Engineered cell and use thereof

Abstract

The present invention relates to an engineered cell, expressing: (i) a PD1 binding molecule, comprising a PD1 binding domain and a transmembrane domain but not comprising an intracellular domain other than a PD1 ligand; and (ii) an immunosuppressive molecule, comprising an immunosuppressive protein binding domain and a transmembrane domain but not comprising a primary signaling domain, wherein an immunosuppressive protein is selected from NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1 or a combination thereof. The present invention further relates to a composition comprising the engineered cell and a use of the engineered cell.

Description

Engineered cells and their uses Technical Field

[0001] The present invention belongs to the field of cell therapy. More specifically, the present invention relates to an engineered cell expressing a PD1 binding molecule and an immunosuppressive molecule and its use. Background Art

[0002] As an emerging immunotherapy, adoptive cell therapy has developed rapidly in recent years. Chimeric antigen receptor therapy (CAR-T therapy), in particular, has seen numerous products marketed and demonstrating excellent clinical efficacy. However, while CAR-T therapy exhibits significant efficacy, it is also associated with a variety of adverse reactions, including cytokine release syndrome (CRS, also known as cytokine storm), neurotoxicity, tumor lysis syndrome, cytopenia, infection, hypoimmunoglobulinemia, and hepatitis B virus reactivation.

[0003] CRS refers to the massive release of cytokines during the interaction between immune cells and tumor cells. These cytokines in turn trigger further chain reactions, such as excessive inflammation, capillary leakage, coagulation cascades, etc., which in turn lead to organ damage and brain swelling. CRS is the most frequent and most symptomatic acute toxic reaction in CAR-T cell therapy. It is reported that in clinical trials, more than 90% of patients with B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin's B-cell lymphoma treated with anti-CD19 CAR-T cells developed CRS. Therefore, it is still necessary to improve existing CAR-T therapies to reduce the occurrence of adverse reactions and achieve better therapeutic effects.

[0004] Summary of the Invention

[0005] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0006] In a first aspect, the present invention provides an engineered cell expressing:

[0007] (i) a PD1 binding molecule comprising a PD1 binding domain and a transmembrane domain and not comprising an intracellular domain other than a PD1 ligand;

[0008] (ii) an immunosuppressive molecule comprising an immunosuppressive protein binding domain and a transmembrane domain and not comprising a primary signaling domain, wherein the immunosuppressive protein is selected from NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1 or a combination thereof.

[0009] In some embodiments, the immunosuppressive molecule further comprises a costimulatory domain. In other embodiments, the immunosuppressive molecule does not comprise a costimulatory domain.

[0010] PD1 binding molecules and immunosuppressive molecules

[0011] The term "immunosuppressive molecule" refers to a molecule that can bind to an immunosuppressive protein (such as NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1, etc.) to inhibit the subject's immune rejection of exogenous cells, such as reducing the killing function of immune cells (such as T cells, NK cells, etc.) in the subject's body or inhibiting the excessive proliferation of immune cells. The term "immunosuppressive protein" refers to a ligand or receptor that inhibits the immune response, which plays an important role in maintaining autoimmune tolerance and regulating the duration and amplitude of the immune response, thereby avoiding damage and destruction of normal tissues by the immune system. The immunosuppressive protein in the present invention is selected from NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1 or a combination thereof.

[0012] The term "PD1 binding domain" refers to any structure (such as an antibody, ligand, or receptor) that can bind to PD1, or a functional variant thereof. The term "immunosuppressive protein binding domain" refers to any structure (such as an antibody, ligand, or receptor) that can bind to an immunosuppressive protein, or a functional variant thereof.

[0013] In some embodiments, the binding domain in the present invention is selected from an antibody. The term "antibody" has the broadest meaning understood by those skilled in the art, and includes complete antibodies such as monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit desired biological activity, which can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). The term "antibody fragment" refers to at least a portion of a complete antibody or a variant thereof, and refers to a binding domain (e.g., an antigen variable region of a complete antibody) sufficient to confer recognition and specific binding to a target (e.g., an antigen) on the antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), linear antibodies, "diabodies" with two antigen-binding sites, single-domain antibodies (sdAb) (e.g., the heavy chain variable region VH, the light chain variable region VL, nanobodies VHH, etc. of an antibody).

[0014] In some embodiments, the "PD1 binding domain" in the present invention is selected from ligands, receptors and functional fragments thereof that bind to PD1 (i.e., functional fragments with PD1 binding ability, such as extracellular regions). In one embodiment, the PD1 binding domain and transmembrane domain contained in the PD1 binding molecule are from the same molecule or different molecules and do not contain any intracellular domains (such as costimulatory domains and / or primary signaling domains). In another embodiment, the PD1 binding domain and transmembrane domain contained in the PD1 binding molecule are from the same molecule or different molecules and contain an intracellular domain from a PD1 ligand. In other words, the PD1 binding molecule of the present invention can be the PD1 ligand itself.

[0015] In some embodiments, the "immunosuppressive protein binding domain" in the present invention is selected from ligands, receptors and functional fragments thereof that bind to immunosuppressive proteins (i.e., functional fragments with immunosuppressive protein binding ability, such as extracellular regions). In one embodiment, the three domains of immunosuppressive protein binding domain, transmembrane domain and co-stimulatory domain contained in the immunosuppressive molecule are not from the same molecule at the same time. For example, two of the domains are from the same molecule, while the other domain is from a different molecule, or all three domains are from different molecules. In other words, the immunosuppressive molecule of the present invention is not a naturally occurring full-length ligand or receptor itself.

[0016] The term "ligand or receptor" refers to any molecule or atom that can interact with an immunosuppressive protein after binding. A ligand or receptor can be a naturally occurring molecule, such as an organic or inorganic molecule, or a synthetic molecule. For example, known ligands for NKG2A include HLA-E, known ligands for TIM3 include Galectin9, HMGB1, and CEACAM1, known ligands for LAG3 include Galectin3, LSECtin, FGL1, and some MHC class II molecules, known ligands for TIM3 include CD112, CD113, CD155, and Nectin4, known ligands for CTLA4 include CD80 and CD86, known ligands for PD1 include PDL1 and PDL2, known ligands for SIRPα include CD47, and known ligands for LILRB1 include HLA-G.

[0017] Unless the context clearly indicates otherwise, the "binding domain" of the present invention encompasses antibodies, ligands, and functional fragments thereof as described above. Therefore, the binding domain described in the present invention is selected from the group consisting of intact antibodies, Fab, Fab', F(ab')2, Fd fragments, Fd', Fv fragments, scFv, sdFv, linear antibodies, diabodies, sdAbs, ligands, or functional fragments of receptors.

[0018] The term "functional variant" or "functional fragment" refers to a variant or fragment that contains at least one amino acid modification (i.e., substitution, deletion, or insertion) compared to the parent amino acid sequence but retains the biological activity of the parent amino acid sequence. For example, a functional fragment of a ligand or receptor in the present invention generally refers to a fragment of the ligand or receptor that is capable of binding to the corresponding PD1 or immunosuppressive protein, such as the extracellular domain.

[0019] The term "heavy chain" refers to the larger of the two types of polypeptide chains found in naturally occurring conformations in antibody molecules and generally determines the class to which the antibody belongs. The term "light chain" refers to the smaller of the two types of polypeptide chains found in naturally occurring conformations in antibody molecules. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0020] The term "complementarity determining region" or "CDR" refers to the amino acid sequence in the antibody variable region that imparts antigen specificity and binding affinity. For example, in general, there are three CDRs (e.g., CDR1-H, CDR2-H, and CDR3-H) in each heavy chain variable region, and three CDRs (CDR1-L, CDR2-L, and CDR3-L) in each light chain variable region. The precise amino acid sequence boundaries of CDRs can be determined using any of the many well-known schemes, including: Kabat numbering scheme, Chothia numbering scheme, IMGT numbering scheme, AHo numbering scheme, AbM numbering scheme. The precise amino acid sequence of a given CDR or FR may be different due to the different numbering schemes selected. It should be understood that "CDR" or "FR" of a given antibody or its region (e.g., its variable region) encompasses CDRs or FRs defined by any of the above-mentioned schemes or other known schemes. In the case where a specified CDR or FR contains a given amino acid sequence, it should be understood that such CDR or FR can also have the sequence of the corresponding CDR or FR defined by any of the above-mentioned schemes or other known schemes. The numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.

[0021] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., connected via a linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise indicated, the scFv herein may have the VL and VH in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus. The term "linker" refers to a molecular sequence that connects two molecules or two sequences on the same molecule. In some embodiments, the linker is a peptide linker. Preferably, the linker does not adversely affect the expression, secretion, or biological activity of the polypeptide. In addition, the linker is preferably not antigenic and does not elicit an immune response. In some embodiments, the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a GS-rich sequence), or a non-peptide chemical linker. In one embodiment, the linker of the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 111 or 112.

[0022] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs), including full-length antibodies (e.g., HCAbs) and antigen-binding fragments thereof (e.g., VH, VL, VHH). In some cases, the single domain antibody is selected from or engineered from HCAbs of camel or shark origin, and its heavy chain variable domain is referred to herein as "VHH". VHH has the following basic structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0023] PD1

[0024] In some embodiments, the PD1 binding molecule of the present invention comprises a PD1 binding domain, which is an antibody, ligand, or functional fragment thereof that targets PD1. PD1, also known as CD279, is a member of the immunoglobulin superfamily and a type I transmembrane protein primarily expressed on activated T cells, NK cells, B cells, macrophages, dendritic cells, and monocytes. It comprises an extracellular immunoglobulin variable domain, a transmembrane domain, and a cytoplasmic tail domain. The cytoplasmic tail domain contains two tyrosine motifs: an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor inhibitory tyrosine-based switch motif (ITSM). Studies have shown that the ITSM is essential for PD1's immunosuppressive function on active T cells. PD1's ligands include PDL1 (B7-H1, CD274) and PDL2 (B7-DC, CD273), both of which belong to the B7 family of proteins. PD1 exerts negative immune regulatory effects by interacting with its ligands, PDL1 and PDL2. When PD1 interacts with its ligand, its intracellular ITSM becomes phosphorylated and recruits the corresponding phosphatases SHP-1 and SHP-2, leading to dephosphorylation of downstream signaling molecules and thus downregulating immune cell responses. This negative regulatory mechanism of the immune system is a key molecular basis for maintaining immune tolerance. Numerous studies have shown that the overexpression of negative immune regulatory molecules such as PD1 and their interaction with the receptors PD-L1 / PD-L2, which induce an immunosuppressive state, play a crucial role in the pathogenesis of cancer and chronic infectious diseases such as HIV, HCV, and HBV.

[0025] In some embodiments, the PD1 binding domain is an antibody targeting PD1. Anti-PD1 antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 75, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 76. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:69, CDR2-H as shown in SEQ ID NO:70, and CDR3-H as shown in SEQ ID NO:71, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:72, CDR2-L as shown in SEQ ID NO:73, and CDR3-L as shown in SEQ ID NO:74.

[0026] In some embodiments, the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 75, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 76. Preferably, the antibody targeting PD1 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 75 and a light chain variable region as set forth in SEQ ID NO: 76.

[0027] In some embodiments, the antibody targeting PD1 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 77. Preferably, the antibody targeting PD1 is as shown in SEQ ID NO: 77.

[0028] In other embodiments, the PD1 binding domain contained in the PD1 binding molecule of the present invention is a ligand that binds to PD1, such as PDL1, PDL2, or a functional fragment thereof (i.e., a fragment having PD1 binding function, such as the extracellular region of PDL1 or PDL2). Preferably, the PD1 binding ligand comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 78 or 79. More preferably, the PD1 binding ligand comprises the amino acid sequence shown in SEQ ID NO: 78 or 79. In this embodiment, the transmembrane region of the PD1 binding molecule is preferably the transmembrane region of PDL1 or PDL2, and optionally, the PD1 binding molecule comprises the intracellular region of PDL1 or PDL2.

[0029] NKG2A

[0030] In some embodiments, the immunosuppressive molecule of the present invention comprises an NKG2A binding domain, which is an antibody, ligand, or functional fragment thereof that targets NKG2A. NKG2A, also known as KLRC1 or CD159A, is a member of the C-type lectin superfamily. It is a type II transmembrane protein primarily expressed in cytotoxic lymphocytes (such as CD8+ T cells and NK cells), comprising a cytoplasmic domain, a transmembrane domain, and an extracellular lectin-like domain. Its intracellular portion has two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that participate in inhibitory signal transduction. NKG2A can form a heterodimeric receptor with CD94, and NKG2A / CD94 transmits inhibitory signals to NK cells and CD8+ T cells. The natural ligand of NKG2A is HLA-E. The interaction between NKG2A and HLA-E contributes to tumor immune escape, and disrupting the interaction between NKG2A and HLA-E has been shown to effectively enhance anti-tumor immune responses. In addition, more and more research results show that NKG2A also plays an important role in other immune-related diseases, including viral infections, autoimmune diseases, inflammatory diseases, parasitic infections, etc.

[0031] In some embodiments, the NKG2A binding domain is an antibody targeting NKG2A. Anti-NKG2A antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO:7, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO:8. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 1, CDR2-H as shown in SEQ ID NO: 2, and CDR3-H as shown in SEQ ID NO: 3; the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 4, CDR2-L as shown in SEQ ID NO: 5, and CDR3-L as shown in SEQ ID NO: 6.

[0032] In some embodiments, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. Preferably, the antibody targeting NKG2A of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 8.

[0033] In some embodiments, the antibody targeting NKG2A is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9. Preferably, the antibody targeting NKG2A is as shown in SEQ ID NO: 9.

[0034] In other embodiments, the NKG2A binding domain comprised by the immunosuppressive molecule of the present invention is a ligand that binds NKG2A, such as HLA-E or a functional fragment thereof (i.e., a fragment having NKG2A binding function, such as the extracellular domain of HLA-E). Preferably, the NKG2A binding ligand comprises amino acids that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10 or 11. More preferably, the NKG2A binding ligand comprises the amino acid sequence set forth in SEQ ID NO: 10 or 11. In this embodiment, the transmembrane region of the immunosuppressive molecule may be the transmembrane region of HLA-E. However, typically, the immunosuppressive molecule in this embodiment does not comprise the intracellular domain of HLA-E. In other words, the immunosuppressive molecule is not full-length HLA-E.

[0035] TIM3

[0036] In some embodiments, the immunosuppressive molecule of the present invention comprises a TIM3 binding domain, which is an antibody, ligand, or functional fragment thereof that targets TIM3. TIM3, also known as HAVCR2 or CD366, is a member of the TIM family. It is a type I transmembrane protein that can be expressed in a variety of immune cells (such as Th1 cells, Th17 cells, monocytes, dendritic cells, macrophages, etc.), and comprises an extracellular immunoglobulin variable domain, a mucin domain, a transmembrane domain, and an intracellular domain. Studies have found that TIM3 binds to different ligands (such as Galectin9, HMGB1, CEACAM1), causing signal transduction of immune cells. Galectin9 has a high affinity for the TIM3IgV domain, and the TIM3 / Galectin9 signaling pathway can mediate T cell exhaustion. In addition, Galectin9 can promote the maturation of TIM3+ antigen-presenting cells, thereby enhancing adaptive immunity. Therefore, the TIM3 / Galectin9 signaling pathway plays an important role in the body's immune regulation. HMGB1 is a DNA-binding protein that acts as a chromatin factor and plays a crucial role in inflammation, immune responses, DC differentiation, genomic stability maintenance, and nuclear transcriptional regulation. HMGB1 is a ligand for TIM3. In the tumor microenvironment, tumor-infiltrating DCs, which highly express TIM3, compete with nucleic acids released by tumor cells for HMGB1 binding, preventing nucleic acids from entering endosomes and thereby inhibiting nucleic acid-stimulated innate immune responses. Consequently, the activation and function of innate immune cells are impaired. Blocking TIM3-mediated nucleic acid signaling may be beneficial for DNA vaccine research. CEACAM1, a TIM3 ligand, binds to TIM3 through its N-terminal domain to form a cis-heterodimer, promoting stable TIM3 expression on the surface of immune cells. However, trans-interactions between CEACAM1 and TIM3 can inhibit immune cell function. Both cis and trans interactions between CEACAM1 and TIM3 maintain immune cell tolerance. Previous studies have shown that combined blockade of the CEACAM1 and TIM3 pathways can enhance antitumor immune responses and improve tumor clearance in patients with colorectal cancer. Therefore, the interaction between CEACAM1 and TIM3 plays a key role in regulating autoimmunity and anti-tumor immunity.

[0037] In some embodiments, the TIM3 binding domain is an antibody targeting TIM3. Any anti-TIM3 antibody known in the art can be used in the present invention. In some embodiments, the antibody targeting TIM3 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 19, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 20. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 13, CDR2-H as shown in SEQ ID NO: 14, and CDR3-H as shown in SEQ ID NO: 15, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 16, CDR2-L as shown in SEQ ID NO: 17, and CDR3-L as shown in SEQ ID NO: 18.

[0038] In some embodiments, the antibody targeting TIM3 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20. Preferably, the antibody targeting TIM3 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 19 and a light chain variable region as set forth in SEQ ID NO: 20.

[0039] In some embodiments, the antibody targeting TIM3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 21. Preferably, the antibody targeting TIM3 is as shown in SEQ ID NO: 21.

[0040] In other embodiments, the TIM3 binding domain contained in the immunosuppressive molecule of the present invention is a ligand that binds to TIM3, such as Galectin 9, HMGB1, CEACAM1, or a functional fragment thereof (i.e., a fragment having TIM3 binding function, such as the extracellular region of CEACAM1). Preferably, the TIM3 binding ligand comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 22-24. More preferably, the TIM3 binding ligand comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-24. In a specific embodiment, the TIM3 binding ligand comprises the extracellular region of CEACAM1 set forth in SEQ ID NO: 24. In this embodiment, the transmembrane region of the immunosuppressive molecule is preferably the transmembrane region of CEACAM1. Typically, however, the immunosuppressive molecule in this embodiment does not comprise the intracellular region of CEACAM 1. In other words, the immunosuppressive molecule is not full-length CEACAM 1.

[0041] LAG3

[0042] In some embodiments, the immunosuppressive molecules of the present invention comprise a LAG3 binding domain, which is an antibody, ligand, or functional fragment thereof that targets LAG3. LAG3, also known as CD223, is a type I transmembrane protein that belongs to the immunoglobulin superfamily (IgSF). It is expressed as a dimer or oligomer on the surface of activated CD4+ and CD8+ T cells, regulatory T cells, Tr1 cells, NK cells, and plasmacytoid dendritic cells (pDCs). The LAG3 protein has a homologous structure to CD4, with both possessing four Ig-like domains in their extracellular regions. However, LAG3 contains an additional loop structure within the distal Ig-like domain of the membrane, which CD4 lacks. This additional ring structure enables the LAG3 protein to bind to MHC class II molecules (such as HLA-DR, HLA-DP, and HLA-DQ antigens) expressed on antigen-presenting cells (APCs) or tumor cells with significantly higher affinity than CD4, and can negatively regulate T cell receptor (TCR) signaling. In addition to MHC class II molecules, Galectin3, LSECtin, and FGL1 are also ligands for LAG3. These three proteins are expressed on various tumor cells and inhibit T cell responses. Studies have shown that LAG3 can negatively regulate T cell activation, proliferation, and cytokine production, inhibit pDC activation, and enhance the suppressive activity of regulatory T cells.

[0043] In some embodiments, the LAG3 binding domain is an antibody targeting LAG3. Any anti-LAG3 antibody known in the art can be used in the present invention. In some embodiments, the antibody targeting LAG3 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 32, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 33. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:26, CDR2-H as shown in SEQ ID NO:27, and CDR3-H as shown in SEQ ID NO:28, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:29, CDR2-L as shown in SEQ ID NO:30, and CDR3-L as shown in SEQ ID NO:31.

[0044] In some embodiments, the antibody targeting LAG3 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 33. Preferably, the antibody targeting LAG3 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 32 and a light chain variable region as set forth in SEQ ID NO: 33.

[0045] In some embodiments, the antibody targeting LAG3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 34. Preferably, the antibody targeting LAG3 is as set forth in SEQ ID NO: 34.

[0046] In other embodiments, the LAG3-binding domain comprised by the immunosuppressive molecules of the present invention is a LAG3-binding ligand, such as an MHC class II molecule, Galectin 3, LSECtin, FGL1, or a functional fragment thereof (i.e., a fragment having LAG3-binding function, such as the extracellular domain of LSECtin). Preferably, the LAG3-binding ligand comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 35-37. More preferably, the LAG3-binding ligand comprises the amino acid sequence set forth in any one of SEQ ID NOs: 35-37. In a specific embodiment, the LAG3-binding ligand comprises the extracellular domain of LSECtin set forth in SEQ ID NO: 37. In this embodiment, the transmembrane region of the immunosuppressive molecule is preferably the transmembrane region of LSECtin. Typically, however, the immunosuppressive molecule in this embodiment does not comprise the intracellular region of LSECtin. In other words, the immunosuppressive molecule is not the full-length LSECtin.

[0047] TIGIT

[0048] In some embodiments, the immunosuppressive molecule of the present invention comprises a TIGIT binding domain, which is an antibody, ligand or functional fragment thereof targeting TIGIT. TIGIT, also known as VSTM3, VSIG9 or WUCAM, is a member of the PVR-like protein family. It is a type I transmembrane protein mainly expressed in NK cells and T cells (such as CD4+T cells, CD8+T cells and Treg cells, etc.), comprising an extracellular immunoglobulin variable domain, a transmembrane domain and a short intracellular domain, with an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT)-like motif. TIGIT has four ligands: CD112, CD113, CD155 and Nectin4, all of which belong to the Nectin / NECL family, which is involved in mediating processes such as cell adhesion and cell polarization. The binding affinity of TIGIT to CD155 and Nectin4 is much higher than that of CD112 and CD113. CD155 also acts as a ligand for the activating receptor CD226. When CD155 binds to CD226, it transmits an activation signal to immune cells. When CD155 binds to TIGIT, it recruits the SHP1 phosphatase to the membrane via its ITIM motif, transmitting an inhibitory signal and subsequently inactivating numerous proteins involved in T cell effector functions. Nectin4 is the only member of the nectin / NECL family of proteins that specifically binds to TIGIT without interacting with CD226, CD96, or CD112R.

[0049] In some embodiments, the TIGIT binding domain is an antibody targeting TIGIT. Anti-TIGIT antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting TIGIT comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 45, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 46. In some embodiments, the light chain variable region comprises CDR1-L as shown in SEQ ID NO:39, CDR2-L as shown in SEQ ID NO:40, and CDR3-L as shown in SEQ ID NO:41; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:42, CDR2-H as shown in SEQ ID NO:43, and CDR3-H as shown in SEQ ID NO:44.

[0050] In some embodiments, the antibody targeting TIGIT comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 46. Preferably, the antibody targeting TIGIT in the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 45 and a light chain variable region as shown in SEQ ID NO: 46.

[0051] In some embodiments, the antibody targeting TIGIT is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 47. Preferably, the antibody targeting TIGIT is as shown in SEQ ID NO: 47.

[0052] In other embodiments, the TIGIT binding domain contained in the immunosuppressive molecule of the present invention is a ligand that binds to TIGIT, such as CD155, CD112, CD113, Nectin4 or a functional fragment thereof (i.e., a fragment having TIGIT binding function, such as the extracellular region of CD155, CD112, CD113 or Nectin4). Preferably, the ligand that binds to TIGIT comprises an amino acid that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 48-51. More preferably, the ligand that binds to TIGIT comprises an amino acid sequence shown in any one of SEQ ID NOs: 48-51. In this embodiment, the transmembrane region of the immunosuppressive molecule can be the corresponding transmembrane region of CD155, CD112, CD113 or Nectin4. Typically, however, the immunosuppressive molecule in this embodiment does not comprise the corresponding intracellular region of CD155, CD112, CD113 or Nectin 4. In other words, the immunosuppressive molecule is not full-length CD155, CD112, CD113 or Nectin 4.

[0053] CTLA4

[0054] In some embodiments, the immunosuppressive molecule of the present invention comprises a CTLA4 binding domain, which is an antibody, ligand, or functional fragment thereof that targets CTLA4. CTLA4, also known as CD152 or CELIAC3, is a member of the CD28 immunoglobulin subfamily. It is a type I transmembrane protein that is primarily expressed on T cells and comprises an extracellular immunoglobulin variable domain, a transmembrane domain, and a conserved cytoplasmic tail. There is a very conserved motif (MYPPPY) on the extracellular domain of the CD28 immunoglobulin subfamily, which is crucial for its binding to ligands CD80 and CD86. CTLA4's ligands CD80 and CD86 are typically found on the surface of antigen-presenting cells and can bind to CD28 or CTLA4 to produce co-stimulatory or co-inhibitory responses, respectively, but CD80 and CD86 have higher binding affinity to CTLA4. Due to this inhibitory effect, CTLA4 is a key regulator in regulating T cell homeostasis and self-tolerance.

[0055] In some embodiments, the CTLA4 binding domain is an antibody targeting CTLA4. Anti-CTLA4 antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 62, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 63. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:56, CDR2-H as shown in SEQ ID NO:57, and CDR3-H as shown in SEQ ID NO:58, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:59, CDR2-L as shown in SEQ ID NO:60, and CDR3-L as shown in SEQ ID NO:61.

[0056] In some embodiments, the antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 62, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 63. Preferably, the antibody targeting CTLA4 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 62 and a light chain variable region as set forth in SEQ ID NO: 63.

[0057] In some embodiments, the antibody targeting CTLA4 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 64. Preferably, the antibody targeting CTLA4 is as shown in SEQ ID NO: 64.

[0058] In other embodiments, the CTLA4 binding domain contained in the immunosuppressive molecule of the present invention is a ligand that binds to CTLA4, such as CD80, CD86, or a functional fragment thereof (i.e., a fragment having CTLA4 binding function, such as the extracellular region of CD80 or CD86). Preferably, the CTLA4-binding ligand comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 65 or 66. More preferably, the CTLA4-binding ligand comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66. In this embodiment, the transmembrane region of the immunosuppressive molecule is preferably the transmembrane region of CD80 or CD86. However, typically, the immunosuppressive molecule in this embodiment does not comprise the intracellular region of CD80 or CD86. In other words, the immunosuppressive molecule is not full-length CD80 or CD86.

[0059] IRP60

[0060] In some embodiments, the immunosuppressive molecule of the present invention comprises an IRP60 binding domain, which is an antibody, ligand, or functional fragment thereof that targets IRP60. IRP60, also known as CD300a, is a type I transmembrane inhibitory receptor. IRP60 is widely expressed on the surface of myeloid cells (e.g., dendritic cells, mast cells, granulocytes, monocytes) and lymphocytes (T cells, B cells, etc.), and its expression level varies depending on the cell type and degree of differentiation. IRP60 contains four ITIMs, and the initiation of its inhibitory signal depends on the phosphorylation of tyrosine residues in the ITIMs. By interacting with ligand aminophospholipids (e.g., PtdEth and / or PtdSer), IRP60 participates in the regulation of various cellular activities, such as cell growth, proliferation, apoptosis, differentiation, and immune response. It has been reported that IRP60 is closely related to the development of diseases such as hematological malignancies (e.g., leukemia, lymphoma, etc.), infectious diseases, allergic reactions, and chronic inflammatory reactions.

[0061] In some embodiments, the IRP60 binding domain is an antibody targeting IRP60, such as TX41, TX49, etc. Any anti-IRP60 antibody known in the art can be used in the present invention. In some embodiments, the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 150, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 151. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 144, CDR2-H as shown in SEQ ID NO: 145, and CDR3-H as shown in SEQ ID NO: 146, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 147, CDR2-L as shown in SEQ ID NO: 148, and CDR3-L as shown in SEQ ID NO: 149.

[0062] In some embodiments, the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 150, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 151. Preferably, the antibody targeting IRP60 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 150 and a light chain variable region as set forth in SEQ ID NO: 151.

[0063] In some embodiments, the antibody targeting IRP60 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 152. Preferably, the antibody targeting IRP60 is as shown in SEQ ID NO: 152.

[0064] SIRPα

[0065] In some embodiments, the immunosuppressive molecule of the present invention comprises a SIRPα binding domain, which is an antibody, ligand, or functional fragment thereof that targets SIRPα. SIRPα, also known as signal regulatory protein a or CD172a, is a transmembrane protein expressed on the surface of macrophages, dendritic cells, NK cells, and nerve cells, and its intracellular region contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The ligand of SIRPα is CD47, a cell surface protein that transmits a "don't eat me" signal on healthy cells. In addition, CD47 is also widely expressed on various tumor cells as a mechanism to evade immune detection. Many studies have found that inhibiting the CD47-SIRPα signaling pathway in cancer cells (for example, by anti-CD47 antibodies or anti-SIRPα antibodies) can promote the phagocytosis of tumor cells by macrophages, ultimately limiting tumor growth.

[0066] In some embodiments, the SIRPα binding domain is an antibody targeting SIRPα. Anti-SIRPα antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting SIRPα comprises a light chain variable region and a heavy chain variable region, and the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 159, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 160. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 153, CDR2-H as shown in SEQ ID NO: 154, and CDR3-H as shown in SEQ ID NO: 155, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 156, CDR2-L as shown in SEQ ID NO: 157, and CDR3-L as shown in SEQ ID NO: 158.

[0067] In some embodiments, the antibody targeting SIRPα comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 159, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 160. Preferably, the antibody targeting SIRPα of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 159 and a light chain variable region as shown in SEQ ID NO: 160.

[0068] In some embodiments, the antibody targeting SIRPα is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 161. Preferably, the antibody targeting SIRPα is as shown in SEQ ID NO: 161.

[0069] In other embodiments, the SIRPα binding domain contained in the immunosuppressive molecule of the present invention is a ligand that binds to SIRPα, such as CD47 or a functional fragment thereof (i.e., a fragment having SIRPα binding function, such as the extracellular region of CD47). Preferably, the ligand that binds to SIRPα comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 163. More preferably, the ligand that binds to SIRPα comprises the amino acid sequence shown in SEQ ID NO: 163. In this embodiment, the transmembrane region of the immunosuppressive molecule is preferably the transmembrane region of CD47. However, generally, the immunosuppressive molecule in this embodiment does not comprise the intracellular region of CD47. In other words, the immunosuppressive molecule is not full-length CD47.

[0070] KIR2DL1 / 2 / 3

[0071] In some embodiments, the immunosuppressive molecule of the present invention comprises a KIR2DL1 / 2 / 3 binding domain, and the KIR2DL1 / 2 / 3 binding domain is an antibody, ligand or a functional fragment thereof that targets KIR2DL1, KIR2DL2 or KIR2DL3 (i.e., KIR2DL1 / 2 / 3). According to the number of Ig-like domains contained in the extracellular region, KIR can be divided into KIR2D and KIR3D subfamilies. According to the length of the cytoplasmic region, KIR can also be divided into long type (L) and short type (S), such as KIR2DL, KIR2DS, KIR3DL, KIR3DS. Among them, the cytoplasmic region of KIR2DL1, KIR2DL2, and KIR2DL3 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and belongs to inhibitory KIR receptors. Specifically, when the ITIM contained in the cytoplasmic region of KIR2DL1, KIR2DL2, and KIR2DL3 is phosphorylated, it can recruit the phosphatases SHP1 and SHP2, leading to dephosphorylation of cellular substrates and ultimately inhibiting or terminating the effector functions of NK cells, such as cytotoxicity and cytokine secretion.

[0072] In some embodiments, the KIR2DL1 / 2 / 3 binding domain is an antibody targeting KIR2DL1 / 2 / 3. Anti-KIR2DL1 / 2 / 3 antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting KIR2DL1 / 2 / 3 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 119 or 122, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 120 or 123. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 113, CDR2-H as shown in SEQ ID NO: 114, and CDR3-H as shown in SEQ ID NO: 115, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 116, CDR2-L as shown in SEQ ID NO: 117, and CDR3-L as shown in SEQ ID NO: 118.

[0073] In some embodiments, the antibody targeting KIR2DL1 / 2 / 3 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 119 or 122, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 120 or 123. Preferably, the antibody targeting KIR2DL1 / 2 / 3 of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 119 and a light chain variable region as shown in SEQ ID NO: 120, or a heavy chain variable region as shown in SEQ ID NO: 122 and a light chain variable region as shown in SEQ ID NO: 123.

[0074] In some embodiments, the antibody targeting KIR2DL1 / 2 / 3 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 121 or 124. Preferably, the antibody targeting KIR2DL1 / 2 / 3 is as shown in SEQ ID NO: 121 or 124.

[0075] LILRB1

[0076] In some embodiments, the immunosuppressive molecule of the present invention comprises a LILRB1 binding domain, which is an antibody, ligand or functional fragment thereof that targets LILRB1. LILRB1, also known as ILT2 or CD85j, is an immunoglobulin-like receptor belonging to the LILR family. LILRB1 is usually expressed on the surface of immune cells, particularly on monocytes, dendritic cells, T cells and B cells. The ligands of LILRB1 include molecules such as HLA-G. HLA-G is a type of MHC class I molecule that is expressed on the surface of placental tissue, fetus and certain tumor cells. LILRB1 participates in regulating the activation state of immune cells by binding to its ligand, and may play a role in immunoregulation, tolerance and anti-inflammatory processes. This interaction is crucial for regulating immune responses and maintaining the balance of the immune system.

[0077] In some embodiments, the LILRB1 binding domain is an antibody targeting LILRB1. Anti-LILRB1 antibodies known in the art can be used in the present invention. In some embodiments, the antibody targeting LILRB1 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 131 or 140, and the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 132 or 141. In some embodiments, the heavy chain variable region comprises a CDR1-H as shown in SEQ ID NO: 134, a CDR2-H as shown in SEQ ID NO: 135, and a CDR3-H as shown in SEQ ID NO: 136, and the light chain variable region comprises a CDR1-L as shown in SEQ ID NO: 137, a CDR2-L as shown in SEQ ID NO: 138, and a CDR3-L as shown in SEQ ID NO: 139; or the heavy chain variable region comprises a CDR1-H as shown in SEQ ID NO: 125, a CDR2-H as shown in SEQ ID NO: 126, and a CDR3-H as shown in SEQ ID NO: 127, and the light chain variable region comprises a CDR1-L as shown in SEQ ID NO: 128, a CDR2-L as shown in SEQ ID NO: 129, and a CDR3-L as shown in SEQ ID NO: 130.

[0078] In some embodiments, the antibody targeting LILRB1 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 131 or 140, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 132 or 141. Preferably, the antibody targeting KIR2DL1 / 2 / 3 of the present invention comprises a heavy chain variable region as shown in SEQ ID NO: 131 and a light chain variable region as shown in SEQ ID NO: 132, or a heavy chain variable region as shown in SEQ ID NO: 140 and a light chain variable region as shown in SEQ ID NO: 141.

[0079] In some embodiments, the antibody targeting LILRB1 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 133 or 142. Preferably, the antibody targeting KIR2DL1 / 2 / 3 is as shown in SEQ ID NO: 133 or 142.

[0080] The term sequence "identity" refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity. It is known to those skilled in the art that sequence identity can be determined using algorithms such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.

[0081] The term "transmembrane domain" refers to a polypeptide structure that enables protein expression on the cell surface and anchors the target-binding domain to the cell membrane. Transmembrane domains can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. When the target-binding domain binds to its target, the transmembrane domain enables signal transduction. Particularly suitable transmembrane domains for use in the present invention can be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD28, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD112, CD113, CD134, CD137, CD154, CD155, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, BCMA, IL-2Rβ, IL-2Rγ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, LSECtin, PDL1, PDL2, CEACAM1, MHC class I molecules, MHC class II molecules, CRT AM, Ly9, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. In some embodiments, the transmembrane domain is derived from the following molecules: CD8α, CD4, CD28 or 4-1BB, or the transmembrane domain can be synthetic and can mainly contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 82, or the transmembrane domain is derived from CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 83 or 84.In other embodiments, the transmembrane domain contained in the PD1 binding molecules or immunosuppressive molecules of the present invention is derived from a ligand or receptor that targets the PD1 binding molecules or immunosuppressive proteins, such as HLA-E, CEACAM1, LSECtin, CD47, CD112, CD113, CD155, Nectin4, CD80, CD86, PDL1, PDL2, and some MHC class II molecules. Preferably, the transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 12, 25, 38, 52, 53, 54, 55, 67, 68, 80, 81, and 162. More preferably, the transmembrane domain is connected to the extracellular domain of the corresponding ligand or receptor, preferably directly connected.

[0082] In some embodiments, a hinge region is further included between the binding domain and the transmembrane domain. The term "hinge region" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antibody. The hinge region can contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from natural molecules, such as whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it can be a fully synthetic hinge sequence. Preferably, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably a CD8α, CD28 or IgG4 hinge. In some embodiments, the hinge region is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the hinge region is from CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 86 or 87. In some embodiments, the hinge region is from IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 88.

[0083] The term "costimulatory domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function, and is an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular region of the costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (e.g., proliferation and survival). The costimulatory signaling domain of any costimulatory molecule is suitable for use in immunosuppressive molecules as described herein. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. The costimulatory domains of the present invention include but are not limited to the intracellular region derived from the following proteins:LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70 and their combinations. Preferably, the costimulatory domains of CAR of the present invention are 4-1BB and / or CD28. In some embodiments, the costimulatory domain is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 89. In some embodiments, the costimulatory domain is from 4-1BB, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 90 or 91.

[0084] The term "primary signaling domain" refers to a protein structure that works together to initiate primary signaling after antigen-receptor binding, which is generally an intracellular sequence of a T cell receptor and a co-receptor. The primary signaling domain generally contains one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The primary signaling domain of the present invention includes, but is not limited to, intracellular regions derived from the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d. In some embodiments, the immunosuppressive molecules of the present invention do not contain a CD3ζ intracellular region, for example, a CD3ζ intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 92 or 93.

[0085] In some embodiments, the PD1-binding molecules or immunosuppressive molecules of the present invention further comprise a signal peptide, so that when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long, hydrophobic amino acid stretch that has a tendency to form a single α-helix. At the end of the signal peptide, there is typically an amino acid stretch that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after translocation to produce a free signal peptide and the mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, such as those derived from B2M, CD8α, IgG1, GM-CSFRα, and the like. In some embodiments, the signal peptide useful in the present invention is derived from B2M and has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:94. In some embodiments, the signal peptide useful in the present invention is derived from CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 95 or 96.

[0086] In some embodiments, the engineered cells of the invention also express a functional exogenous receptor (as defined below).

[0087] Functional xenobiotic receptors

[0088] In some embodiments, the engineered cells expressing immunosuppressive molecules of the present invention may also express functional exogenous receptors. The functional exogenous receptors described in the present invention include chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell receptor fusion proteins (TFPs), T cell antigen couplers (TACs), or immune mobilization monoclonal T cell receptors (ImmTACs), etc., preferably chimeric antigen receptors or T cell receptors, more preferably chimeric antigen receptors.

[0089] The term "T cell receptor" or "TCR" refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen-presenting cells, which present antigenic peptides to T cells and bind to the TCR complex to induce a series of intracellular signaling. The TCR consists of six peptide chains that form heterodimers, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to a specific particular antigen and MHC molecule. The variable region of the TCR may include a ligand binding domain or be operably linked to a ligand binding domain, wherein the definition of the ligand binding domain is as described below.

[0090] The term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen (e.g., tumor antigen) binding domain (e.g., a ligand for an antibody or antigen), a transmembrane domain, an optional costimulatory domain, and a primary signaling domain, with each domain connected by a linker. CAR is capable of redirecting the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC restricted manner. In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor comprising a tumor antigen binding domain, a transmembrane domain, and a primary signaling domain, and optionally, one or more costimulatory domains. In some embodiments, the chimeric antigen receptor further comprises one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, and the like.

[0091] The term "T cell antigen coupler" or "TAC" includes three functional domains: (1) a tumor targeting domain, which can be a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, which is linked to the protein kinase LCK and catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.

[0092] The term "T cell receptor fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of a TCR, typically consisting of a TCR subunit and an antigen-binding region attached thereto, and expressed on the cell surface. The TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

[0093] The term "immune mobilizing monoclonal T cell receptor" or "ImmTAC" is composed of an engineered T cell receptor (TCR) and an anti-CD3 scFv, wherein: the engineered TCR can specifically recognize and bind to the HLA-peptide complex on the surface of tumor cells with significantly improved affinity, and promote T cell-mediated effector function through the interaction of the scFv antibody fragment with CD3.

[0094] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen (e.g., a tumor antigen). In some embodiments, the extracellular domain comprises an antibody that specifically binds to an antigen or a ligand for the antigen. In some embodiments, the antigen is selected from the group consisting of ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2,EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variants of tumor necrosis, and any combination thereof. Preferably,The antigen is selected from CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor α, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from CD19, Claudin18.2, MSLN, GPRC5D, ROR1, CD7 and BCMA.

[0095] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD19, such as an antibody targeting CD19. Antibodies targeting CD19 known in the art can be used in the present invention. In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO: 103; wherein the CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO: 104. In some embodiments, the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:97, CDR2-H as shown in SEQ ID NO:98, and CDR3-H as shown in SEQ ID NO:99, and the light chain variable region comprises CDR1-L as shown in SEQ ID NO:100, CDR2-L as shown in SEQ ID NO:101, and CDR3-L as shown in SEQ ID NO:102.

[0096] In some embodiments, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 103, and the light chain variable region is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 104. Preferably, the antibody targeting CD19 of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 103 and a light chain variable region as set forth in SEQ ID NO: 104.

[0097] In some embodiments, the antibody targeting CD19 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 105. Preferably, the antibody targeting CD19 is as shown in SEQ ID NO: 105.

[0098] In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically recognizes Claudin 18.2, such as an antibody targeting Claudin 18.2. Antibodies targeting Claudin 18.2 known in the art can be used in the present invention. In some embodiments, the antibody targeting Claudin 18.2 comprises a VHH, wherein the CDR1, CDR2, and CDR3 comprised by the VHH are identical to the CDR1, CDR2, and CDR3 comprised by SEQ ID NO: 109. In some embodiments, the CDR1 comprised by the VHH is as shown in SEQ ID NO: 106, the CDR2 is as shown in SEQ ID NO: 107, and the CDR3 is as shown in SEQ ID NO: 108.

[0099] In some embodiments, the antibody targeting Claudin 18.2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 109. Preferably, the antibody targeting Claudin 18.2 of the present invention comprises the amino acid sequence shown in SEQ ID NO: 109.

[0100] In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain, and optionally, one or more costimulatory domains. In some embodiments, the functional exogenous receptor of the present invention is a chimeric antigen receptor that targets CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor α, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, or any combination thereof, more preferably targeting CD19, Claudin 18.2, MSLN, ROR1, GPRC5D, CD7, BCMA, or any combination thereof. In some embodiments, the chimeric antigen receptor further comprises a signal peptide, a hinge region, or both. For the definitions of the transmembrane domain, co-stimulatory domain, primary signaling domain, and optional hinge region, signal peptide and other structures that can be used in the CAR of the present invention, please refer to the above section "PD1 binding molecules and immunosuppressive molecules".

[0101] In some embodiments, the CAR of the present invention may further comprise a switch structure to regulate the expression time of CAR. For example, the switch structure may be in the form of a dimerization domain, which causes a conformational change by binding to its corresponding ligand, exposing the extracellular binding domain so that it binds to the targeted antigen, thereby activating the signal transduction pathway. Alternatively, the switch domain may be used to connect the binding domain and the signal transduction domain respectively, and only when the switch domains bind to each other (for example, in the presence of an inducing compound) can the binding domain and the signal transduction domain be connected together through a dimer, thereby activating the signal pathway. The switch structure may also be in the form of a masking peptide. The masking peptide may shield the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain may be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art may be used in the present invention.

[0102] In some embodiments, the CAR of the present invention may also include a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to remove the CAR cells when needed (e.g., when serious toxic side effects are produced). For example, the suicide gene can be in the form of an inserted epitope, such as a CD20 epitope, RQR8, etc., and when needed, the CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which can cause cells to die under the induction of ganciclovir treatment. The suicide gene can also be iCaspase-9, which can be induced by chemical induction drugs such as AP1903, AP20187, etc. to dimerize iCaspase-9, thereby activating downstream Caspase3 molecules, leading to cell apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0103] cell

[0104] In some embodiments, the engineered cells of the invention are engineered immune cells.

[0105] The term "immune cell" refers to any cell with one or more effector functions (for example, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC) of the immune system. For example, immune cell can be a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell. Immune cell can be obtained from a variety of sources, for example, from a subject (for example, from the subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue, tumor etc. separation and obtain), or from a cell line (such as Jurkat, SupT1, NK92 etc.) cultured in vitro, or from stem cell differentiation (for example, derived from cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSC etc.). Preferably, immune cell is a T cell or a NK cell, more preferably a T cell. T cell can also be concentrated or purified. T cells may be at any developmental stage, including but not limited to: CD4+CD8+T cells, CD4+T cells (e.g., Th1 and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), CD4-CD8-T cells, tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. Preferably, the immune cells are human T cells, which can be obtained using a variety of techniques known to those skilled in the art, such as isolating T cells from the subject's blood using Ficoll.

[0106] In other embodiments, the cells are stem cells.

[0107] The term "stem cell" is a primitive cell with the potential for self-replication, multidirectional differentiation and homing. It is the origin cell of the body and the ancestral cell that forms various tissues and organs of the human body. In the process of cell differentiation, due to the uneven distribution of regulatory differentiation proteins in the cytoplasm, a daughter cell irreversibly moves towards the terminal of differentiation and becomes a differentiated cell with a specific function, until it completely loses the ability to divide again and eventually ages and dies. In order to make up for this deficiency, the body retains a part of the undifferentiated primitive cells, which retain the characteristics of the parent. This part of the undifferentiated primitive cells that are retained are called stem cells. Once needed, these stem cells can produce differentiated cells by dividing according to the developmental pathway. The stem cells described in the present invention can be embryonic stem cells, adult stem cells (such as umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells, etc.) and pluripotent stem cells (such as induced pluripotent stem cells iPSC, etc.).

[0108] In some embodiments, the expression of endogenous HLA-I and / or HLA-II genes in the engineered cells of the present invention is not modified. That is, the expression level of any endogenous HLA-I and / or HLA-II genes is not altered by any artificial intervention method (gene editing or non-gene editing).

[0109] In some embodiments, the expression of at least one endogenous HLA-class I gene of the engineered cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-class II gene of the engineered cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene of the engineered cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-class I gene of the engineered cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous HLA-class I and HLA-class II gene of the engineered cells of the present invention is suppressed or silenced. In some embodiments, the expression of at least one endogenous TCR / CD3 gene, at least one endogenous HLA-class I gene, and at least one endogenous HLA-class II gene of the engineered cells of the present invention is suppressed or silenced. Preferably, the HLA-class I gene is selected from HLA-A, HLA-B, HLA-C, and B2M. Preferably, the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA. Preferably, the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε and CD3ζ.

[0110] In some embodiments, the expression of one or more endogenous genes selected from the group consisting of CD52, GR, dCK, and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, and CASP10 is suppressed or silenced in the engineered cells of the present invention. , CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, I L10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

[0111] Methods for inhibiting gene expression or silencing genes are well known to those skilled in the art, including but not limited to, for example, DNA or RNA breakage mediated by large-range nucleases, zinc finger nucleases, TALENs, CRISPR / Cas systems, base editors, or gene inactivation by antisense oligonucleotides, RNAi, shRNA, transposons, mutations, and the like.

[0112] In some embodiments, the engineered cells of the present invention are allogeneic cells. As used herein, the term "allogeneic" refers to any material derived from a different animal or patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to one another when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may differ genetically enough to allow antigenic interaction.

[0113] Pharmaceutical composition

[0114] The present invention also provides a pharmaceutical composition comprising the engineered cells of the present invention as an active agent and one or more pharmaceutically acceptable excipients. Therefore, the present invention also encompasses the use of the engineered cells in the preparation of a pharmaceutical composition or a medicament.

[0115] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavorings, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coatings, isotonic agents, absorption delaying agents, stabilizers, and tension modifiers. It is known to those skilled in the art to select suitable excipients to prepare the pharmaceutical compositions desired in the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. In general, the choice of suitable excipients depends on, inter alia, the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

[0116] The pharmaceutical composition according to the present invention can be applied in a variety of ways. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0117] The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The processes known in the present invention for producing drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions comprising, for example, immune cells as described herein are typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

[0118] The pharmaceutical composition according to the present invention can also be used in combination with one or more other medicaments (biological agents such as antibody reagents, and / or small molecules) or therapeutic methods (such as surgery, chemotherapy or radiotherapy) that are applicable to the treatment and / or prevention of the disease to be treated. Preferred examples of the medicament suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium porphyrin II (sorfimer sodium photofrin II), temozolomide, topotecan, trimetreate glucuronate (trimetreate glucuronate), auristatin E (auristatin E), vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes and RNA enzymes; radionuclides such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as platelet factor 4 and melanoma growth stimulating protein; antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains and viral / bacterial peptides. In addition, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0119] Uses and treatment methods

[0120] The present invention also provides uses of the pharmaceutical composition or engineered cells described above in the preparation of drugs for diseases such as cancer, infection or autoimmunity, as well as methods for treating cancer, infection or autoimmune diseases.

[0121] In some embodiments, the cancer is a cancer associated with expression of a target to which a functional exogenous receptor binds. For example, the cancer includes, but is not limited to, brain gliomas, blastomas, sarcomas, leukemias, basal cell carcinomas, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancers of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumors, lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous lung cancer), lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancers of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignancies of the urinary system, vulvar cancer, and other carcinomas and sarcomas, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL) ), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large mass disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myeloid leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell neoplasm, and post-transplantation lymphoproliferative disorder (PTLD); and other diseases related to target expression. Preferably, the disease that can be treated with the engineered cells or pharmaceutical compositions of the present invention is selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, etc.

[0122] In some embodiments, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0123] In some embodiments, the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus.

[0124] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with examples. It should be noted that those skilled in the art should understand that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and do not constitute any limitation to the present invention. The embodiments of the present invention and the features therein may be combined with each other unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1: CAR expression levels in CAR19T, CAR19-P28A T, and CAR19-PNRA T cells.

[0126] Figure 2: Anti-NKG2A antibody expression levels of CAR19T, CAR19-P28A T, and CAR19-PNRA T cells.

[0127] Figure 3: PD-L1 expression levels of CAR19T, CAR19-P28A T, and CAR19-PNRA T cells.

[0128] Figure 4: In vitro killing effect of CAR19T, CAR19-P28A T and CAR19-PNRA T cells on target cells.

[0129] Figure 5: Cytokine release levels after CAR19T, CAR19-P28A T, and CAR19-PNRA T cells were co-incubated with target cells.

[0130] Figure 6: Inhibitory effects of CAR19T, CAR19-P28A T, and CAR19-PNRA T cells on tumors in vivo. DETAILED DESCRIPTION

[0131] Example

[0132] Example 1: Construction of anti-CD19 CAR-T cells expressing PD-L1 and NKG2A binding molecules and verification of their function

[0133] The following coding sequences were synthesized and cloned into the pGEM-T Easy vector (Promega) in sequence: anti-CD19scFv (SEQ ID NO: 105), CD8α hinge region (SEQ ID NO: 86), CD8α transmembrane region (SEQ ID NO: 83), 4-1BB co-stimulatory domain (SEQ ID NO: 90), CD3ζ intracellular region (SEQ ID NO: 92) to obtain the CAR19 plasmid, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0134] The CAR19 plasmid prepared in Example 1 further includes a PD1 binding molecule and an NKG2A binding molecule (connected by a 2A peptide). The structure of the PD1 binding molecule is as follows: PD-L1 signal peptide (SEQ ID NO: 164), PD-L1 extracellular domain (SEQ ID NO: 78), PD-L1 transmembrane domain (SEQ ID NO: 80), and the structure of the NKG2A binding molecule is as follows: B2M signal peptide (SEQ ID NO: 94), NKG2A binding domain (SEQ ID NO: 9), IgG4 hinge region (SEQ ID NO: 88), CD28 transmembrane region (SEQ ID NO: 82), CD28 costimulatory domain (SEQ ID NO: 89). The CAR19-PNRA plasmid was obtained, and the correct insertion of the target sequence in the plasmid was confirmed by sequencing.

[0135] The PD1 binding molecule of the CAR19-PNRA plasmid further includes a CD28 costimulatory domain (SEQ ID NO: 89) to obtain the CAR19-P28A plasmid, and the correct insertion of the target sequence in the plasmid is confirmed by sequencing.

[0136] 1.1 Construction of CAR19-PNRA T cells and verification of their function

[0137] 1.1.1 Construction of CAR19-PNRA T cells

[0138] After diluting the CAR19, CAR19-P28A, and CAR19-PNRA plasmids separately in 3 ml of Opti-MEM (Gibco) in a sterile tube, the packaging vector psPAX2 (Addgene) and the envelope vector pMD2.G (Addgene) were added at a ratio of 4:2:1 between plasmid:viral packaging vector:viral envelope vector. 120 μl of X-treme GENE HP DNA transfection reagent (Roche) was then added, mixed immediately, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to the 293T cell culture flask. Viruses were collected at 24 and 48 hours, combined, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours).

[0139] T cells were activated with DynaBeads CD3 / CD28CTSTM (Gibco) and cultured for 1 day at 37°C and 5% CO2. The next day, concentrated lentivirus was added, and after 3 days of culture, T cells expressing CAR19 (and PD1 binding molecules and NKG2A binding molecules) (CAR19T, CAR19-P28A T, and CAR19-PNRA T cells) were obtained.

[0140] 1.1.2 Detection of CAR expression levels and PD1 / NKG2A binding domain expression levels in each CAR-T cell

[0141] Using flow cytometry, FITC goat anti-human Fab (Thermo Fisher) was used to detect the expression of anti-CD19scFv in CAR19T, CAR19-P28A T, and CAR19-PNRA T cells. It can be seen that the CAR in the CAR-T cells prepared in this example can be effectively expressed (Figure 1).

[0142] Using flow cytometry, the expression of anti-NKG2A scFv in CAR19-P28A T and CAR19-PNRA T cells was detected by NKG2A & CD94 protein (ACRO), and the expression of PDL1 in CAR19-P28A T and CAR19-PNRA T cells was detected by PD1 protein (ACRO). It can be seen that anti-NKG2A scFv and PDL1 in CAR19-P28A T and CAR19-PNRA T cells can be effectively expressed (Figures 2 and 3).

[0143] 1.1.3 Detection of the in vitro killing effect of each CAR-T cell on target cells

[0144] In order to detect the killing ability of CAR-T cells on target cells, 1×104 Raji target cells carrying the luciferase gene were plated into each well of a 96-well plate. Three types of CAR T cells and NT cells were then co-cultured in the 96-well plate at effector-target ratios of 4:1, 2:1, and 1:1. After 8 hours, fluorescence values ​​were measured using a microplate reader. Killing efficiency was calculated using the formula: (target cell mean fluorescence - sample mean fluorescence) / target cell mean fluorescence × 100%, as shown in Figure 4.

[0145] It can be seen that at the effector-target ratios of 4:1 and 2:1, there was no significant difference in the in vitro killing effect of CAR19T, CAR19-P28A T and CAR19-PNRA T cells on target cells; at the effector-target ratio of 1:1, the in vitro killing effect of CAR19-PNRA T cells on target cells was better than that of CAR19T and CAR19-P28A T cells.

[0146] 1.1.4 Detection of cytokine release levels of each CAR-T cell

[0147] Considering that intracellular CD28 co-activation signals may lead to rapid proliferation of CAR-T cells in vivo and thus increase the risk of CRS in patients, the inventors investigated the secretion of CRS-related cytokines by CAR-T cells with two structures in the presence of macrophages and tumor cells.

[0148] PBMCs from the same donor as the CAR-T cells were resuscitated and 20 ng / mL M-CSF (PEPROTECH) was added to induce macrophage differentiation. After 7 days of induction culture, they were used for subsequent experiments.

[0149] First, the induced differentiated macrophages were cultured at a rate of 2 × 10 4 The target cells Raji were plated at a concentration of 1×10 5 Cells / well were also plated in a 96-well plate, and finally CAR19-A T cells, CAR19-AT3T, CAR19-AL3 T, CAR19-ATT T, and NT cells were added at a ratio of target cells: CAR-T = 1:1. After co-culture for 18-24 hours, the cell co-culture supernatant was collected.

[0150] The levels of IL6, IL10, IL2, and IFN-γ in the co-culture supernatant were detected using Human IL-6 DuoSet ELISA Kit (R&D systems), Human IL-10 DuoSet ELISA Kit (R&D systems), Human IL-2 DuoSet ELISA Kit (R&D systems), and Human IFN-gamma DuoSet ELISA Kit (R&D systems), respectively. The results are shown in Figure 5.

[0151] It can be seen that compared with CAR19T and CAR19-P28A T, CAR19-PNRA T cells can significantly reduce the secretion of CRS-related cytokines (IL6, IL10, IL2 and IFN-γ). This indicates that CAR19-PNRA T cells can reduce the risk of CRS in patients.

[0152] 1.1.5 Detection of the in vivo inhibitory effect of each CAR-T cell on tumors

[0153] Fifteen healthy female NPI mice aged 6-8 weeks were divided into three groups, with 5 mice in each group: CAR19 T cell group, CAR19-P28A T cell group, and CAR19-PNRA T cell group. On day 0, 5×10 5 After 6 days, 2×10 Raji cells were injected into the tail vein of each mouse according to the grouping situation. 6 The corresponding CAR-T cells were added. The mice were evaluated weekly. The mouse survival curves are shown in Figure 6.

[0154] It can be seen that compared with the CAR19T cell group and the CAR19-P28A T cell group, the CAR19-PNRA T cell group significantly improved the survival rate of mice and had a significant tumor inhibition effect. This shows that the CAR19-PNRA T cells prepared by the present invention can enhance the in vivo killing activity of CAR-T cells.

[0155] In summary, the simultaneous expression of PD1 binding molecules and immunosuppressive molecules will not have an adverse effect on the in vitro killing activity of CAR-T cells, but can significantly reduce the risk of CRS in patients and enhance the in vivo inhibitory effect of CAR-T cells.

[0156] It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may be modified and varied in various ways. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. An engineered cell expressing: (i) a PD1 binding molecule comprising a PD1 binding domain and a transmembrane domain and not comprising an intracellular domain other than a PD1 ligand; (ii) an immunosuppressive molecule comprising an immunosuppressive protein binding domain and a transmembrane domain and not comprising a primary signaling domain, wherein the immunosuppressive protein is selected from NKG2A, TIM3, LAG3, TIGIT, CTLA4, IRP60, SIRPα, KIR2DL1 / 2 / 3, LILRB1 or a combination thereof.

2. The engineered cell of claim 1, wherein the binding domain is selected from the group consisting of a complete antibody, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, sdFv, linear antibody, diabody, sdAb, and a functional fragment of a ligand or receptor.

3. The engineered cell according to claim 1 or 2, wherein the PD1 binding domain is selected from an antibody, a ligand or a functional fragment thereof targeting PD1.

4. The engineered cell according to claim 3, wherein the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, the CDR1-H, CDR2-H and CDR3-H comprised in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H comprised in SEQ ID NO: 75, and the CDR1-L, CDR2-L and CDR3-L comprised in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L comprised in SEQ ID NO: 76; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 75, the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 76, and preferably, the PD1 binding domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

77. 5 . The engineered cell of claim 3 , wherein the ligand targeting PD1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 78 or 79.

6. The engineered cell according to any one of claims 1 to 5, wherein the immunosuppressive molecule comprises an NKG2A binding domain, a TIM3 binding domain, a LAG3 binding domain, a TIGIT binding domain, a CTLA4 binding domain, an IRP60 binding domain, a SIRPα binding domain, a KIR2DL1 / 2 / 3 binding domain, a LILRB1 binding domain, or a combination thereof; wherein, (1) The NKG2A binding domain is an antibody, ligand, or a functional fragment thereof targeting NKG2A; (2) The TIM3 binding domain is an antibody, ligand, or functional fragment thereof targeting TIM3; (3) The LAG3 binding domain is an antibody, ligand, or a functional fragment thereof that targets LAG3; (4) The TIGIT binding domain is an antibody, ligand, or a functional fragment thereof targeting TIGIT; (5) The CTLA4 binding domain is an antibody, ligand, or functional fragment thereof that targets CTLA4; (6) The IRP60 binding domain is an antibody, ligand, or a functional fragment thereof that targets IRP60; (7) The SIRPα binding domain is an antibody, ligand, or a functional fragment thereof targeting SIRPα; (8) The KIR2DL1 / 2 / 3 binding domain is an antibody, receptor, or functional fragment thereof targeting KIR2DL1 / 2 / 3; or (9) The LILRB1 binding domain is an antibody, receptor or functional fragment thereof targeting LILRB1.

7. The engineered cell according to claim 6, wherein: (1) The antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO:7, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO:8; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:8; (2) The antibody targeting TIM3 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 19, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 20; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 20; (3) The LAG3-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H comprised in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H comprised in SEQ ID NO: 32, and the CDR1-L, CDR2-L, and CDR3-L comprised in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L comprised in SEQ ID NO: 33; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 32, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 33; (4) The antibody targeting TIGIT comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 45, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 46; preferably, the heavy chain variable region the light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:45, the light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:46; (5) The antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 62, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 63; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 62, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 63; (6) The IRP60-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H, and CDR3-H comprised in the heavy chain variable region are identical to the CDR1-H, CDR2-H, and CDR3-H comprised in SEQ ID NO: 150, and the CDR1-L, CDR2-L, and CDR3-L comprised in the light chain variable region are identical to the CDR1-L, CDR2-L, and CDR3-L comprised in SEQ ID NO: 151; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 150, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 151; (7) The antibody targeting SIRPα comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 159, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 160; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 159, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 160; (8) The antibody targeting KIR2DL1 / 2 / 3 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 119 or 122, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 120 or 123; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 119 or 122, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 120 or 123; or (9) The antibody targeting LILRB1 comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are identical to the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 131 or 140, and the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are identical to the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 132 or 141; preferably, the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 131 or 140, and the light chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 132 or 141.

8. The engineered cell according to claim 6, wherein: (1) The ligand targeting NKG2A comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 10 or 11; (2) the TIM3-targeting ligand comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22, 23, or 24; (3) the LAG3-targeting ligand comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 35, 36, or 37; (4) the TIGIT-targeting ligand comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 48, 49, 50, or 51; (5) the CTLA4-targeting ligand comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 65 or 66; (6) the ligand targeting SIRPα comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 163; or (7) The ligand targeting LILRB1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

143.

9. The engineered cell of any one of claims 1-8, wherein the immunosuppressive molecule further comprises a co-stimulatory domain.

10. The engineered cell according to any one of claims 1 to 9, wherein the engineered cell further expresses: (iii) a functional exogenous receptor that specifically recognizes an antigen.

11. The engineered cell according to claim 10, wherein the functional exogenous receptor is selected from the group consisting of a chimeric antigen receptor, a T cell receptor, a T cell receptor fusion protein, a T cell antigen coupler, and an immune mobilizing monoclonal T cell receptor, preferably a chimeric antigen receptor or a T cell receptor.

12. The engineered cell of claim 11, wherein the functional exogenous receptor is selected from a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain, and optionally, the chimeric antigen receptor further comprises a co-stimulatory domain.

13. The engineered cell according to claim 10, wherein the antigen specifically recognized by the functional exogenous receptor is selected from the group consisting of: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138. CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin 6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS Fusion gene, ETV6-AML, EMR2, EGP2, EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, g p100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, ephrin B2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor β, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variant of tumor necrosis area, and any combination thereof.

14. The engineered cell according to any one of claims 1 to 13, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD28, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD112, CD113, CD134 , CD137, CD154, CD155, Nectin4, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp 80. CD160, BCMA, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl Id, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, LSECtin, PDL1, PDL2, CEACAM1, MHC class I molecules, MHC class II molecules, CRT AM, Ly9, CD160, PSGL1, CD100, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

15. The engineered cell of any one of claims 1-14, wherein the costimulatory domain is selected from the intracellular region of the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70, and any combination thereof.

16. The engineered cell of any one of claims 1-15, wherein the primary signaling domain is selected from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d. 17 . The engineered cell according to claim 1 , wherein the binding domain and the transmembrane domain further comprise a hinge region derived from the following proteins: CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1.

18. The engineered cell according to any one of claims 1 to 17, wherein the expression of one or more of endogenous HLA-I class genes, HLA-II class genes, and TCR / CD3 genes is inhibited or silenced in the engineered cell.

19. The engineered cell according to claim 18, wherein the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C, B2M and any combination thereof; the HLA-II class gene is selected from HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and any combination thereof; and the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ and any combination thereof.

20. The engineered cell according to any one of claims 1 to 19, wherein the expression of one or more endogenous genes selected from the group consisting of CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10 , CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, I L10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

21. The engineered cell according to any one of claims 1-20, wherein the cell is an immune cell selected from the group consisting of a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, and a NKT cell.

22. The engineered cell of claim 21, wherein the cell is selected from the group consisting of CD4+CD8+T cells, CD4+T cells, CD8+T cells, CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, and αβ-T cells.

23. The engineered cell of claim 21, wherein the cell is a stem cell selected from the group consisting of embryonic stem cells, umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells, and induced pluripotent stem cells.

24. A pharmaceutical composition comprising the engineered cell of any one of claims 1-23, and one or more pharmaceutically acceptable excipients.

25. Use of the engineered cell according to any one of claims 1 to 23 or the pharmaceutical composition according to claim 24 in the preparation of a medicament for preventing or treating cancer, infection or autoimmune disease.

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