Enhanced function cell therapy

Abstract

The present invention relates to an engineered cell characterized in that: (i) the expression of at least one endogenous HLA-II related gene is down-regulated; and (ii) the expression of the endogenous HLA-I related gene is not altered. The invention also relates to a composition comprising the cell.

Description

Function-enhanced cell therapy

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number CN2023100919886, entitled “Function-enhanced cell therapy,” filed with the Patent Office of China on February 9, 2023, the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present invention belongs to the field of immunotherapy. Specifically, the present invention relates to engineered cells and compositions comprising the cells that reduce the risk of rejection by downregulating the expression of endogenous HLA-II-related genes while retaining the expression of endogenous HLA-I-related genes. Background Art

[0004] As an emerging immunotherapy, adoptive cell therapy has developed very rapidly in recent years. In particular, chimeric antigen receptor therapy has a number of products on the market and has shown excellent therapeutic effects in clinical practice. However, the products currently on the market are all autologous therapies, which have disadvantages such as long preparation time, high cost, and high preparation failure rate. In response to this, allogeneic cell therapy has been developed to improve patient accessibility. However, allogeneic cell therapy also faces a series of challenges, such as the risk of immune rejection and poor cell persistence. Therefore, there is still a need to improve existing allogeneic cell therapies to reduce the risk of immune rejection and enhance cell persistence.

[0005] Summary of the Invention

[0006] 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.

[0007] In a first aspect, the present invention provides an engineered cell having the following characteristics: (i) expression of at least one endogenous HLA-II-related gene is downregulated; and (ii) expression of endogenous HLA-I-related genes is not altered.

[0008] I. Expression of endogenous genes

[0009] A. Expression of endogenous HLA genes

[0010] The major histocompatibility complex (MHC) is a collective term for a group of tightly linked and diverse genes in the vertebrate genome that encode cell surface proteins that are crucial to the immune system. Because human MHC genes were first discovered in white blood cells, they are also called human leukocyte antigens (HLA). HLA is by far the most polymorphic region in human chromosomes and is key to the immune system's recognition of "self" and "non-self." Clinically, HLA is the main immune barrier that affects the survival of organ or cell transplants. HLA mismatch between the recipient and donor will trigger cellular and antibody-mediated immune rejection reactions. HLA is mainly divided into class I and class II.

[0011] As used herein, the term "HLA-I-related gene" refers to a gene involved in regulating the expression of the HLA-I complex. The HLA-I complex is composed of an α chain encoded by the HLA-I class gene and a β chain encoded by the B2M gene, which are bound by non-covalent bonds. The HLA-I complex is expressed in most types of cells in the body and can present endogenous antigen peptides (mostly 8-12 amino acids in length) to CD8+ T cells and induce their killing function. Among them, the α chain includes an extracellular region (including three domains, α1, α2, and α3), a transmembrane domain, and a cytoplasmic region. The α1 and α2 domains form an antigen binding groove, which is responsible for binding to the antigen peptide entering the groove; α3 is homologous to the constant region domain of immunoglobulins and binds to the T cell surface molecule CD8. HLA-I class genes mainly include HLA-A, HLA-B, and HLA-C. In the engineered cells of the present invention, the expression of endogenous HLA-I-related genes is not changed. In other words, the expression level of any endogenous HLA-I-associated gene in the cell is not altered (e.g., inhibited, knocked down, knocked out, or silenced) by any gene editing or other non-gene editing means. In one embodiment, the endogenous HLA-I-associated genes are HLA-A, HLA-B, HLA-C, and B2M.

[0012] As used herein, the term "HLA-II related genes" includes the HLA-II class genes themselves and genes involved in or regulating the expression of HLA-II class genes. HLA-II class genes are mainly expressed in immune cells such as macrophages, dendritic cells, activated T cells, and B cells, and can present exogenous antigen peptides (mostly 13-28 amino acids in length) to CD4+ T cells and induce their function. HLA-II class molecules are composed of α chains and β chains, each of which contains three parts: an extracellular region, a transmembrane domain, and a cytoplasmic region, wherein the extracellular region contains two domains. The α1 and β1 domains form an antigen binding groove, which is responsible for binding to the antigen peptide entering the groove, and the β2 domain binds to the T cell surface molecule CD4. HLA-II class genes mainly include HLA-DP, HLA-DQ, and HLA-DR. In addition, some genes regulate the expression of HLA-II class genes by affecting antigen processing and transport, including, for example, LMP2, LMP7, TAP1, TAP2, HLA-DM, HLA-DN, and HLA-DO. Other genes regulate the expression of HLA-II class genes by binding to HLA-II transcription factors, including, for example, RFX5, RFXAP, RFXANK, and CIITA. In the engineered cells of the present invention, the expression of at least one endogenous HLA-II-associated gene is downregulated. In one embodiment, the HLA-II-associated gene is selected from the group consisting of HLA-DP, HLA-DQ, HLA-DR, LMP2, LMP7, TAP1, TAP2, HLA-DM, HLA-DN, HLA-DO, RFX5, RFXAP, RFXANK, and CIITA.

[0013] Since HLA-I and HLA-II related genes are recognized by T cell receptors (TCR), they activate T cells and then induce the killing activity of T cells. Therefore, it is particularly important to lower the expression level of such genes, especially HLA-I related genes, for reducing immune rejection, because CD8+ T cells are the most important killer T cell subsets. In fact, in the preparation of allogeneic cell therapy, knocking out HLA-I related genes has become a more common practice. However, the inventors unexpectedly found that compared with the combination of knocking out HLA-I related genes and HLA-II related genes, only knocking out HLA-II related genes (i.e., retaining the expression of HLA-I related genes) can achieve a more significant effect of reducing immune rejection. In addition, most of the current research on HLA focuses on its role in immune rejection. The inventors of the present invention unexpectedly found that knocking out HLA-II related genes (i.e., retaining the expression of HLA-I related genes) can also improve the proliferation ability of CAR-T cells, thereby enhancing its tumor suppression effect.

[0014] As used herein, gene expression is “downregulated” means that the expression level of the product of the modified gene decreases significantly or is hardly expressed relative to the expression level of the unmodified wild-type gene. This means that for the gene whose expression is “downregulated”, the content of its expression product (such as mRNA or protein) is reduced or the expression product is not functional. Therefore, downregulation can be achieved in different dimensions such as gene level, transcription level, translation level. Generally, gene expression is downregulated by methods well known to those skilled in the art, including but not limited to, for example, by technologies such as meganucleases, zinc finger nucleases, TALEN, CRISPR / Cas systems, base editors, lead editors, etc. to knock down or knock out the target gene itself or the regulatory gene of the target gene, or by technologies such as antisense oligonucleotides, RNAi, shRNA, transposons, siRNA, antigomer RNA, mutations, antibodies or chemical inhibitors to reduce the expression level of the target gene itself or the regulatory gene of the target gene.

[0015] B. Expression of other endogenous genes

[0016] In one embodiment, the engineered cells of the present invention further comprise down-regulation of the expression levels of other endogenous genes to further reduce the risk of cell rejection or enhance the efficacy of the cells, such as increasing the proliferation activity / killing activity of the cells, prolonging the persistence of the cells, etc.

[0017] In one embodiment, the expression of at least one endogenous TCR / CD3 gene of the engineered cells is downregulated. The T cell surface receptor (T cell receptor, TCR) is a characteristic marker on the surface of all T cells. It binds to CD3 with a non-covalent bond to form a TCR / CD3 complex, and generates a specific antigen stimulation signal by binding to the specific MHC-antigen peptide complex on the surface of the antigen presenting cell, activating T cells and exerting a killing effect. Therefore, inhibiting or silencing the expression of endogenous TCR / CD3 genes in CAR-T cells can avoid their attack on normal cells or tissues in the patient's body, thereby avoiding or reducing the risk of graft-versus-host disease (GvHD). In one embodiment, the TCR / CD3 gene is selected from: TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ.

[0018] In one embodiment, the engineered cells have downregulated expression of at least one endogenous gene selected from the group consisting of CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFR1, DR3, DR4, DR5, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7 , FADD, FAS, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3, more preferably selected from FAS, TGFBR1, TGFBR2, TNFR1, DR3, DR4 and DR5.

[0019] Fas, TNFR1, DR3, DR4 and DR5 all belong to the death receptor (DR) family and share a Cys-rich extracellular domain and an intracellular death domain (DD). When these death receptors bind to their specific death ligands, they receive extracellular death signals, activate the intracellular apoptosis mechanism, and induce cell apoptosis. TGFBR1 and TGFBR2 are both receptors for TGFB1, and their intracellular regions contain serine / threonine kinase activity. They mediate Smad-dependent and -independent signaling pathways and play an important regulatory role in processes such as cell proliferation, differentiation, wound healing, and tumor formation and metastasis. The inventors have found that reducing the expression of these endogenous genes in cells can significantly prolong cell persistence and improve cell survival.

[0020] II. Immunosuppressive molecules (IIMs)

[0021] The engineered cells provided by the present invention further express exogenous immunosuppressive molecules, wherein the immunosuppressive molecules can reduce the immunogenicity of the engineered cells.

[0022] As used herein, the term "immunosuppressive molecule" refers to a molecule that can bind to an immunosuppressive protein and thereby inhibit the subject's immune rejection of foreign cells, such as reducing the killing function of immune cells (such as T cells, NK cells) in the subject's body or inhibiting the excessive proliferation of immune cells. In one embodiment, the immunosuppressive molecule comprises one or more immunosuppressive protein binding domains, a transmembrane domain, and a co-stimulatory domain and does not comprise a primary signaling domain.

[0023] As used herein, the term "immunosuppressive protein binding domain" refers to a domain that is capable of specifically binding to an immunosuppressive protein. In one embodiment, the immunosuppressive protein binding domain is an antibody that targets an immunosuppressive protein, or the extracellular region of a ligand of an immunosuppressive protein. In one embodiment, such immunosuppressive proteins include, but are not limited to: NKG2 / CD94 components (e.g., NKG2A, NKG2B, CD94); killer cell Ig-like receptor (KIR) family members (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); leukocyte Ig-like receptor (LIR) family members (e.g., LIR1, LIR2, LIR3, LIR5, and LIR8); NK cell receptor protein 1 (NKR-P1) family members (e.g., NKR-P1B and NKR-P1D); immune checkpoint receptors (e.g., PD-1, TIGIT, CD96, TIM3, LAG3, CTLA4); carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1); sialic acid-binding immunoglobulin-like lectin (SIGLEC) family members (e.g., SIGLEC7 and SIGLEC9); leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Ly49 family members (e.g., Ly49A, Ly49C, Ly49F, Ly49G1, and Ly49G4), killer cell lectin-like receptor G1 (KLRG1), and FASL. Preferably, the immunosuppressive protein is selected from PD1, TIGIT, CD96, TIM3, LAG3, CTLA4, FASL, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, SIRP-α, IRp60 and KLRG1.

[0024] In one embodiment, the immunosuppressive protein binding domain is an antibody targeting an immunosuppressive protein, such as an antibody that specifically binds to the following targets: PD1, TIGIT, CD96, TIM3, LAG3, CTLA4, FASL, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, SIRP-α, IRp60, and KLRG1. Known antibodies to the above targets can be used in the present invention. In one embodiment, the immunosuppressive protein binding domain is an extracellular region of an immunosuppressive protein ligand, for example, an extracellular region selected from the following proteins: HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PDL1, PDL2, CD155, CD112, CD113, Gal-9, FGL1, FAS, CD80, CD86, CD47.

[0025] As used herein, the term "antibody" has the broadest meaning understood by those skilled in the art, and includes monoclonal antibodies (including complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit desired biological activity. The antibodies of the present invention can be of any kind (such as IgG, IgE, IgM, IgD, IgA, etc.) or subclass (such as IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). As used herein, the term "antibody fragment" refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments in the present invention include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, single-chain antibody (scFv), disulfide bond-linked Fv (sdFv), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, "diabodies" with two antigen binding sites, single domain antibodies (sdAb), nanobodies, and the like. Therefore, unless the context clearly indicates otherwise, the "antibody" of the present invention encompasses antibody fragments as defined above. Therefore, in a preferred embodiment, the antibody of the present invention is selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, diabodies, sdAb or nanobodies, preferably scFv, sdAb or nanobodies. In one embodiment, the antibody of the present invention is a murine antibody, a chimeric antibody, a camel antibody, a humanized antibody or a human antibody.

[0026] Typically, a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds. Each light chain is connected to its own heavy chain by disulfide bonds, forming a "Y"-shaped structure. Each heavy chain comprises a heavy chain variable region (VH), which contains three complementarity-determining regions (CDRs): CDR1-H, CDR2-H, and CDR3-H, and a heavy chain constant region, which contains three constant domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL), which contains three CDRs: CDR1-L, CDR2-L, and CDR3-L, and a light chain constant region, which contains one constant domain, CL. Within the heavy and light chain variable regions, the CDRs are separated by more conserved framework regions (FRs). The variable regions of the heavy and light chains are responsible for antigen recognition and binding, while the constant regions mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0027] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using a number of numbering schemes well known in the art, including: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding sitetopography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGTunique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0028] In some embodiments, the boundary of CDR or FR is different depending on the scheme used to define CDR or FR. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of Kabat and Chothia schemes is all based on the most common antibody region sequence length, wherein insertion (such as "30a") is provided by inserting letters and disappearance occurs in some antibodies. These two schemes are placed in different positions with certain insertions and deletions (indel), thereby producing different numbering. The Contact scheme is based on the analysis of complex crystal structure, and is similar to the Chothia numbering scheme in many aspects. The AbM scheme is a compromise between Kabat and Chothia definitions, and it is based on the scheme used by the AbM antibody modeling software of Oxford Molecular.

[0029] Therefore, unless otherwise specified, it should be understood that the "CDR" of a given antibody or its region (such as its variable region) covers the CDR defined by any of the above schemes or other known schemes. For example, when specifying that a specific CDR (such as CDR3) contains a given amino acid sequence, it should be understood that such CDR can also have the sequence of the corresponding CDR (such as CDR3) defined by any of the above schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) covers the FR defined by any of the above schemes or other known schemes. Unless otherwise specified, the numbering scheme used to define the boundaries of CDR and FR in this article adopts the Chothia scheme.

[0030] As used herein, 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.

[0031] In one embodiment, the immunosuppressive protein binding domain of the present invention is an antibody targeting PD1.

[0032] Any anti-PD1 antibody known in the art can be used in the present invention. In one embodiment, the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, 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: 60; 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: 59. In one embodiment, the CDR1-L contained in the light chain variable region is as shown in SEQ ID NO: 56, the CDR2-L is as shown in SEQ ID NO: 57, and the CDR3-L is as shown in SEQ ID NO: 58; the CDR1-H contained in the heavy chain variable region is as shown in SEQ ID NO: 53, the CDR2-H is as shown in SEQ ID NO: 54, and the CDR3-H is as shown in SEQ ID NO: 55. In one embodiment, the antibody targeting PD1 comprises a light chain variable region and a heavy chain variable region, wherein 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: 60; 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: 59. In a preferred embodiment, the anti-PD1 antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 59 and a light chain variable region as set forth in SEQ ID NO: 60. In one embodiment, 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: 61. Preferably, the antibody targeting PD1 is as shown in SEQ ID NO: 61.

[0033] In one embodiment, the immunosuppressive protein binding domain contained in the immunosuppressive molecule of the present invention is the extracellular region of the PD1 ligand, such as the extracellular region of PDL1 or PDL2. In this embodiment, the immunosuppressive molecule may also contain the corresponding transmembrane region of PDL1 or PDL2, but generally does not contain its corresponding intracellular region at the same time. That is, the immunosuppressive molecule is not the full-length PDL1 or PDL2. In one embodiment, the extracellular region of PDL1 has 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% identity with SEQ ID NO: 64; the extracellular region of PDL2 has 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% identity with SEQ ID NO: 67. The transmembrane region of PDL1 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; the transmembrane region of PDL2 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: 66. In this embodiment, the immunosuppressive molecule may further comprise a PDL1 signal peptide or a PDL2 signal peptide. The PDL1 signal peptide 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; the PDL2 signal peptide 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.

[0034] In one embodiment, the immunosuppressive protein binding domain contained in the immunosuppressive molecule of the present invention is an antibody targeting NKG2A. Known anti-NKG2A antibodies can be used in the present invention. In one embodiment, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, 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: 49; 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: 48. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 45, CDR2-L as shown in SEQ ID NO: 46, and CDR3-L as shown in SEQ ID NO: 47; 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. In one embodiment, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, wherein 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: 49; and 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: 48. In a preferred embodiment, the anti-NKG2A antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 48 and a light chain variable region as set forth in SEQ ID NO: 49. In one embodiment, 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: 50. Preferably, the antibody targeting NKG2A is as shown in SEQ ID NO: 50.

[0035] In one embodiment, the immunosuppressive protein binding domain of the present invention comprises an extracellular region of an NKG2A ligand, such as an extracellular region of HLA-E. In one embodiment, the HLA-E extracellular 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: 51 or 52. In this embodiment, the immunosuppressive molecule may also comprise the corresponding transmembrane region of HLA-E, but generally does not also comprise the corresponding intracellular region thereof. That is, the immunosuppressive molecule is not full-length HLA-E.

[0036] In one embodiment, the immunosuppressive protein binding domain of the present invention is an antibody targeting FasL. Any anti-FasL antibody known in the art can be used in the present invention. In one embodiment, the antibody targeting FasL comprises a light chain variable region and a heavy chain variable region, 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: 75; 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: 74. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO:71, CDR2-L as shown in SEQ ID NO:72, and CDR3-L as shown in SEQ ID NO:73; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:68, CDR2-H as shown in SEQ ID NO:69, and CDR3-H as shown in SEQ ID NO:70. In one embodiment, the antibody targeting FasL comprises a light chain variable region and a heavy chain variable region, wherein 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: 75; and 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: 74. In a preferred embodiment, the anti-FasL antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 74 and a light chain variable region as set forth in SEQ ID NO: 75. In one embodiment, the antibody targeting FasL 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 FasL is as shown in SEQ ID NO: 76.

[0037] In one embodiment, the immunosuppressive protein binding domain of the present invention comprises the extracellular region of a FasL ligand, such as the extracellular region of Fas. In one embodiment, the Fas extracellular 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: 77. In this embodiment, the immunosuppressive molecule may also comprise the corresponding transmembrane region of Fas, but generally does not also comprise its corresponding intracellular region. That is, the immunosuppressive molecule is not full-length Fas. In one embodiment, the Fas transmembrane 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: 78.

[0038] In one embodiment, the immunosuppressive protein binding domain of the present invention is an antibody targeting CTLA4. Any anti-CTLA4 antibody known in the art can be used in the present invention. In one embodiment, the antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, 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: 86; 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: 85. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO:82, CDR2-L as shown in SEQ ID NO:83, and CDR3-L as shown in SEQ ID NO:84; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:79, CDR2-H as shown in SEQ ID NO:80, and CDR3-H as shown in SEQ ID NO:81; or CDR1-H as shown in SEQ ID NO:10, CDR2-H as shown in SEQ ID NO:11, and CDR3-H as shown in SEQ ID NO:12. In one embodiment, the antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, wherein 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: 86; 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: 85. In a preferred embodiment, the anti-CTLA4 antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 85 and a light chain variable region as set forth in SEQ ID NO: 86. In one embodiment, 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: 87. Preferably, the antibody targeting CTLA4 is as shown in SEQ ID NO: 87.

[0039] In one embodiment, the immunosuppressive protein binding domain of the present invention comprises an extracellular region of a CTLA4 ligand, such as an extracellular region of CD80 or CD86. In one embodiment, the CD80 extracellular 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: 88; the CD86 extracellular 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: 89. In this embodiment, the immunosuppressive molecule may also comprise the corresponding transmembrane region of CD80 or CD86, but generally does not simultaneously comprise its corresponding intracellular region. That is, the immunosuppressive molecule is not full-length CD80 or CD86. In one embodiment, the CD80 transmembrane 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: 90. In one embodiment, the CD86 transmembrane 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: 91.

[0040] In one embodiment, the immunosuppressive protein binding domain of the present invention is an antibody targeting SIRPα. Anti-SIRPα antibodies known in the art can be used in the present invention. In one embodiment, the antibody targeting SIRPα comprises a light chain variable region and a heavy chain variable region, wherein 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: 99; wherein 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: 98. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 95, CDR2-L as shown in SEQ ID NO: 96, and CDR3-L as shown in SEQ ID NO: 97; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 92, CDR2-H as shown in SEQ ID NO: 93, and CDR3-H as shown in SEQ ID NO: 94. In one embodiment, the antibody targeting SIRPα comprises a light chain variable region and a heavy chain variable region, wherein 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: 99; 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: 98. In a preferred embodiment, the anti-SIRPα antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 98 and a light chain variable region as set forth in SEQ ID NO: 99. In one embodiment, 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: 100. Preferably, the antibody targeting SIRPα is as shown in SEQ ID NO: 100.

[0041] In one embodiment, the immunosuppressive protein binding domain of the present invention comprises the extracellular region of a SIRPα ligand, such as the extracellular region of CD47. In this embodiment, the immunosuppressive molecule may also comprise the corresponding transmembrane region of CD47, or even a portion of the intracellular region, but generally does not comprise the complete intracellular region at the same time. That is, the immunosuppressive molecule is not full-length CD47. In one embodiment, the immunosuppressive molecule comprises an amino acid sequence having 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% identity to SEQ ID NO: 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119.

[0042] In one embodiment, the immunosuppressive protein binding domain of the present invention is an antibody targeting IRP60. Any anti-IRP60 antibody known in the art can be used in the present invention. In one embodiment, the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, 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: 108; 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: 107. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 104, CDR2-L as shown in SEQ ID NO: 105, and CDR3-L as shown in SEQ ID NO: 106; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 101, CDR2-H as shown in SEQ ID NO: 102, and CDR3-H as shown in SEQ ID NO: 103. In one embodiment, the antibody targeting IRP60 comprises a light chain variable region and a heavy chain variable region, wherein 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: 108; 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: 107. In a preferred embodiment, the anti-IRP60 antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 107 and a light chain variable region as set forth in SEQ ID NO: 108. In one embodiment, 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: 109. Preferably, the antibody targeting IRP60 is as shown in SEQ ID NO: 109.

[0043] In one embodiment, the engineered cells express an exogenous immunosuppressive molecule, which comprises one or more binding domains that specifically bind to the following immunosuppressive proteins: PD1, NKG2A, FasL, CTLA4, IRP60, SIRPα. In one embodiment, the binding domain is an antibody targeting the immunosuppressive protein or the extracellular region of the ligand of the immunosuppressive protein. For example, the immunosuppressive molecule comprises anti-PD1 antibody, anti-NKG2A antibody, anti-FasL antibody, anti-CTLA4 antibody, anti-SIRPα antibody, anti-IRP60 antibody, Fas extracellular region, CD80 extracellular region, CD86 extracellular region, PDL1 extracellular region, PDL2 extracellular region, HLA-E extracellular region, CD47 extracellular region or a combination thereof, such as anti-NKG2A antibody (or HLA-E extracellular region) and anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region). Antibodies include anti-NKG2A antibody (or HLA-E extracellular region) and anti-FasL antibody (or Fas extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-CTLA4 antibody (or CD80 extracellular region, CD86 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-SIRPα antibody (or CD47 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-IRP60 antibody, anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-FasL antibody (or Fas extracellular region), anti-P D1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-CTLA4 antibody (or CD80 extracellular region, CD86 extracellular region), anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-SIRPα antibody (or CD47 extracellular region), anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-IRP60 antibody, anti-FasL antibody (or Fas extracellular region) and anti-CTLA4 antibody (or CD80 extracellular region, CD86 extracellular region), anti-FasL antibody (or Fas The invention relates to a combination of an anti-SIRPα antibody (or an extracellular region of CD47), an anti-FasL antibody (or an extracellular region of Fas) and an anti-IRP60 antibody, an anti-CTLA4 antibody (or an extracellular region of CD80, an extracellular region of CD86) and an anti-SIRPα antibody (or an extracellular region of CD47), an anti-CTLA4 antibody (or an extracellular region of CD80, an extracellular region of CD86) and an anti-IRP60 antibody, or an anti-SIRPα antibody (or an extracellular region of CD47) and an anti-IRP60 antibody (the two binding domains in the combination can be connected by a linker).

[0044] In one embodiment, the engineered cells express two or more exogenous immunosuppressive molecules, each of which comprises a binding domain that binds to a different immunosuppressive protein selected from the group consisting of PD1, NKG2A, FasL, CTLA4, IRP60, and SIRPα. For example, the two exogenous immunosuppressive molecules bind to NKG2A and PD1, NKG2A and CTLA4, NKG2A and FasL, NKG2A and SIRPα, NKG2A and IRP60, PD1 and CTLA4, PD1 and FasL, PD1 and SIRPα, PD1 and IRP60, CTLA4PD1 and SIRPα, CTLA4 and IRP60, FasL and SIRPα, FasL and IRP60, SIRPα and IRP60, or CTLA4 and FasL. For example, in one embodiment, the engineered cells express two exogenous immunosuppressive molecules, which respectively include anti-NKG2A antibody (or HLA-E extracellular region) and anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-FasL antibody (or Fas extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-CTLA4 antibody (or CD80 extracellular region, CD86 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-SIRPα antibody (or CD47 extracellular region), anti-NKG2A antibody (or HLA-E extracellular region) and anti-IRP60 antibody, anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-FasL antibody (or Fas extracellular region), anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-CTLA4 antibody (or CD80 extracellular region, CD86 extracellular region), anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-SIRPα antibody (or CD47 extracellular region), anti-PD1 antibody (or PDL1 extracellular region, PDL2 extracellular region) and anti-IRP60 antibody, anti-FasL antibody (or Fas extracellular region) and anti-CTLA4 antibody (or C In some embodiments, the two exogenous immunosuppressive molecules may be linked to each other via a 2A peptide (e.g., F2A, T2A, etc.).As used herein, "A (or B) and C (or D, E)" encompasses various combinations of A and C, A and D, A and E, B and C, B and D, and B and E.

[0045] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables expression of an immunosuppressive molecule on the cell surface and anchors the immunosuppressive protein-binding domain to the cell membrane. A transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. When the target-binding domain binds to a target, the transmembrane domain enables signal transduction. Transmembrane domains particularly suitable 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, CD8a, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD134, CD137, CD154, 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, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRT AM, Ly9, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D or NKG2C. In some embodiments, the transmembrane domain is derived from the following molecules: CD8a, CD4, CD28, 4-1BB, CD47, CD80, CD86, CD152 and PD1. Alternatively, 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: 14. Preferably, the transmembrane domain is derived from CD8a, 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: 15 or 16.

[0046] In one embodiment, the immunosuppressive molecule further comprises a hinge region between the immunosuppressive protein binding domain and the transmembrane domain. As used herein, 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 comprise 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 can be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises the hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably CD8α, CD28 or IgG4 hinge. In one embodiment, 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: 26. In one embodiment, the hinge region is from CD8a, 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: 27 or 28. In one embodiment, 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: 29.

[0047] As used herein, "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, which is an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular region of the costimulatory molecule, or a functional fragment thereof. "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, class 1 MHC molecules, BTLA, and Toll ligand receptors. The non-limiting examples of the costimulatory domains of the present invention include but are not limited to the intracellular region derived from following protein: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, PD-1, LIGHT, TRIM, ZAP70 and their combination.Preferably, the costimulatory domains of CAR of the present invention are 4-1BB, CD28 or 4-1BB+CD28. In one embodiment, the costimulatory domain is from 4-1BB, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 18 or 19. In one embodiment, the costimulatory domain is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 17.

[0048] The immunosuppressive molecules of the present invention do not include a primary signaling domain. As used herein, the term "primary signaling domain" refers to a protein structure that works together to trigger 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 includes one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of the primary signaling domain of the present invention include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, NFAM1, STAM1, STAM2, and CD66d. In one embodiment, the immunosuppressive molecules of the present invention do not include a CD3ζ intracellular region. For example, the CD3ζ intracellular region 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: 21, 22 or 20.

[0049] In one embodiment, the immunosuppressive molecule of the present invention further comprises a signal peptide so that when it is expressed in a cell, such as a T cell, 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 segment that has a tendency to form a single α-helix. At the end of the signal peptide, there is usually an amino acid segment 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 a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide that can be used in the present invention is derived from B2M and has a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% to the amino acid sequence shown in SEQ ID NO: 23. In one embodiment, the signal peptide useful in the present invention is derived from CD8a and 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: 24 or 25.

[0050] III. Functional exogenous receptors

[0051] In one embodiment, the engineered cells of the present invention may further express a functional exogenous receptor. Preferably, the functional exogenous receptor is selected from a recombinant T cell receptor, a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler, more preferably a chimeric antigen receptor.

[0052] As used herein, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from various components of a TCR, typically consisting of a TCR subunit and an antibody linked thereto, and expressed on the cell surface. The TCR subunit includes at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

[0053] As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: 1. a tumor targeting domain, including a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting groups; 2. an extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3. a transmembrane region and an intracellular region of the CD4 co-receptor, wherein the intracellular region is connected to the protein kinase LCK, which catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.

[0054] As used herein, the term "T cell receptor" or "TCR" is a characteristic marker on the surface of T cells, which binds to CD3 with a non-covalent bond to form a complex. Antigen presenting cells present antigen peptides to T cells through major histocompatibility complex molecules (MHC) and bind to the TCR complex to induce a series of intracellular signal transductions. 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 specific antigens and MHC molecules. The term "recombinant TCR receptor" refers to an artificially constructed T cell receptor that further comprises an antigen (e.g., tumor antigen) binding domain.

[0055] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an extracellular domain (such as a ligand of an antibody or antigen, or a functional fragment thereof) that specifically binds to an antigen (such as a tumor antigen), a transmembrane domain, an optional costimulatory domain, and a primary signaling domain, each domain being connected by a joint. 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 one embodiment, the functional exogenous receptor of the present invention is a chimeric antigen receptor comprising a tumor antigen binding domain, a transmembrane domain, one or more costimulatory domains, and a primary signaling domain. In one embodiment, 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.

[0056] Various linker sequences known in the art can be used in the present invention to connect the various domains contained in the functional exogenous receptor or immunosuppressive molecule. Examples of linkers include, but are not limited to, (GS) linkers, such as (GS)n, (GSGGS)n, (GGGS)n, (GGGGS)n, where n is an integer greater than 1. In one embodiment, the linker sequence is as shown in SEQ ID NO: 31. In one embodiment, the linker sequence is as shown in SEQ ID NO: 32.

[0057] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen (e.g., a tumor antigen). In one embodiment, the extracellular domain comprises an antibody that specifically binds to an antigen or a ligand of the antigen, or a functional fragment of the antigen ligand (i.e., an antigen binding region). In one embodiment, 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, CLEC12A, CDH16, CSPG4, CS1, CLL-1, Claudin 6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, 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, or 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, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from CD19, Claudin18.2, MSLN, GPRC5D, CD7 and BCMA.

[0058] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD19, such as an antibody targeting CD19. Anti-CD19 antibodies known in the art can be used in the present invention. In one embodiment, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, 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: 12; 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: 11. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 8, CDR2-L as shown in SEQ ID NO: 9, and CDR3-L as shown in SEQ ID NO: 10; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 5, CDR2-H as shown in SEQ ID NO: 6, and CDR3-H as shown in SEQ ID NO: 7. In one embodiment, the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region, wherein 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: 12; 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: 11. In a preferred embodiment, the anti-CD19 antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 12. In one embodiment, 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: 13. Preferably, the antibody targeting CD19 is as shown in SEQ ID NO: 13.

[0059] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes Claudin18.2, such as an antibody targeting Claudin18.2. Antibodies against Claudin18.2 known in the art can be used in the present invention. In one embodiment, the antibody targeting Claudin18.2 is a single-domain antibody comprising CDR1, CDR2, and CDR3 identical to the CDR1, CDR2, and CDR3 contained in SEQ ID NO: 4. In one embodiment, CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2, and CDR3 is as shown in SEQ ID NO: 3. In one embodiment, the antibody targeting Claudin18.2 has 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% identity to SEQ ID NO: 4. Preferably, the antibody targeting SIRPα is as shown in SEQ ID NO: 4.

[0060] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes ROR1, such as an antibody targeting ROR1. Anti-ROR1 antibodies known in the art can be used in the present invention. In one embodiment, the antibody targeting ROR1 comprises a light chain variable region and a heavy chain variable region, wherein 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: 40; wherein 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: 39. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 36, CDR2-L as shown in SEQ ID NO: 37, and CDR3-L as shown in SEQ ID NO: 38; the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 33, CDR2-H as shown in SEQ ID NO: 34, and CDR3-H as shown in SEQ ID NO: 35. In one embodiment, the antibody targeting ROR1 comprises a light chain variable region and a heavy chain variable region, wherein 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: 40; 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: 39. In a preferred embodiment, the anti-ROR1 antibody of the present invention comprises a heavy chain variable region as set forth in SEQ ID NO: 39 and a light chain variable region as set forth in SEQ ID NO: 40. In one embodiment, the antibody targeting ROR1 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: 41. Preferably, the antibody targeting ROR1 is as shown in SEQ ID NO: 41.

[0061] In one embodiment, the functional exogenous receptor of the present invention is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, one or more costimulatory domains, and a primary signaling domain. In one embodiment, the functional exogenous receptor of the present invention is a chimeric antigen receptor that targets CD19, 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, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA, or GPRC5D, more preferably targeting CD19, Claudin18.2, MSLN, ROR1, GPRC5D, CD7, and BCMA. In one embodiment, the chimeric antigen receptor further comprises a signal peptide, a hinge region, or both. The definitions of the transmembrane domain, costimulatory domain, primary signaling domain, and optional hinge region, signal peptide, etc. of the CAR of the present invention are shown in the "immunosuppressive molecule" section above. In one embodiment, the transmembrane domain included in the CAR of the present invention may be the same as or different from the transmembrane domain included in the immunosuppressive molecule. In one embodiment, the costimulatory domain included in the CAR of the present invention may be the same as or different from the costimulatory domain included in the immunosuppressive molecule.

[0062] In one embodiment, 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.

[0063] In one embodiment, the CAR of the present invention may also include a suicide gene, that is, it is expressed by a cell death signal that can be induced by an exogenous substance, so as to remove the CAR cells when needed (for example, 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 apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

[0064] IV. Engineered Cells

[0065] In one embodiment, the engineered cells of the invention are engineered immune cells.

[0066] As used herein, the term "immune cell" refers to any cell with one or more effector functions (e.g., 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 (e.g., from 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.), or from a cell line (e.g., Jurkat, SupT1, NK92 etc.) cultured in vitro, or from stem cell differentiation (e.g., 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 can be in 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, immature T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll separation.

[0067] In one embodiment, the engineered cells of the present invention are engineered stem cells. Examples of stem cells include, but are not limited to, cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs, and the like.

[0068] In one embodiment, 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 each other 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 cause antigenic interaction.

[0069] V. Pharmaceutical Compositions

[0070] 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.

[0071] As used herein, 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), which 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] In one embodiment, the pharmaceutical compositions of the present invention are used to treat a subject suffering from cancer, infection, or autoimmune disease.

[0076] In one embodiment, 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 diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, etc.

[0077] In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0078] In one embodiment, 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.

[0079] 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 and features in the embodiments of the present application may be combined with each other unless there is any contradiction. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1: Cytotoxic activity of CD19 CAR-T cells against target cells.

[0081] Figure 2: Cytokine release levels after co-incubation of CD19 CAR-T cells with target cells.

[0082] Figure 3: Survival curves of tumor-bearing mice treated with CD19 CAR-T cells.

[0083] Figure 4: Cytotoxic activity of CD19 CAR-T cells against target cells with knockout of HLA-II, Fas, TGFBR1, TGFBR2 or their combination.

[0084] Figure 5: Cytokine release levels after CD19 CAR-T cells with HLA-II, Fas, TGFBR1, TGFBR2 knockout or their combination were co-incubated with target cells.

[0085] Figure 6: In vivo tumor suppression effect of CD19 CAR-T cells with HLA-II, Fas, TGFBR1, TGFBR2 knockout or their combination.

[0086] Figure 7: Cytotoxic activity of CLDN18.2 CAR-T cells against target cells.

[0087] Figure 8: Expansion level of CLDN18.2 CAR-T cells.

[0088] Figure 9: Cytotoxic activity of ROR1 CAR-T cells against target cells.

[0089] Figure 10: Expansion level of ROR1 CAR-T cells.

[0090] Figure 11: Inhibitory effect of engineered B cells on the cytotoxic activity of NK92 cells. Specific implementation plan

[0091] Example 1: Structures of exemplary CARs and immunosuppressive molecules used in the examples

[0092] The CAR structure comprises an extracellular domain, a CD8α hinge region (SEQ ID NO: 27), a CD8α transmembrane region (SEQ ID NO: 15), a 4-1BB costimulatory domain (SEQ ID NO: 18), and a CD3ζ intracellular region (SEQ ID NO: 21) connected in sequence. The extracellular domain included in the CD19 CAR structure is an anti-CD19 scFv (SEQ ID NO: 13). The extracellular domain included in the Claudin18.2 CAR structure is an anti-Claudin18.2 vhh (SEQ ID NO: 4). The extracellular domain included in the ROR1 CAR structure is an anti-ROR1 scFv (SEQ ID NO: 44).

[0093] The structure of the PD1-targeting immunosuppressive molecule IIM-PDL1 comprises a sequentially linked PDL1 signal peptide (SEQ ID NO: 62), a PDL1 extracellular region (SEQ ID NO: 64), a PDL1 transmembrane region (SEQ ID NO: 63), and a CD28 costimulatory domain (SEQ ID NO: 17). The structure of the PD1-targeting immunosuppressive molecule IIM-PDL2 comprises a sequentially linked PDL2 signal peptide (SEQ ID NO: 65), a PDL2 extracellular region (SEQ ID NO: 67), a PDL2 transmembrane region (SEQ ID NO: 66), and a CD28 costimulatory domain (SEQ ID NO: 17).

[0094] The structure of the NKG2A-targeting immunosuppressive molecule IIM-NKG2A comprises an anti-NKG2A scFv (SEQ ID NO: 50), an IgG4 hinge region (SEQ ID NO: 29), a CD28 transmembrane region (SEQ ID NO: 14), and a CD28 costimulatory domain (SEQ ID NO: 17) connected in sequence.

[0095] Example 2. Preparation of CD19 CAR-T cells

[0096] The CD19 CAR coding sequence was synthesized and cloned into the pGEM-T Easy plasmid (Promega). After diluting the plasmid 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 the plasmid:viral packaging vector:viral envelope vector. Next, 120 μl of X-treme GENE HP DNA transfection reagent (Roche) was added, mixed immediately, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to a 293T cell culture flask. Viruses were harvested at 24 and 48 hours, pooled, and concentrated by ultracentrifugation (25,000 g, 4°C, 2.5 hours) to obtain the CD19 CAR lentivirus.

[0097] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco) and cultured for 1 day at 37 ° C and 5% CO2. The next day, concentrated lentivirus was added, and after continuous culture for 3 days, T cells expressing CD19 CAR (19T cells) were obtained. In addition, T cells in which CIITA was knocked out with the CRISPR / Cas9 system were transfected with the above-mentioned lentivirus to obtain 19-CII KO T cells; or T cells in which CIITA and B2M were knocked out with the CRISPR / Cas9 system to obtain 19-CI+CII KO T cells. Wild-type T cells (NT cells) that were not transduced with CAR and not genetically modified were used as a control.

[0098] Example 3. Killing effect of CD19 CAR-T cells on target cells

[0099] 1×10 4 Target Nalm6 cells were plated into 96-well plates at a concentration of 10 cells / well. NT cells, 19T cells, 19-CII KO T cells, and 19-CI+CII KO T cells were then co-cultured in 96-well plates at effector-target ratios of 4:1, 2:1, 1:1, or 0.5:1. Fluorescence was measured 18 hours later using a microplate reader. Killing efficiency was calculated using the formula: (mean fluorescence of target cells - mean fluorescence of sample) / mean fluorescence of target cells × 100%. The results are shown in Figure 1.

[0100] It can be seen that the three tested CAR-T cells targeting CD19 all showed effective and specific killing of target cells, and the killing activity was comparable. This indicates that downregulating HLA-II-related genes or further downregulating HLA-I-related genes will not adversely affect the in vitro killing activity of CAR-T cells.

[0101] Example 4. Cytokine release levels after co-incubation of CD19 CAR-T cells with target cells

[0102] 1×10 5 Target cells (Nalm6 cells) were plated in a 96-well plate at a concentration of 10 cells / well. NT cells, 19T cells, 19-CII KO T cells, and 19-CI+CII KO T cells were added at a 1:1 ratio. After 18-24 hours of co-culture, the cell co-culture supernatant was collected. The levels of IL-2 and IFN-γ in the co-culture supernatant were measured using the Human IL-2 DuoSet ELISA Kit (R&D systems) and the Human IFN-gamma DuoSet ELISA Kit (R&D systems), respectively. The results are shown in Figure 2. As can be seen, compared with NT cells, the release levels of the cytokines IL-2 and IFN-γ were significantly increased after co-culture with target cells by the three CAR-T cells, indicating that this cytokine release is specific. In addition, the release levels of IL-2 and IFN-γ by the three CAR-T cells were comparable, indicating that downregulating HLA-II-associated genes or further downregulating HLA-I-associated genes did not affect the cytokine release levels of CAR-T cells.

[0103] Example 5. In vivo inhibitory effect of CD19 CAR-T cells on tumors

[0104] Twenty healthy female NPI mice (a severe immunodeficient mouse model) aged 6-8 weeks were divided into 4 groups, with 5 mice in each group. On day 0, 5×10 5 After 6 days, 2x10 Raji cells were injected into the tail vein of each mouse according to the grouping situation. 6 NT cells, 19T cells, 19-CII KO T cells, or 19-CI+CII KO T cells were used. The survival status of the mice was assessed weekly, and the mouse survival curves are shown in Figure 3.

[0105] The inventors unexpectedly found that the in vivo tumor suppression effect of 19-CI+CII KO T cells was comparable to that of 19T cells, indicating that simultaneous knockout of HLA-I-related genes and HLA-II-related genes had no effect on the killing activity of CD19 CAR-T cells. However, it was observed that 19-CII KO T cells showed the best tumor suppression effect. This may be because knocking out HLA-II-related genes enhances the expansion ability of CAR-T cells, thereby prolonging their killing effect time in vivo. In short, this shows that compared with knocking out HLA-I-related genes and HLA-II-related genes at the same time, knocking out only HLA-II-related genes can better enhance the efficacy of CAR-T cells.

[0106] Example 6. Preparation of CD19 CAR-T cells with multiple endogenous gene knockouts and verification of their function

[0107] The CD19 CAR lentivirus prepared in Example 1 was used to transfect T cells in which CIITA and Fas were knocked out by the CRISPR / Cas9 system to obtain 19-CII+Fas KO T cells; or T cells in which CIITA and TGFBR1 were knocked out by the CRISPR / Cas9 system to obtain 19-CII+TR1 KO T cells; or T cells in which CIITA and TGFBR2 were knocked out by the CRISPR / Cas9 system to obtain 19-CII+TR2 KO T cells.

[0108] The method described in Example 2 was used to test the cytotoxic activity of the CAR-T cells against target cells Nalm6 at effector-to-target ratios of 4:1, 2:1, and 1:1, and the results are shown in Figure 4. It can be seen that compared with 19-CII KO T cells, additional knockout of Fas, TGFBR1, or TGFBR2 genes did not negatively affect and even significantly improved the cytotoxic activity of CAR-T cells.

[0109] The cytokine release levels after co-incubation of the above-mentioned CAR-T cells with target cells were detected using the method described in Example 3, and the results are shown in Figure 5. It can be seen that compared with 19-CII KO T cells, additional knockout of Fas and TGFBR1 genes did not significantly affect the cytokine release levels, while knockout of TGFBR2 gene could significantly increase the release level of INF-γ cytokine.

[0110] Twenty-five healthy female NPI mice aged 6-8 weeks were divided into five groups, with five mice in each group. On day 0, 4 × 10 6 After 6 days, 2x10 Raji cells were injected into the tail vein of each mouse according to the grouping situation. 6 The mice were treated with either NT cells, 19-CII knockout T cells, 19-CII+Fas knockout T cells, 19-CII+TR1 knockout T cells, or 19-CII+TR2 knockout T cells. The mice were assessed weekly. The progression of tumor burden in the mice is shown in Figure 6. As can be seen, further knockout of Fas, TGFBR1, or TGFBR2 genes significantly enhanced the in vivo cytotoxic activity of CD19 CAR-T cells compared to CAR-T cells with only HLA-II-related gene knockout.

[0111] Example 7. Preparation of CLDN18.2 CAR-T cells and verification of their function

[0112] The coding sequence of Claudin18.2 CAR was synthesized and cloned into the pGEM-T Easy plasmid (Promega) to obtain the CAR18.2 plasmid. The plasmid was packaged into lentivirus according to the method described in Example 1 and then transfected into T cells in which CIITA and B2M were knocked out using the CRISPR / Cas9 system to obtain 18.2-CI+CII KO T cells.

[0113] The CAR18.2 plasmid further comprises a coding sequence for the immunosuppressive molecule IIM-PDL1 targeting PD1 linked by T2A (SEQ ID NO: 31) to obtain a CAR18.2P plasmid, and the correct insertion of the target sequence in the plasmid is confirmed by sequencing. After the plasmid is packaged into a lentivirus according to the method described in Example 1, T cells in which CIITA and B2M are knocked out using the CRISPR / Cas9 system are transfected to obtain 18.2P-CI+CII KO T cells; or T cells in which CIITA is knocked out using the CRISPR / Cas9 system are transfected to obtain 18.2P-CII KO T cells.

[0114] The cytotoxicity of the CAR-T cells against target cells NUGC4-18.2 was tested using the method described in Example 2, and the results are shown in Figure 7. It can be seen that the cytotoxicity of the three tested CAR-T cells targeting Claudin18.2 was essentially equivalent at various effector-target ratios.

[0115] The proliferation levels of the above-mentioned CAR-T cells were detected by cell counting, and the results are shown in Figure 8. It can be seen that the proliferation level of 18.2-CI+CII KO T cells is the lowest, followed by 18.2P-CI+CII KO T cells, and the highest is 18.2P-CII KO T cells. This indicates that further expression of immunosuppressive molecules in CAR-T cells and replacement of HLA-I+HLA-II knockout with HLA-II knockout can significantly improve cell proliferation activity.

[0116] Example 8. Preparation of ROR1 CAR-T cells and verification of their function

[0117] The coding sequence of ROR1 CAR was synthesized and cloned into pGEM-T Easy plasmid (Promega) to obtain ROR1 plasmid. The plasmid was packaged into lentivirus according to the method described in Example 1, and then transfected into T cells in which CIITA and B2M were knocked out using the CRISPR / Cas9 system to obtain ROR1-CI+CII KO T cells.

[0118] The ROR1 plasmid further comprises the coding sequence of the immunosuppressive molecule IIM-PDL2 targeting PD1 and the coding sequence of the immunosuppressive molecule IIM-NKG2A targeting NKG2A, respectively, linked by T2A, to obtain the ROR1PA plasmid, and the correct insertion of the target sequence in the plasmid is confirmed by sequencing. After the above plasmid is packaged into a lentivirus according to the method described in Example 1, it is transfected into T cells in which CIITA and B2M are knocked out using the CRISPR / Cas9 system to obtain ROR1PA-CI+CII KO T cells; or transfected into T cells in which CIITA is knocked out using the CRISPR / Cas9 system to obtain ROR1PA-CII KO T cells.

[0119] The cytotoxicity of the CAR-T cells against target cells MDA-MB-231 was tested using the method described in Example 2, and the results are shown in Figure 9. It can be seen that the cytotoxicity of the three ROR1-targeting CAR-T cells tested was essentially equivalent at various effector-target ratios.

[0120] The proliferation level of the above-mentioned CAR-T cells was detected by cell counting, and the results are shown in Figure 10. It can be seen that the proliferation level of ROR1-CI+CII KO T cells is comparable to that of ROR1PA-CI+CII KO T cells, while the proliferation level of ROR1PA-CII KO T cells is the highest. This indicates that the simultaneous expression of immunosuppressive molecules in CAR-T cells and the knockout of HLA-II-related genes (with retention of HLA-I) can significantly enhance the proliferation activity of cells.

[0121] Example 9. Preparation of engineered B cells and verification of their function

[0122] Use the CRISPR / Cas9 system to knock out CIITA in B cells to obtain CII KO B cells; or knock out CIITA and B2M to obtain CI+CII KO B cells.

[0123] The coding sequence of the PD1-targeting immunosuppressive molecule IIM-PD1 was cloned into the pGEM-T Easy plasmid. This plasmid was packaged into lentivirus according to the method described in Example 1 and then transfected into B cells in which CIITA and B2M were knocked out using the CRISPR / Cas9 system to obtain P-CI+CII KO B cells; or transfected into B cells in which CIITA was knocked out using the CRISPR / Cas9 system to obtain P-CII KO B cells.

[0124] The inhibitory effect of the engineered B cells on the cytotoxic activity of NK92 cells was detected. Specifically, the B cells of CI+CII KO, CII KO, P-CI+CII KO and P-CII KO prepared by the present invention were labeled with Far-Red (Invitrogen, Catalog No. C34564). Then, 1x10 4 The labeled B cells were plated into 96-well plates at a concentration of 10 cells / well and co-cultured with NK92 cells at effector-target ratios of 4:1, 2:1, 1:1, and 0.5:1. After 16-18 hours, the number of surviving B cells in the culture was detected by flow cytometry using trucount, and the results are shown in Figure 11. It can be seen that compared with the knockout of HLA-I + HLA-II, the knockout of HLA-II alone significantly increased the inhibitory effect of B cells on NK cell killing, and the additional expression of immunosuppressive molecules can further enhance this inhibitory effect, thereby increasing the number of surviving B cells.

[0125] In summary, compared with knocking out both HLA-I and HLA-II simultaneously, knocking out only HLA-II (i.e., retaining HLA-I) will not adversely affect the in vitro cytotoxic activity or cytokine release levels of the engineered cells of the present invention (e.g., T cells, B cells, etc.). Instead, it can enhance the in vivo cytotoxic activity of the engineered cells (possibly by enhancing their ability to proliferate in vivo, thereby prolonging their survival), thereby enhancing the tumor suppression effect of the engineered cells. On the other hand, it can further inhibit the killing of engineered cells by endogenous immune cells, thereby reducing the risk of immune rejection. Secondly, the inventors have also found that, on the basis of knocking out HLA-II (i.e., retaining HLA-I), further knocking out genes such as Fas, TGFBR1, and TGFBR2 can enhance the in vitro and in vivo cytotoxic activity of engineered cells, or additionally expressing immunosuppressive molecules can further inhibit the killing of other immune cells, ultimately achieving a synergistic effect of enhancing the function of engineered cells.

[0126] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Those skilled in the art will appreciate that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An engineered cell, characterized in that: (i) expression of at least one endogenous HLA-II-associated gene is downregulated; and (ii) The expression of endogenous HLA-I-associated genes was not altered.

2. The engineered cell of claim 1, wherein the HLA-II-related gene is selected from the group consisting of HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof.

3. The engineered cell of claim 1, wherein the HLA-I-related genes are HLA-A, HLA-B, HLA-C, and B2M.

4. The engineered cell of any one of claims 1-3, wherein the expression of at least one endogenous TCR / CD3 gene of the engineered cell is downregulated.

5. The engineered cell of claim 4, wherein the TCR / CD3 gene is selected from the group consisting of TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

6. The engineered cell of any one of claims 1-5, wherein the expression of at least one endogenous gene selected from the group consisting of CD52, GR, dCK, PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFR1, DR3, DR4, DR5, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP 6, CASP7, FADD, FAS, TGFBR1, TGFBR2, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3, more preferably selected from FAS, TGFBR1, TGFBR2, TNFR1, DR3, DR4 and DR5.

7. The engineered cell of any one of claims 1-6, wherein the engineered cell further expresses an immunosuppressive molecule capable of reducing the immunogenicity of the engineered cell.

8. The engineered cell of claim 7, wherein the immunosuppressive molecule comprises an immunosuppressive protein binding domain, a transmembrane domain, and a co-stimulatory domain and does not comprise a primary signaling domain.

9. The engineered cell of claim 8, wherein the immunosuppressive protein binding domain comprises: (i) antibodies that specifically bind to the following targets: PD1, TIGIT, CD96, TIM3, LAG3, CTLA4, FASL, NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2 / 3, KIR3DL1, CEACAM1, LAIR1, SIGLEC7, SIGLEC9, SIRP-α, IRp60, and KLRG1; and / or (ii) an extracellular region selected from the group consisting of HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PDL1, PDL2, CD155, CD112, CD113, Gal-9, FGL1, FAS, CD80, CD86, and CD47.

10. The engineered cell of claim 9, wherein the immunosuppressive protein binding domain comprises an anti-PD1 antibody, an anti-NKG2A antibody, an anti-FasL antibody, an anti-CTLA4 antibody, a PDL1 extracellular region, a PDL2 extracellular region, an HLA-E extracellular region, a Fas extracellular region, a CD80 extracellular region, a CD86 extracellular region, or a combination thereof.

11. The engineered cell of claim 8, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD28, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD47, CD64, CD80, CD86, CD94, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, CD18, ICOS, 4-1BB, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, BCMA, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRT AM, Ly9, CD160, PSGL1, CDIOO, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D or NKG2C.

12. The engineered cell of claim 8, wherein the co-stimulatory 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, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.

13. The engineered cell of claim 8, wherein the immunosuppressive molecule does not comprise a CD3ζ intracellular domain.

14. The engineered cell of any one of claims 1-13, further expressing a functional exogenous receptor.

15. The engineered cell of claim 14, wherein the functional exogenous receptor is selected from the group consisting of a chimeric antigen receptor, a chimeric T cell receptor, a T cell antigen coupler, and a T cell fusion protein, preferably a chimeric antigen receptor.

16. The engineered cell of claim 14, wherein the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen.

17. The engineered cell of claim 16, wherein the extracellular domain comprises an antibody that specifically recognizes the antigen or a ligand of the antigen.

18. The engineered cell of claim 16 or 17, wherein 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, CD69, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138, CD151, CD171, CD179a, CD300LF, CLEC12A, CDH16, CSPG4, CS1, CLL-1, Claudin 6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, 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, PDL1, PDL2, 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, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2VEGFR, 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 region, or any combination thereof. , 19. The engineered cell of claim 18, wherein the functional exogenous receptor is a chimeric antigen receptor that targets B7H3, CD19, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD69, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA, or GPRC5D.

20. The engineered cell of any one of claims 1-19, wherein the engineered cell is a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, or a NKT cell.

21. The engineered immune cell of claim 20, wherein the engineered cell is a CD4+CD8+T cell, a CD4+T cell, a CD8+T cell, a CD4-CD8-T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell or an αβ-T cell.

22. The engineered cell of any one of claims 1-19, wherein the engineered cell is selected from or differentiated from the following stem cells: cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs.

23. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-22, and one or more pharmaceutically acceptable excipients.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is used to treat cancer, infection or autoimmune disease.

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