allogeneic cells.

In one example, the immune cells are selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof.

In one example, the engineered cell is a T cell. In one example, the engineered cell is an allogeneic T cell. In one example, the engineered cell is a stem cell derived T cell. T cells can be cytotoxic T cells, helper T cells, or γδT, CD4+/CD8+ double positive T cells, CD4+T cells, CD8+T cells, CD4/CD8 double negative T cells, CD3+T cells, initial T cells, effector T cells, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like central memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells or γδT cells. In one example, T cells are cytotoxic T cells. In some embodiments, the genetically engineered T cells provided by the present invention are separated. In one example, the genetically engineered T cells provided by the present invention are substantially purified.

In one example, the engineered cells provided by the present invention are derived from cells isolated from a subject. As used in the present invention, genetically engineered cells derived from source cells refer to genetically engineered cells obtained by obtaining source cells and genetically manipulating the source cells. The source cells may be from natural sources. For example, the source cells may be primary cells isolated from a subject. The source cells may also be cells that have been passaged or genetically manipulated in vitro.

In one example, the genetically engineered cells provided by the invention are derived from cells separated from human body.Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue of infection site, ascites, pleural effusion, spleen tissue and tumor. In one example, T cell lines available in the art can be used. In one example, the genetically engineered cells provided by the invention are derived from cells separated from peripheral blood. In one example, the genetically engineered cells provided by the invention are derived from cells separated from bone marrow. In one example, the genetically engineered cells provided by the invention are derived from cells separated from peripheral blood mononuclear cells (PBMC).

In one example, the engineered cells provided by the present invention are derived from cells differentiated in vitro from stem cells or progenitor cells. In one example, stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem/progenitor cells, hematopoietic multipotent progenitor cells, embryonic stem cells and induced pluripotent cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from T cell progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from hematopoietic stem/progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from hematopoietic multipotent progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from embryonic stem cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from induced pluripotent cells.

Due to the immunogenetic differences between the donor and the recipient (or host), when an exogenous donor is transplanted, the donor as an exogenous transplant will be recognized and attacked by immune cells (such as NK cells) in the host, thereby inhibiting or eliminating the donor, resulting in a host-versus-graft reaction (HVGR). For example, in allogeneic cell transplantation, when the HLA-I class molecules of allogeneic cells are missing, the host CD8+-mediated cellular immune rejection can be reduced; in one example, the absence of HLA-I class molecules of allogeneic cells will enhance the host NK cell-mediated cellular immune rejection. In one example, the present invention provides engineered cells with low or no endogenous TCR and/or B2M expression.

Graft-versus-host disease (GVHD) is due to the diversity of TCRs of exogenous transplant donor T lymphocytes and incompatibility with host HLA molecules. Donor T lymphocytes recognize antigens on normal host tissues, amplify and release a series of cytokines, greatly enhance the immune response of the graft to host antigens, attack host cells, and knock out TCRs of exogenous transplant donors (e.g., T cells) to avoid graft-versus-host disease. In one example, the present invention provides engineered cells with low or no endogenous HLA-I/TCR expression. In one example, the present invention uses a CRISPR system to knock out the gene TRAC of the α chain of endogenous TCR to prepare cells with low or no endogenous TCR expression. In one example, the present invention uses a CRISPR system to knock out endogenous B2M to prepare cells with endogenous HLA-I deletion. In one example, the present invention provides cells with low or no expression of HLA-A and HLA-B and/or TCR. In one example, the present invention provides an engineered cell with low or no expression of endogenous HLA-I/TCR/and or HLA-II. In one example, the present invention provides an engineered cell with low or no expression of endogenous HLA-I/TCR/FAS. In one example, the present invention provides an engineered cell with low or no expression of endogenous HLA-I/TCR/CD58.

In one example, the engineering cell also includes: low expression or non-expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS and/or CD58. In one example, the engineering cell also includes low expression or non-expression of endogenous TCR and B2M. In one example, the engineering cell also includes low expression or non-expression of endogenous FAS. In one example, the engineering cell also includes: low expression or non-expression of endogenous CD58. In one example, the engineering cell also includes: low expression or non-expression of endogenous NKG2A. In one example, the engineering cell also includes: non-expression of endogenous TCR and B2M. In one example, the engineering cell also includes: non-expression of endogenous TCR/B2M/FAS. In one example, the engineering cell also includes: non-expression of endogenous TCR/B2M/CD58.

In one example, the present invention provides a method for transferring the polynucleotide provided by the present invention into a cell for genetic engineering by using gene editing. If necessary, techniques such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPRs), homologous recombination, nonhomologous end joining, microhomology-mediated end joining, homology-mediated end joining, etc. can be used to achieve site-specific integration (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011); Vasileva, et al. Cell Death Dis. 6: e1831. (Jul 23 2015); Sontheimer, Hum. Gene Ther. 26 (7): 413-424 (2015); Yao et al. Cell Research volume 27, 801-814 (2017)).

In one example, endogenous TCR/B2M, TCR/B2M/FAS, or TCR/B2M/CD58 is knocked out by CRISPR/Cas9 technology. In one example, the sgRNA sequences targeting TRAC, B2M, FAS, and CD58 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

For example, in some embodiments, the present invention provides an engineered T cell expressing a fusion protein, the engineered T cell including the non-expression of endogenous TCR/B2M, and optionally, the non-expression of FAS and/or CD58; the fusion protein includes, from the N-terminus to the C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain; wherein the extracellular region includes an extracellular domain that binds to an anti-FasL antibody or Fas disclosed herein, the transmembrane domain includes, for example, a transmembrane domain of CD28, wherein the intracellular signal transduction domain includes, for example, a CD3ζ intracellular signaling domain and a CD28 intracellular signaling domain. Its extracellular domain and transmembrane domain may be connected by, for example, a CD28 hinge region or an IgG spacer region or a fragment thereof. The fusion protein may also include a domain that binds to an immune cell marker other than FasL and/or a domain that binds to a target antigen on a pathological cell.

In one example, the engineering cell expressing the fusion protein of the present application can bind to the target cell expressing FasL. For example, the engineering cell of the present application can bind to NK cells. In one example, the engineering cell of the present application can bind to the target cell expressing FasL and an immune cell marker. For example, the engineering cell of the present application can bind to the target cell expressing FasL and an NK cell marker. For example, the engineering cell of the present application can bind to the target cell expressing FasL and NK inhibitory receptor. For example, the engineering cell of the present application can bind to the target cell expressing FasL and NKG2A. For example, the engineering cell of the present application can bind to the target cell expressing FasL and CD38. For example, the engineering cell of the present application can bind to the target cell expressing FasL and TIGIT. For example, the engineering cell of the present application can bind to the target cell expressing FasL and CS1. For example, the engineering cell of the present application can bind to the target cell expressing FasL and CD94. For example, the engineering cell of the present application can bind to the target cell expressing FasL and NKG2DL.

In one example, the engineered cells expressing fusion proteins of the present application do not cause host rejection of graft reactions. In one example, when there are host immune cells (e.g., NK cells), the engineered cells expressing fusion proteins of the present application have longer survival time and/or amplification capacity. For example, the engineered cells can kill the host's immune cells. For example, the engineered cells can kill the host's NK cells. For example, the engineered cells can kill allogeneic NK cells. For example, the engineered cells can resist the killing of host NK cells. For example, the engineered cells can resist the killing of allogeneic NK cells. For example, the engineered cells that do not express the fusion proteins of the present application have a significantly improved ability to kill pathological cells.

In one example, the engineered cells have a prolonged survival time or enhanced proliferation ability in the presence of host immune cells. In one example, the engineered cells have an inhibitory or killing function on the host's immune cells. In one example, the engineered cells have an inhibitory or killing function on the host's T cells and NK cells. In one example, the engineered cells have an inhibitory or killing function on the host's NK cells. In one example, the engineered cells can enhance the survival, proliferation, and killing of pathological cells of another engineered cell that is introduced into the subject previously, simultaneously, or later.

In one example, in the presence of host immune cells (e.g., NK cells), the dual-target engineered cells of the present application that express and bind FasL and NK cell markers (e.g., NKG2A, CD38, CD94, CS1, TIGIT, NKG2DL) have longer survival time and/or amplification ability. For example, the engineered cells have a stronger ability to kill host NK cells. For example, the engineered cells have a stronger ability to kill allogeneic NK cells. For example, the engineered cells have a stronger ability to resist killing host NK cells. For example, the engineered cells have a stronger ability to resist killing allogeneic NK cells.

In one example, in the presence of host immune cells (e.g., NK cells), the dual-target engineered cells of the present application that express and bind FasL and target antigens on pathological cells have a longer survival time and/or expansion ability. For example, the engineered cells exhibit a stronger cell-killing effect on pathological cells carrying target antigens in vivo and in vitro.

In one example, the engineered cells expressing the fusion protein also express immune checkpoint inhibitors. Immune checkpoint inhibitors include any agent that blocks, inhibits or reduces the activity or function of the inhibitory pathway of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies or their antigen binding fragments, which bind to and block or inhibit immune checkpoint receptors, ligands and/or receptor-ligand interactions. In some embodiments, the regulation, enhancement and/or stimulation of a specific receptor can outperform immune checkpoint pathway components. Exemplary immune checkpoint molecules that can be targeted for blocking, inhibition, regulation, enhancement and/or stimulation include, but are not limited to: PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor associated antigen (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and transforming growth factor receptor (TGFR; e.g., TGFRβ). Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to and block or inhibit and/or enhance or stimulate the activity of one or more of any of the described molecules.

Composition

The present invention also provides a pharmaceutical composition comprising the above-mentioned fusion protein. The present invention also provides a cell composition comprising the above-mentioned engineered cells. In one example, the pharmaceutical composition comprises an effective amount of the fusion protein disclosed in the present invention and a pharmaceutically acceptable carrier. In one example, the pharmaceutical composition comprises an effective amount of the engineered cells disclosed in the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful in suppressing host rejection of graft reactions. In some embodiments, the pharmaceutical composition is useful in immunotherapy. In some embodiments, the pharmaceutical composition is useful in immuno-oncology. In one example, the pharmaceutical composition is useful in suppressing tumor growth in a subject (e.g., a human patient). In one example, the pharmaceutical composition is useful in treating cancer in a subject (e.g., a human patient). In one example, the pharmaceutical composition is useful in treating viral infections. In one example, the pharmaceutical composition is useful in treating autoimmune diseases.

In one example, the present application provides a cell composition including a first engineered cell and a second engineered cell. In one example, the first engineered cell in the cell composition provided by the present application includes a fusion protein of a domain binding to FasL disclosed in the present application, and the second engineered cell binds to a target antigen (e.g., CD19, BCMA) on a pathological cell (e.g., a tumor cell, a pathological cell of an autoimmune disease). In one example, the first engineered cell in the cell composition provided by the present application includes a fusion protein of a domain binding to FasL and other NK cell markers (e.g., NKG2A, CD38) disclosed in the present application, and the second engineered cell binds to a target antigen (e.g., CD19, BCMA) on a pathological cell (e.g., a tumor cell, a pathological cell of an autoimmune disease). In one example, the first engineered cell in the cell composition provided by the present application includes a fusion protein of a domain binding to FasL disclosed in the present application and a chimeric receptor 1 of a domain binding to other NK cell markers (e.g., NKG2A, CD38), and the second engineered cell binds to a target antigen (e.g., CD19, BCMA) on a pathological cell (e.g., a tumor cell, a pathological cell of an autoimmune disease). The second engineered cell can be an engineered immune cell used in the prior art for tumor treatment. In one example, the second engineered cell can be a CART cell in the prior art.

In one example, the first engineered cell in the cell composition provided by the present application includes the fusion protein disclosed in the present application that includes a domain that binds to FasL, the second engineered cell binds to other NK cell markers (e.g., NKG2A, CD38), and the third engineered cell binds to target antigens (e.g., CD19, BCMA) on pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases).

In one example, the types of cells in the cell composition provided by the present application can be the same or different. For example, the first engineering cell or the second engineering cell can be any known suitable immune cell, and the type of the first engineering cell does not affect the type of the second engineering cell. For example, the first engineering cell is T, NK, NKT cell, and the second engineering cell can be T, NK, NKT cell or any other suitable immune cell.

In one example, the invention provides a pharmaceutical composition containing a fusion protein or cell provided by the invention. In one example, the composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one example, the composition is suitable for topical administration. In some aspects, topical administration includes intratumoral injection, peritumoral injection, juxtatumoral injection, intralesional injection and/or injection into tumor draining lymph nodes, or substantially any tumor targeted injection, wherein the anti-tumor agent is expected to leak into the primary lymph nodes adjacent to the targeted solid tumor.

According to the route of administration, active ingredient (i.e. fusion protein or engineered cell) can be coated in material to protect active ingredient from the effect of acid and other natural conditions that can inactivate active ingredient. Pharmaceutically acceptable carriers that can be used for compositions provided by the invention or preparations include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents and other materials with physiological compatibility. In some embodiments, carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion). According to the route of administration, active ingredient (i.e. fusion protein or cell of genetic engineering) can be coated in material to protect active ingredient from the effect of acid and other natural conditions that can inactivate active ingredient.

The present invention also provides a kit for preparing a pharmaceutical composition containing the fusion protein disclosed in the present invention. In some embodiments, the kit includes the fusion protein disclosed in the present invention and a pharmaceutically acceptable excipient present in one or more containers. In another embodiment, the kit may include the fusion protein disclosed in the present invention for administration to a subject. In a specific embodiment, the kit includes instructions for the preparation and/or administration of the fusion protein.

The present invention also provides a kit for preparing the cells disclosed in the present invention. In one embodiment, the kit includes one or more vectors for producing engineered cells (e.g., T cells) expressing the fusion protein disclosed in the present invention. The kit can be used to produce genetically engineered cells from autologous or non-autologous cells for administration to compatible subjects. In another embodiment, the kit can include cells disclosed in the present invention for administration to subjects. In a specific embodiment, the kit includes cells disclosed in the present invention in one or more containers. In a specific embodiment, the kit includes instructions for the preparation and/or administration of genetically engineered cells.

The present invention also provides a kit for inducing and/or enhancing an immune response and/or treating and/or preventing tumor or pathogen infection, autoimmune disease, inflammatory disease in a subject. For example, the kit includes an effective amount of the fusion protein of the present application and/or a composition and a pharmaceutical composition of a chimeric receptor that binds to a pathological cell. For example, the kit includes a sterile container; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister package, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil or other materials suitable for containing drugs. For example, the kit includes molecules encoding the fusion protein, chimeric receptor, recombinant TCR, exogenous cytokine and/or therapeutic monoclonal antibody of the present application, which can be optionally included in one or more carriers.

Methods and uses

The present invention provides the use of the fusion protein or engineered cell disclosed in the present invention. In one example, the fusion protein or engineered cell disclosed in the present invention can be used to prepare a drug for preventing or resisting transplant immune rejection. In one example, the fusion protein or engineered cell disclosed in the present invention is used to prevent or resist transplant immune rejection.

In one example, the present invention provides a method for preventing, alleviating and/or treating tumors, autoimmune diseases, and inflammatory diseases, which comprises administering the fusion protein, engineered cells, or pharmaceutical composition to a subject in need. The pharmaceutical composition provided in this application has been described above, and the method for preventing, alleviating and/or treating tumors, autoimmune diseases, and inflammatory diseases provided in this application includes all technical solutions thereof.

In one example, the fusion protein or engineered cell disclosed in the present invention can induce and/or increase an immune response in a subject. The present application provides a method for inhibiting the activity of pathological cells in a subject, the method comprising: administering to a subject a therapeutically effective amount of an engineered cell expressing the fusion protein of the present application, the engineered cell inhibiting the activity of pathological cells in the subject and/or killing pathological cells. For example, the pathological cell is a tumor cell. For example, the pathological cell includes, but is not limited to, acute myeloma leukemia cells, anaplastic lymphoma cells, astrocytoma cells, B cell cancer cells, breast cancer cells, colon cancer cells, ependymoma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, hepatocarcinoma cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T cell lymphoma cells, renal cancer cells, sarcoma cells, gastric cancer cells, hepatocarcinoma cells, mesothelioma cells or sarcoma cells. For example, the target cell is an immune cell.

In one example, the fusion protein or engineered cell disclosed in the present invention can be used to treat and/or prevent tumors in subjects. The disclosed fusion protein or engineered cell can be used to prolong the survival of subjects with tumors. The disclosed fusion protein or engineered cell can also be used to treat and/or prevent pathogen infection or other infectious diseases in human subjects such as immunocompromised subjects. This method includes administering an effective amount of the composition of the present invention to achieve the desired effect, whether to alleviate existing symptoms or prevent recurrence. For treatment, the amount administered is an amount that effectively produces the desired effect. The effective amount can be provided by one or more administrations. The effective amount can be provided in large doses or by continuous perfusion.

In one embodiment, the tumor is a solid tumor or a blood tumor. In one embodiment, the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; blood tumor is selected from: leukemia, lymphoma, myeloma.

In one example, the fusion protein or engineered cell disclosed in the present invention is used to prepare a drug for treating an autoimmune disease (AID) or an inflammatory disease. In one example, the fusion protein or engineered cell disclosed in the present invention can be used to treat, prevent or improve an autoimmune disease or an inflammatory disease in a subject in need thereof.

In one embodiment, the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis/dermatomyositis.

In one example, the inflammatory disease associated with an autoimmune disease is selected from, such as arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis (arthritis chronica progrediente) and osteoarthritis) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarthropathies (including ankylosing spondylitis), Reiter's syndrome, reactive arthritis, psoriatic arthritis, juvenile idiopathic arthritis and enteropathic arthritis, enthesitis, hypersensitivity reactions (including airway hypersensitivity reactions and skin hypersensitivity reactions) and allergies. The engineered T cells provided by the present invention are used to treat and prevent autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus (SLE), lupus nephritis, inflammatory muscle disease (dermatomyositis), periodontitis, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens Johnson syndrome, spontaneous sprue, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis, Crohn's disease and irritable bowel syndrome), endocrine eye 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, periprosthetic osteolysis, Glomerulonephritis (with and without nephrotic syndrome, including, for example, idiopathic nephrotic syndrome or minimal change 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 disease (including Hashimoto's thyroiditis), primary vasculitis of small and medium vessels, large vessel vasculitis including giant cell arteritis, hidradenitis suppurativa, neuromyelitis optica, Sjogren's syndrome, Behcet's disease, atopic and contact dermatitis, bronchiolitis, inflammatory muscle disease, autoimmune peripheral neuropathies, immune kidney, liver and thyroid diseases, inflammation and atherosclerosis, autoinflammatory febrile syndrome, immune hematological disorders, and bullous diseases of the skin and mucous membranes. For example, AID refers to a group of diseases that invade multiple tissues, organs or systems, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis/dermatomyositis, etc.

In one example, the fusion protein or engineered cell disclosed in the present invention can be used to treat, prevent or improve asthma, bronchitis, bronchiolitis, idiopathic interstitial pneumonia, pneumoconiosis, emphysema and other obstructive or inflammatory diseases of the airways.

In one example, the present invention provides a method for treating a disease in a subject suffering from a tumor, an immune disease (e.g., an autoimmune disease), or an inflammatory disease, the method comprising: i) genetically modifying autologous or allogeneic T cells, NK cells, and/or NKT cells using a vector comprising a fusion protein encoding the present application, wherein the fusion protein can specifically bind to immune cells (e.g., NK cells) and pathological cells (e.g., tumor cells, pathological cells of autoimmune diseases, pathological cells of inflammatory diseases) in the subject; ii) introducing the genetically modified T cells, NK cells, and/or NKT cells into the subject, wherein the genetically modified T cells, NK cells, and/or NKT cells recognize and kill tumor cells, and the genetically modified cells can resist attacks by the subject's immune cells.

In the present application, the methods described in the present application can also be interpreted as therapeutic uses, that is, the methods described in the present application can be considered as the therapeutic uses of the compositions, nucleic acid molecules or engineered cells of the present application or the pharmaceutical uses for preparing corresponding therapeutic uses. For example, the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for regulating the activity of engineered cells or the use of the above-mentioned engineered cells for preparing drugs for regulating the activity of engineered cells; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for activating engineered cells or the use of the above-mentioned engineered cells for preparing drugs for activating engineered cells; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for inhibiting the activity of target cells in a subject or for preparing drugs for inhibiting the activity of target cells in a subject; the present application relates to the use of the above-mentioned engineered cells or nucleic acid molecules for improving or treating the health status of subjects in need or for preparing drugs for improving or treating the health status of subjects in need. All the contents described in the above-mentioned present application can also be applied to therapeutic uses or pharmaceutical uses.

The engineered cells of the invention can be administered as the sole active ingredient or in combination with other drugs such as immunosuppressants or immunomodulators or other anti-inflammatory or other cytotoxic or anti-cancer agents (e.g., as adjuvants or in combination therewith), for example, to treat or prevent diseases related to immune disorders. For example, the antibodies of the invention can 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; regulators 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 with immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; Cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homologues, analogs or derivatives; immunosuppressive monoclonal antibodies, for example, monoclonal antibodies against leukocyte receptors, for example, MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Example

Example 1: Screening of targets for cell tolerance to NK cell killing based on CRISPR-GeCKO-v2 library

Human CRISPR Knockout Pooled Library (GeCKO v2) library (purchased from Suzhou Anshengda Biotechnology Co., Ltd.) was used for screening. B2M-KO Jurkat was used as the target cell to prepare GeCKOv2 lentivirus; 3.5×10^7GeCKOv2 lentivirus was used to infect 1×10^8 Jurkat (B2M-KO) target cells. Under this condition, the infection efficiency can reach nearly 30% and the coverage of each gRNA can reach 300-500×; after 8 hours of infection, the Jurkat target cells were replaced with medium, and after 48 hours of culture, 4μg/mL puromycin was added for pressure screening; the surviving cells are Jurkat library cells.

Use NK cells to kill library cells: take 1×10^8 Jurkat library cells, add NK cells to co-culture for 8 to 12 hours, then add 4μg/mL puromycin to remove NK, and continue to culture for 48 hours. Count the surviving library cells and control the number within 1×10^7. The obtained cells are the NK first round killing library cells. Then repeat the killing experiment to obtain the NK second round killing library cells. Repeat the experiment based on the second round to obtain the NK third round killing library cells.

High-throughput sequencing and analysis: DNA was extracted from the library cells using ZOYMO research Quick-DNA Midiprep Plus Kit, and the gRNA sequence was amplified using primers to obtain amplicons. The amplicons of various samples were mixed and then handed over to Suzhou Anshengda for library construction and sequencing. The raw data were sequenced and analyzed using the open source software MAGeCK (Genome Biol. 2014; 15(12):554.), and the obtained gene target list was visualized and analyzed using R language.

According to the enrichment score (beta.score) of the screening, a heat map analysis was performed on the top 70 genes in the three rounds of killing. It can be seen that all genes were significantly enriched in the second and third rounds of killing, which is consistent with the expansion of the enrichment multiple of positive screening genes under pressure screening. From the data of the heat map, genes such as FAS appeared. According to these genes screened for NK tolerance, the FAS-FADD-BID-BAK1 signal axis may play an important role.

Example 2. Detection of FasL expression in human primary NK and aNK by flow cytometry

Peripheral blood PBMC cells from donors were collected, and human primary NK cells were screened using CD56 magnetic beads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions. 1×10 ⁵ NK cells were selected for each flow cytometry sample, and the sample cells were resuspended in 100 μL MACS solution (Miltenyi Biotec, 130-091-221). 1 μL FasL antibody (Biolegend, 306421) was added to each flow cytometry sample at a ratio of 1:100. PBS was used as a negative control, and the cells were incubated at room temperature for 20 minutes. The expression of FasL was detected by flow cytometry. Figure 1A shows that among NK cells from different donors, about 57.8% to 66% of NK cells expressed FasL, and the median MFI value of the fluorescence intensity of FasL was ～300.

Peripheral blood PBMC cells from donors were collected, and NK cells were screened using CD56 magnetic beads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions. After stimulation and expansion with cytokines IL2 and IL15, activated NK (active NK, aNK) was obtained. 1 μL FasL antibody (Biolegend, 306421) was added to 1×10 ⁵ aNK cells, and PBS was used as a negative control. The cells were incubated at room temperature for 20 minutes, and the expression of FasL was detected by flow cytometry. Figure 1B shows that among aNK cells from different donors, about 92.9% to 99.5% of aNK cells expressed FasL, and the median fluorescence intensity MFI of FasL was 1000 to 1200. After NK activation, FasL expression is upregulated.

Example 3. Vector construction

The vector expressing the fusion protein disclosed in the present application was constructed using conventional molecular biological techniques (Fig. 2A). The sequences comprising the extracellular domain (antibody binding to FasL or the extracellular segment of Fas), hinge region, transmembrane region, and intracellular domain fragment were synthesized (Suzhou Anshengda Biotechnology Co., Ltd.), and integrated into the pRRLSIN vector (Addgene) to construct the expression vectors 1-28Z, 2-28Z, 3-28Z, 4-28Z, 5-28Z, 10-28Z, 11-28Z, and 12-28Z of FasL-CAR including the antibody binding to FasL as shown in Table 1, and the expression vectors Fas-CAR1, Fas-CAR2, Fas-CAR3, and Fas-CAR4 of the extracellular segment of Fas; the expression vectors 7-28Z of NKG2A-CAR including the binding to NKG2A as shown in Table 2, and the expression vectors 8-28Z of CD38-CAR including the binding to CD38. As shown in Figure 2A, the FasL binding domain can be an anti-FasL antibody (such as scFv, single domain antibody (sdAb), or DDpp antibody), or Fas extracellular segment. The corresponding lentivirus of the above expression vector is prepared by conventional molecular biological techniques.

Example 4. CAR-T cell preparation

The conventional method for preparing CAR-T in the art was adopted: donor PBMC cells were collected, activated by magnetic beads of anti-CD3 and CD28 antibodies (Life Technologies, 40203D), and then cultured to obtain T cells; after infecting T cells with lentivirus containing the above vector, FasL-CAR-T cells were prepared. 1×10 ⁵ cells were collected for each FasL-CAR-T sample, and the sample cells were resuspended in 100 μL MACS solution (Miltenyi Biotec, 130-091-221). 1 μL of FasL antigen protein (Acro, FAL-H5241-50 μg) was added to each flow cytometry sample at a ratio of 1:100 and incubated at room temperature for 1 hour, and anti-His secondary antibody (R&D systems, IC0501G-100UG) was added and incubated at room temperature for 30 minutes. The positive rate of FasL-CAR detected by flow cytometry was 82% to 92%.

Example 5. Preparation of FasL-CAR-T cells

sgRNA sequences targeting TRAC, B2M, FAS, and CD58 (shown in SEQ ID NOs: 12, 13, 14, and 15, respectively) were synthesized in vitro (Kaixing Diagnostics), and the endogenous TCR/B2M, TCR/B2M/FAS, or TCR/B2M/CD58 of the FasL-CAR-T cells constructed in Example 4 were knocked out by CRISPR/Cas9 technology (Cas9 protein, Kaixia Biotech (Shanghai), CAS-EE109), and FasL-CAR-T-dko, FasL-CAR-T-tko1, or FasL-CAR-T-tko2 were obtained by magnetic bead sorting (Miltenyi Biopharmaceuticals, 130-048-801). UTD cells that were not transduced but TCR/B2M knocked out were used as controls.

Specifically, FasL-CAR-T cells prepared in Example 4 were collected, and TCR/B2M, TCR/B2M/FAS, or TCR/B2M/CD58 were knocked out by CRISPR/Cas9 technology on D0. On D3, the knockout efficiency and CAR positivity were determined; on D4, the cell survival rate of CAR-T was determined using AO/PI staining solution (CountStar, RE010213). The CAR positivity rate was determined as in Example 4.

Figure 3 shows that the survival rate of T cells expressing FasL-CAR is about 95% to 98%, and the CAR positive rate is about 72% to 92%, which indicates that there is no mutual killing during the culture process of T cells expressing FasL-CAR.

Example 6. In vitro killing of human aNK cells by FasL-CAR-T cells

On D0, 5×10 ⁴ FasL-CAR-T-dko cells and aNK cells (efficacy-target ratio 1:1.5) were collected and co-incubated in an incubator at 37°C. On D0-D3, HLA-ABC (Thermo Fisher, 17-9983-42) was used to label B2M knockout cells (B2M KO-T cells), and CD56 (BD 555516) was used to label NK cells; 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of aNK cells was detected by flow cytometry. On D1, the supernatant was collected, and the release levels of cytokines IL2, TNF-α, and IFN-γ were detected using Cytometric bead array (BD, 558264).

Figure 4 shows that after incubation with NK cells, T cells expressing FasL-CAR secreted IFN-γ, IL-2, and TNF-α; they were able to significantly kill aNK cells, and on D3, about 82% to 94% of aNK cells were killed; in the presence of activated NK cells, the survival rate of CAR-T cells was significantly improved.

Example 7. In vitro killing of human primary NK cells by FasL-CAR-T cells

On D0, 5×10 ⁴ FasL-CAR-T-dko cells and primary NK cells (effector-target ratio 1:1.5) were collected and incubated in an incubator at 37°C. On D0-D3, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (BD 555516); 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of primary NK cells was detected by flow cytometry.

Figure 5 shows that after incubation with NK cells, T cells expressing FasL-CAR can significantly kill primary NK cells. On D3, about 49% to 71% of NK cells were killed. In the presence of primary NK cells, the survival rate of CAR-T cells was significantly improved.

Example 8. Flow cytometry detection of the expression of FasL, NKG2A and CD38 in primary NK and aNK cells from different donors

1×10 ⁵ human primary NK cells or aNK cells from different donors were collected, resuspended in MACS solution, and then 1 μL FasL antibody (Biolegend, Cat#306421), 1 μL NKG2A antibody (BD, Cat#747924), and 1 μL CD38 antibody (CD38-BV421) were added at a ratio of 1:100. PBS was used as a negative control. The cells were incubated at room temperature for 20-30 min, and the expression of FasL, NKG2A, and CD38 was detected by flow cytometry.

FIG6A shows that approximately 58.4% to 66% of primary human NK cells expressed FasL, approximately 16% to 37.8% expressed NKG2A, and approximately 65% to 77% expressed FasL or NKG2A.

FIG6B shows that about 90% to 97% of aNK cells expressed FasL, about 62% to 95% expressed NKG2A, and about 95% to 99% expressed FasL or NKG2A.

FIG. 15A shows that approximately 95.1% to 97.6% of human primary NK cells express CD38.

FIG. 15B shows that approximately 99.5% to 100% of aNK cells expressed CD38.

Example 9. Preparation of CAR-T cells that recognize dual targets

Conventional molecular biology techniques were used to construct CAR-T cells that recognize dual targets (Figure 2B). The sequences of antibodies, hinge regions, transmembrane regions, and intracellular domain fragments that bind to FasL and NKG2A, or sequences of antibodies, hinge regions, transmembrane regions, and intracellular domain fragments that bind to FasL and CD38 were synthesized (Suzhou Anshengda Biotechnology Co., Ltd.), and were respectively integrated into the pRRLSIN vector (Addgene) to construct FasL-NKG2A-CAR expression vectors 6-28Z, 13-28Z, 14-28Z, and 15-28Z (Table 1), and FasL-NKG2A-CAR-T-dko was prepared with reference to Examples 4 and 5. An expression vector comprising an expression vector binding to FasL-CAR and an expression vector of an NKG2A-CAR binding to NKG2A was prepared with reference to Example 3, introduced into T cells, and FasL-NKG2A-CAR-T-dko was prepared with reference to Examples 4 and 5. Referring to Example 3, an expression vector comprising a FasL-CAR binding vector and a CD38-CAR binding vector was prepared, and the expression vector was introduced into T cells. Referring to Examples 4 and 5, an expression vector comprising a FasL-CAR binding vector and a CD38-CAR binding vector was prepared.

FasL-CD38-CAR-T-dko. The CAR-T cell positive rate is about 67% to 75%.

Example 10. In vitro killing of human aNK cells by dual-target CAR-T cells

On D0, 5×10 ⁴ FasL-NKG2A-CAR-T-dko cells and aNK cells (efficacy:target ratio 1:1.5) were collected and incubated in an incubator at 37°C. On D0-D3, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (BD 555516); 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of aNK cells was detected by flow cytometry.

Figure 7 (upper right) shows that after incubation with NK cells, T cells expressing FasL-NKG2A-CAR secreted IFN-γ, IL-2, and TNF-α; dual-target CAR-T cells binding to FasL and NKG2A had significantly higher NK killing ability than single-target CAR-T cells binding to FasL, killing about 82% of aNK cells on D3 (Figure 7, upper left); in the presence of activated NK cells, the survival rate of dual-target CAR-T cells was significantly higher than that of single-target CAR-T cells (Figure 7, lower figure).

Example 11. In vitro killing of primary human NK cells by dual-target CAR-T cells

On D0, 5×10 ⁴ FasL-NKG2A-CAR-T-dko cells and human primary NK cells (effector-target ratio 1:1.5) were collected and incubated in an incubator at 37°C. B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (BD 555516) on D0, 1 and 3 days respectively; 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of primary NK cells was detected by flow cytometry.

Figure 8 shows that the killing effect of dual-target CAR-T cells binding to FasL and NKG2A on primary human NK cells was significantly higher than that of single-target CAR-T cells binding only to FasL, and about 85% to 86% of primary NK cells were killed on D3.

Example 12. In vitro killing of primary human NK cells by dual-target CAR-T cells

On D0, 5×10 ⁴ FasL-CD38-CAR-T-dko cells and primary human NK cells (effector-target ratio 1:1.5) were collected and incubated in an incubator at 37°C. On D0 and 3 days, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42), and NK cells were labeled with CD56 (BD 555516); 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of primary NK cells was detected by flow cytometry.

Figure 9 shows that the killing effect of dual-target CAR-T cells combining FasL and CD38 on primary human NK cells is significantly higher than that of single-target CAR-T cells combining only FasL, and about 93% of primary NK cells were killed on D3.

Example 13. Knockout of endogenous FAS can improve FasL-CAR-T cell survival in the presence of NK cells

On D0, 5×10 ⁴ FasL-CAR-T-dko, FasL-CAR-tko1 and aNK cells (efficacy:target ratio 1:1.5) were collected and incubated in an incubator at 37°C. On D0-D3, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (BD 555516); 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of B2M-KO T cells was detected by flow cytometry.

Figure 10 shows that in the presence of NK cells, knocking out endogenous FAS is beneficial to the survival of CAR-T.

Example 14. Knockout of endogenous CD58 can improve FasL-CAR-T cell survival in the presence of NK cells

On D0, 5×10 ⁴ FasL-CAR-T-dko, FasL-CAR-tko2 and aNK cells (efficacy:target ratio 1:1.5) were collected and incubated in an incubator at 37°C. On D0-D3, B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (PE, BD 555516); 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells, and the number of B2M-KO T cells was detected by flow cytometry.

Figure 11 shows that in the presence of NK cells, knocking out endogenous CD58 is beneficial to the survival of CAR-T.

Example 15. Preparation of Fas-CAR-T cells

The Fas-CAR vector was prepared according to conventional molecular biology. The structures of the fusion proteins Fas-CAR1, Fas-CAR2, Fas-CAR3, Fas-CAR4, Fas-CAR5, Fas-CAR6, and Fas-CAR7 are shown in Table 1.

T cells were obtained by using PBMC cells of donors and prepared by lentivirus infection. The WT group was the control group into which only GFP protein was introduced.

Flow cytometry revealed that the expression of Fas-CAR4 on the cell surface was significantly increased ( FIG. 12A ), and the fluorescence intensity MFI value was significantly increased.

Example 16. Detection of Fas-CAR-T cell proliferation ability

The Fas-CAR-T cells prepared in Example 15 were placed in AIM-V medium containing 2% ABS and 300 UIL2 for 72 h, and the number of Fas-CAR-T cells was measured by flow cytometry every 24 h (Figure 12B). As shown in the figure, the expression of Fas-CAR did not lead to a decrease in the number of T cells.

Example 17. Preparation of B2M knockout Fas-CAR-T (KO) cells

Conventional CRISPR/Cas9 technology was used to knock out the B2M gene in the Fas-CAR-T cells prepared in Example 16 to obtain Fas-CAR-T (KO) cells. The B2M gene in the T cells of the WT group in Example 15 was knocked out to obtain the control group B2M-T (KO) cells. The gRNA-B2M sequence is as shown in SEQ ID NO: 13. Flow cytometry showed that the knockout efficiency of B2M was 85% to 90%.

Example 18. Fas-CAR-T (KO) cells kill NK cells

NK cells were prepared from two donors using conventional biological techniques. The Fas-CAR-T (KO) cells prepared in Example 17 were co-incubated with 1×10^5 NK cells in 100 μL T cell culture medium at a ratio of 1:1, and the number of cells was detected at different time points. The WT group is the T cells in Example 15 without B2M knockout.

The results showed that compared with the B2M-T (KO) group, the number of T cells in the Fas-CAR-T4 (KO) group was significantly increased ( FIG. 13 ), while the number of NK cells was significantly decreased ( FIG. 14 ).

The embodiments of the present invention include the embodiments as any single embodiment or in combination with any other embodiment or part thereof. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Sequence Listing

Claims (90)

1. A fusion protein, which includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus, wherein the extracellular region includes a domain binding to Fas ligand (FasL).
2. The fusion protein according to claim 1, wherein the FasL-binding domain comprises the Fas extracellular segment or a fragment or variant thereof that can bind to FasL, an anti-FasL antibody or a fragment thereof, or a synthetic binding domain.
3. The fusion protein according to claim 2, wherein the FasL-binding domain comprises an anti-FasL antibody or a fragment thereof.
4. The fusion protein as described in claim 3, wherein the anti-FasL antibody or its fragment comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 and VL1 respectively have (1) the amino acid sequences shown in SEQ ID NOs: 16 and 17; (2) the amino acid sequences shown in SEQ ID NOs: 18 and 17; (3) the amino acid sequences shown in SEQ ID NOs: 20 and 21; (4) the amino acid sequences shown in SEQ ID NOs: 22 and 21; (5) the amino acid sequences shown in SEQ ID NOs: 24 and 25; (6) the amino acid sequences shown in SEQ ID NOs: 26 and 27; or (7) the amino acid sequences shown in SEQ ID NOs: 28 and 27.
5. The fusion protein as described in claim 3, wherein the VH1 and VL1 have the amino acid sequences shown in (1) SEQ ID NOs: 18 and 17, respectively.
6. The fusion protein according to any one of claims 3 to 5, wherein the anti-FasL antibody is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.
7. The fusion protein according to claim 6, wherein the anti-FasL antibody is scFv.
8. The fusion protein according to claim 6, wherein the anti-FasL antibody is a single domain antibody.
9. The fusion protein according to claim 6, wherein the anti-FasL antibody is a DDPP antibody.
10. The fusion protein according to claim 2, wherein the FasL-binding domain comprises the extracellular segment of Fas or a variant thereof that can bind to FasL.
11. A fusion protein as described in claim 10, wherein the extracellular segment of Fas comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 29.
12. A fusion protein as described in claim 11, wherein the FasL binding domain comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence shown in SEQ ID NO: 19, 23, or 42.
13. The fusion protein according to any one of claims 1 to 12, wherein the intracellular signal transduction domain comprises an immunoreceptor tyrosine-based activation motif or an ITAM signaling motif.
14. The fusion protein of any one of claims 1 to 13, wherein the intracellular signal transduction domain comprises an intracellular signal transduction domain selected from: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof.
15. The fusion protein as described in any one of claims 1-14, wherein the intracellular signal transduction domain includes the CD3ζ intracellular signal transduction domain (SEQ ID NO: 7).
16. The fusion protein according to any one of claims 1 to 15, wherein the intracellular signal transduction domain further comprises a co-stimulatory domain.
17. The fusion protein of claim 16, wherein the co-stimulatory domain is selected from the intracellular signaling domain of CD137 (4-1BB), CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof.
18. The fusion protein as described in claim 16, wherein the co-stimulatory structure is selected from the intracellular signal transduction domain of CD137 (4-1BB) (SEQ ID NO: 6) or the intracellular signal transduction domain of CD28 (SEQ ID NO: 5).
19. The fusion protein according to any one of claims 1 to 18, wherein the transmembrane domain of the fusion protein is selected from the group consisting of: CD2, CD3ε, CD3δ, CD3ζ, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (P The transmembrane domain of the proteins involved in the invention may be a member of the group consisting of: a) CD45 (CD4+), CD54 (CD5+), CD64 (CD64+), CD72 (CD72+), CD80 (CD80+), CD90 (CD90+), CD100 (CD100+), CD110 (CD110+), CD120 (CD120+), CD130 (CD130+), CD140 (CD140+), CD150 (CD150+), CD160 (CD160+), CD170 (CD170+), CD180 (CD180+), CD190 (CD190+), CD200 (CD190+), CD201 (CD190+), CD202 (CD190+), CD203 (CD190+), CD204 (CD190+), CD205 (CD190+),
20. The fusion protein as described in claim 19, wherein the transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 2).
21. The fusion protein as described in claim 19, wherein the transmembrane domain is the CD8 transmembrane domain (SEQ ID NO: 3).
22. The fusion protein according to any one of claims 1 to 12, wherein the fusion protein comprises a CD28 transmembrane domain (SEQ ID NO: 2), a CD28 intracellular signaling domain (SEQ ID NO: 5), and a CD3ζ intracellular signaling domain (SEQ ID NO: 7).
23. The fusion protein according to any one of claims 1 to 22, wherein the extracellular domain and the transmembrane domain are connected by a connecting polypeptide.
24. The fusion protein of claim 23, wherein the connecting polypeptide is selected from: a CD28 hinge region, a CD8 hinge region, an IgG spacer region or a fragment thereof, or a combination thereof.
25. The fusion protein as described in claim 24, wherein the connecting polypeptide is the CD8 hinge region (SEQ ID NO: 10).
26. The fusion protein as described in claim 24, wherein the connecting polypeptide is an IgG1 spacer region (SEQ ID NO: 8).
27. The fusion protein as described in claim 24, wherein the connecting polypeptide is an IgG4 spacer region (SEQ ID NO: 9).
28. The fusion protein according to any one of claims 1 to 27, wherein the extracellular domain of the fusion protein further comprises a domain that binds to an immune cell marker different from FasL.
29. The fusion protein of claim 28, wherein the immune cell marker is a T cell marker and/or a NK cell marker.
30. The fusion protein as described in claim 28, wherein the immune cell marker is NK inhibitory receptor (NKIR).
31. The fusion protein of claim 28, wherein the immune cell marker is selected from: NKG2/CD94, a KIR family member, a LIR family member, a NKR-P1 family member, an immune checkpoint receptor, a SIGLEC family member, a Ly49 family member, or a combination thereof.
32. The fusion protein of claim 31, wherein the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.
33. The fusion protein of claim 28, wherein the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.
34. The fusion protein of claim 28, wherein the immune cell marker is CD38.
35. The fusion protein of claim 28, wherein the immune cell marker is NKG2A.
36. The fusion protein of any one of claims 28-35, wherein the domain that binds to an immune cell marker other than FasL comprises a ligand, a synthetic binding domain, an antibody or a fragment thereof that binds to the immune cell marker.
37. The fusion protein of claim 36, wherein the extracellular domain comprises an antibody or a fragment thereof that binds to the immune cell marker.
38. The fusion protein of claim 37, wherein the antibody or fragment is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody.
39. The fusion protein of claim 37 or 38, wherein the anti-FasL antibody or fragment thereof comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the antibody or fragment thereof that binds to the immune cell marker comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).
40. The fusion protein of claim 38, wherein the extracellular domain comprises, from N-terminus to C-terminus, (1) VH1-VL1-VH2-VL2, (2) VL1-VH1-VL2-VH2, (3) VH1-VL1-VL2-VH2, (4) VL1-VH1-VH2-VL2, (5) VH1-VH2-VL2-VL1, (6) VL 1-VH2-VL2-VH1, (7)VH1-VL2-VH2-VL1, (8)VL1-VL2-VH2-VH1, (9)VH2-VH1-VL1-VL2, (10)VL2-VH1-VL1-VH2, (11)VH2-VL1-VH1-VL2, or (12)VL2-VL 1-VH1-VH2.
41. The fusion protein of claim 40, wherein the extracellular domain comprises, from N-terminus to C-terminus, VL1-VH2-VL2-VH1.
42. The fusion protein of claim 40, wherein the extracellular domain comprises, from N-terminus to C-terminus, VH1-VL2-VH2-VL1.
43. The fusion protein of claim 40, wherein the extracellular domain comprises, from N-terminus to C-terminus, VL2-VH1-VL1-VH2.
44. The fusion protein of claim 40, wherein the extracellular domain comprises, from N-terminus to C-terminus, VH2-VL1-VH1-VL2.
45. A fusion protein as described in any one of claims 37-44, wherein the extracellular domain comprises an antibody or a fragment thereof that binds to CD38, and the VH2 and VL2 respectively have (1) the amino acid sequences shown in SEQ ID NOs:34 and 35; (2) the amino acid sequences shown in SEQ ID NOs:36 and 37; (3) the amino acid sequences shown in SEQ ID NOs:38 and 39; or (4) the amino acid sequences shown in SEQ ID NOs:40 and 41.
46. The fusion protein as described in claim 45, wherein the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 34 and 35, respectively.
47. The fusion protein as described in any one of claims 37-44, wherein the extracellular domain comprises an antibody or a fragment thereof that binds to NKG2A, and the VH2 and VL2 respectively have (1) the amino acid sequences shown in SEQ ID NOs: 30 and 31; or (2) the amino acid sequences shown in SEQ ID NOs: 32 and 33.
48. The fusion protein as described in claim 47, wherein the VH2 and VL2 have the amino acid sequences shown in SEQ ID NOs: 30 and 31, respectively.
49. The fusion protein according to any one of claims 1 to 48, wherein the fusion protein further comprises a domain that binds to a target antigen on a pathological cell.
50. The fusion protein of claim 49, wherein the pathological cell is selected from a malignant cell or an infected cell.
51. The fusion protein of claim 50, wherein the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases.
52. The fusion protein of claim 51, wherein the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis/dermatomyositis.
53. The fusion protein as described in claim 49, wherein the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.
54. An engineered cell, comprising the fusion protein according to any one of claims 1-53.
55. The engineered cell of claim 55, wherein the engineered cell further comprises a chimeric receptor 1, wherein the chimeric receptor 1 binds to at least one or more immune cell markers different from FasL.
56. The engineered cell according to claim 55, wherein the immune cell marker is selected from: a T cell marker and/or a NK cell marker.
57. The engineered cell as described in claim 55, wherein the immune cell marker is NK inhibitory receptor (NKIR).
58. The engineered cell of claim 55, wherein the immune cell marker is selected from: NKG2/CD94, a KIR family member, a LIR family member, a NKR-P1 family member, an immune checkpoint receptor, a SIGLEC family member, a Ly49 family member, or a combination thereof.
59. The engineered cell of claim 58, wherein the NKG2/CD94 component is selected from NKG2A, NKG2C and CD94; the KIR family member is selected from KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3; the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8; the NKR-P1 family member is selected from NKR-P1B and NKR-P1D; the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3; the SIGLEC family member is selected from SIGLEC7 and SIGLEC9; the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.
60. The engineered cell of claim 55, wherein the immune cell marker is selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, and CD2 26, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.
61. The engineered cell according to any one of claims 55 to 60, wherein the chimeric receptor 1 also binds to a target antigen on a pathological cell.
62. The engineered cell according to any one of claims 54 to 61, wherein the engineered cell further expresses a chimeric receptor 2 that binds to a target antigen on a pathological cell.
63. The engineered cell of claim 61 or 62, wherein the pathological cell is selected from a malignant cell or an infected cell.
64. The engineered cell according to any one of claims 61 to 63, wherein the pathological cells are selected from solid tumor cells, blood tumor cells, and pathological cells of autoimmune diseases.
65. The engineered cell of claim 64, wherein the solid tumor is selected from the group consisting of esophageal cancer, gastric cancer, liver cancer, biliary tract tumors, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, and sarcoma; the blood tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), and polymyositis/dermatomyositis.
66. The engineered cell as described in any one of claims 61-65, wherein the target antigen is selected from: CD19, CD20, CD22, CD38, BCMA, GPRC5D, B7H3, GPC3, Claudin 6, Claudin18.2, FAP, Mesothelin, NKG2D ligand, NKG2A, CD94, FCRH5, EGFR and its mutants, or a combination thereof.
67. The engineered cell according to any one of claims 54 to 66, wherein the engineered cell further comprises: low expression or no expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS and/or CD58.
68. The engineered cell of claim 67, wherein the engineered cell further comprises: no expression of endogenous TCR and B2M.
69. The engineered cell of claim 67, wherein the engineered cell further comprises: no expression of endogenous TCR/B2M/FAS.
70. The engineered cell of claim 67, wherein the engineered cell further comprises: no expression of endogenous TCR/B2M/CD58.
71. The engineered cell according to any one of claims 54 to 70, wherein the engineered cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, a stem cell or a combination thereof.
72. The engineered cell of claim 71, wherein the immune cell is an autologous or allogeneic cell selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof.
73. The engineered cell of claim 71, wherein the engineered cell is an allogeneic T cell.
74. The engineered cells according to any one of claims 54 to 73, wherein the engineered cells have a prolonged survival time or enhanced proliferation capacity in the presence of host immune cells.
75. The engineered cell according to any one of claims 54 to 74, wherein the engineered cell has an inhibitory or killing function on the host's immune cells.
76. The engineered cell according to any one of claims 54 to 75, wherein the engineered cell has an inhibitory or killing function on the host's T cells and NK cells.
77. The engineered cell according to any one of claims 54 to 76, wherein the engineered cell can enhance the survival, proliferation, and killing of pathological cells by another engineered cell that is previously, simultaneously, or subsequently introduced into the subject.
78. A polynucleotide, which is a nucleic acid molecule encoding a fusion protein constructed as described in any one of claims 1-53.
79. A vector comprising the polynucleotide of claim 78.
80. A virus comprising the vector of claim 79.
81. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-53, the engineered cell of any one of claims 54-77, the polynucleotide of claim 78, the vector of claim 79, or the virus of claim 80, and a pharmaceutically acceptable carrier.
82. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, used for preparing a drug for preventing or resisting transplant immune rejection.
83. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, for use in preventing or resisting transplant rejection.
84. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, for use in preparing a medicament for treating an autoimmune disease or an inflammatory disease.
85. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, for treating, preventing or ameliorating an autoimmune disease or inflammatory disease in a subject in need thereof.
86. The fusion protein or engineered cell as described in claim 84 or 85, wherein the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis/dermatomyositis.
87. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, used for preparing a drug for treating tumors.
88. The fusion protein according to any one of claims 1 to 53, or the engineered cell according to any one of claims 54 to 77, for use in treating, preventing or ameliorating a tumor in a subject in need thereof.
89. The fusion protein or engineered cell according to claim 87 or 88, wherein the tumor is a solid tumor or a hematological tumor.
90. The fusion protein or engineered cell as described in claim 89, wherein the solid tumor is selected from: esophageal cancer, gastric cancer, liver cancer, biliary tract tumor, pancreatic cancer, intestinal cancer, laryngeal cancer, lung cancer, breast cancer, head and neck cancer, glioma, thyroid cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, sarcoma; the blood tumor is selected from: leukemia, lymphoma, myeloma.