Enhanced cell therapy

Abstract

The present invention provides engineered cells that express an exogenous immune inhibitory molecule and down-regulate the expression of at least one endogenous gene selected from Fas, TNFR1, DR3, DR4, DR5, TGFBR1, and TGFBR2. The present invention also provides the engineered cells and compositions comprising the cells.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This disclosure claims priority to a Chinese patent application bearing application number CN2023100961489 and entitled "Enhanced Function Cell Therapy," filed with the China Patent Office on February 10, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of immunotherapy. Specifically, the present invention relates to engineered cells and compositions comprising the cells that downregulate the expression of certain endogenous genes and simultaneously express immunoinhibitory molecules, thereby enhancing efficacy and reducing the risk of rejection. [Background technology]

[0003] As an emerging immunotherapy, adoptive cell therapy has developed very rapidly. In particular, chimeric antigen receptor therapy (CART) products have been launched and have demonstrated excellent therapeutic effects in clinical trials. However, all currently available products are autologous therapies, which have disadvantages such as long manufacturing times, high costs, and high manufacturing failure rates. In response to this, allogeneic cell therapy has been developed to increase patient accessibility. However, allogeneic cell therapy also faces a number of challenges, such as the risk of immune rejection and poor cell persistence. Therefore, there is still a need to improve upon conventional allogeneic cell therapy to reduce the risk of immune rejection and enhance cell persistence. Summary of the Invention

[0004] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0005] In a first aspect, the present invention provides engineered cells having the following characteristics: (i) down-regulated expression of at least one endogenous gene selected from Fas, TNFR1, DR3, DR4, DR5, TGFBR1, and TGFBR2; and (ii) expressing an exogenous immune inhibitory molecule comprising one or more immune inhibitory protein binding domains, a transmembrane domain, and a costimulatory domain, but not a primary signaling domain, wherein the immune inhibitory protein binding domain binds to an immune inhibitory protein selected from PD1, NKG2A, FasL, and CTLA4.

[0006] Downregulation of endogenous gene expression The expression of at least one endogenous gene in the engineered cells according to the invention is downregulated, said endogenous gene being selected from Fas, TNFR1, DR3, DR4, DR5, TGFBR1 and TGFBR2.

[0007] 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). After these death receptors bind to their specific death ligands, they receive extracellular death signals, activate the intracellular apoptotic mechanism, and induce apoptosis. TGFBR1 and TGFBR2 are both receptors for TRFb1, and their intracellular domains contain serine / threonine kinase activity. They mediate Smad-dependent and -independent signaling pathways and exert important regulatory roles in processes such as cell proliferation, differentiation, wound healing, and tumor formation and metastasis. The inventors discovered that reducing the expression of these endogenous genes in cells significantly prolongs cell persistence and improves cell survival.

[0008] As used herein, "down-regulation" of gene expression refers to a significant reduction in the expression level of a modified gene product, or to the absence of expression, compared with the expression level of an unmodified wild-type gene. This means that the content of the gene's expression product (e.g., mRNA or protein) is reduced or the expression product is non-functional for the gene whose expression is "down-regulated." Therefore, down-regulation can be achieved at different levels, such as the gene level, transcription level, or translation level. Gene expression is generally down-regulated by methods well known to those skilled in the art, including, but not limited to, knocking down or knocking out a target gene or a regulatory gene for the target gene using technologies such as meganucleases, zinc finger nucleases, TALEN, CRISPR / Cas systems, base editors, and prime editors, or reducing the expression level of a target gene or a regulatory gene for the target gene using technologies such as antisense oligonucleotides, RNAi, shRNA, transposons, siRNA, antagonist RNA, mutations, antibodies, or chemical inhibitors.

[0009] immune inhibitory molecules The engineered cells of the present invention further express an exogenous immune inhibitory molecule, wherein the immune inhibitory molecule comprises one or more immune inhibitory protein binding domains, a transmembrane domain, and a costimulatory domain, and does not comprise a primary signaling domain, wherein the immune inhibitory protein binding domain binds to an immune inhibitory protein selected from PD1, NKG2A, FasL, and CTLA4.

[0010] As used herein, the term "immune inhibitory molecule" refers to a molecule that can bind to an immunoinhibitory protein (e.g., PD1, NKG2A) and inhibit a subject's immune rejection of exogenous cells, for example, by reducing the killing function of immune cells (e.g., T cells, NK cells) or inhibiting the hyperproliferation of immune cells in the subject.

[0011] In one embodiment, the immune inhibitory protein binding domain is an antibody or ligand that specifically binds to an immune inhibitory protein, including an anti-PD1 antibody, an anti-NKG2A antibody, an anti-FasL antibody, an anti-CTLA4 antibody, an extracellular region of PDL1, an extracellular region of PDL2, an extracellular region of HLA-E, an extracellular region of Fas, an extracellular region of CD80, an extracellular region of CD86, or a combination thereof.

[0012] 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 (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides having one or more CDR sequences capable of exhibiting a desired biological activity. Antibodies according to the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., 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 retain the ability to specifically bind to an antigen. It has been previously shown that the antigen-binding function of an antibody can be realized by fragments of a full-length antibody. Examples of antibody fragments of the present invention include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, single-chain antibody (scFv), disulfide-linked Fv (sdFv), antibody heavy chain variable region (VH) or light chain variable region (VL), linear antibody, "diabody" having two antigen-binding sites, single-domain antibody (sdAb), nanobody, etc. Therefore, unless the context clearly indicates otherwise, the "antibody" of the present invention covers antibody fragments as defined above. Therefore, in one preferred embodiment, the antibody of the present invention is selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibody, diabody, sdAb, or nanobody, and is preferably an scFv, sdAb, or nanobody.

[0013] Typically, a complete antibody comprises two heavy chains and two light chains linked by disulfide bonds, with each light chain linked to its respective heavy chain by a disulfide bond, forming a "Y"-shaped structure. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, where the heavy chain variable region comprises three complementarity-determining regions (CDRs), CDR1-H, CDR2-H, and CDR3-H, and the heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region, where the light chain variable region comprises three CDRs, CDR1-L, CDR2-L, and CDR3-L, and the light chain constant region comprises one constant domain, CL. In the heavy / light chain variable regions, the CDRs are separated by more conserved framework regions (FR). The variable regions of the heavy and light chains are responsible for recognizing and binding to antigens, while the constant regions can 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.

[0014] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using many numbering schemes that are well known in the art, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("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 cytotopography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains" domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (the “IMGT” numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun;309(3):657-70 (the “Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989,86(23):9268-9272 (the “AbM” numbering scheme).

[0015] The boundaries of a given CDR or FR may differ depending on the definition scheme. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering schemes for both the Kabat and Chothia schemes are based on the most common antibody region sequence lengths, where alphabetic insertions (e.g., "30a") are provided, and deletions occur in some antibodies. The two schemes generate different numbers by placing some insertions and deletions (indels) in different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.

[0016] Therefore, unless otherwise specified, it should be understood that the "CDR" of a given antibody or region thereof (e.g., its variable region) covers CDRs defined by any of the above or other known schemes. For example, when a particular CDR (e.g., CDR3) is specified to contain a given amino acid sequence, it should be understood that such CDR may further have the sequence of the corresponding CDR (e.g., CDR3) defined by any of the above or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or region thereof (e.g., its variable region) covers FRs defined by any of the above or other known schemes. Unless otherwise indicated, the numbering scheme for defining the boundaries of CDRs and FRs herein employs the Chothia scheme.

[0017] In one embodiment, the antibody of the present invention is a murine antibody, a chimeric antibody, a camelid antibody, a humanized antibody or a human antibody.

[0018] In one embodiment, the immunoinhibitory protein binding domain of the present invention is an antibody targeting PD1. PD1 is a type I transmembrane protein consisting of 288 amino acids, including an extracellular IgV domain, a transmembrane region, and an intracellular region, the latter of which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). PD-1 is expressed on the surface of activated T cells, B cells, and macrophages and is widely involved in the negative regulation of immune responses. Upon binding to its ligand (e.g., PDL1 or PDL2), PD1 upregulates the E3-ubiquitin ligases CBL-b and c-CBL, triggering downregulation of the T cell receptor and inhibiting T cell activation and cytokine release. Research has shown that immunomodulation targeting PD-1 has important implications for anti-tumor, anti-infection, anti-autoimmune disease, and organ transplant survival.

[0019] 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 the same as the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO:51, and the CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO:50. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO: 47, CDR2-L as shown in SEQ ID NO: 48, and CDR3-L as shown in SEQ ID NO: 49, while the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO: 44, CDR2-H as shown in SEQ ID NO: 45, and CDR3-H as shown in SEQ ID NO: 46.

[0020] 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 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:51, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications relative to the amino acid sequence of SEQ ID NO:51, and wherein the heavy chain variable region 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:50, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications relative to the amino acid sequence of SEQ ID NO:51. The anti-PD1 antibody of the invention comprises one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:50. Preferably, the modifications are conservative modifications, such as conservative amino acid substitutions, additions, and deletions. In one preferred embodiment, the anti-PD1 antibody of the invention comprises a heavy chain variable region set forth in SEQ ID NO:50 and a light chain variable region set forth in SEQ ID NO:51.

[0021] In one embodiment, the PD1-targeting antibody 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:52, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:9 or 14. Preferably, the PD1-targeting antibody is as set forth in SEQ ID NO:52.

[0022] In one embodiment, the immunoinhibitory protein-binding domain contained in the immunoinhibitory molecule of the present invention is the extracellular domain of a PD1 ligand, e.g., the extracellular domain of PDL1 or PDL2. In this embodiment, the immunoinhibitory molecule may further contain the corresponding transmembrane domain of PDL1 or PDL2, but generally does not also contain the corresponding intracellular domain. That is, the immunoinhibitory molecule is not full-length PDL1 or PDL2. In this embodiment, the immunoinhibitory molecule may further contain a PDL1 signal peptide or a PDL2 signal peptide.

[0023] 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 to SEQ ID NO:55, and 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 to SEQ ID NO:58. The transmembrane 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 to SEQ ID NO:54, and the transmembrane 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 to SEQ ID NO:57. The PDL1 signal peptide 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:53, and the PDL2 signal peptide 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:56.

[0024] In one embodiment, the immunoinhibitory protein-binding domain contained in the immunoinhibitory molecule of the present invention is an antibody targeting NKG2A. NKG2A is primarily expressed on NK cells and a subset of T cells (CD8+ T cells, Th2 cells, and NKT cells). NKG2A forms a dimeric complex with CD94 and is recognized by its ligand HLA-E (a non-classical HLA-I class molecule). NKG2A recruits SHP1 or SHP-2 via two ITIMs in the cytoplasmic tail, which then induces inhibitory signals and inhibits NK cytotoxic activity and cytokine secretion. Anti-NKG2A antibodies known in the art, such as Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA), and P25 (available from Moretta et al., University of Genova, Italy), can all be used in the present invention.

[0025] In one embodiment, the antibody targeting NKG2A comprises a light chain variable region and a heavy chain variable region, wherein CDR1-L, CDR2-L, and CDR3-L comprised in the light chain variable region are the same as CDR1-L, CDR2-L, and CDR3-L comprised in SEQ ID NO:40, and wherein CDR1-H, CDR2-H, and CDR3-H comprised in the heavy chain variable region are the same as CDR1-H, CDR2-H, and CDR3-H comprised 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, while 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.

[0026] 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 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:40, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:40, and wherein the heavy chain variable region 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:39, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:40. The anti-NKG2A antibody of the invention has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:39. Preferably, the modifications are conservative modifications, such as conservative amino acid substitutions, additions, and deletions. In one preferred embodiment, the anti-NKG2A antibody of the invention comprises a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:40.

[0027] In one embodiment, the NKG2A-targeting antibody 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:41, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:58. Preferably, the NKG2A-targeting antibody is as set forth in SEQ ID NO:41.

[0028] In one embodiment, the immunoinhibitory protein binding domain of the present invention comprises the extracellular region of an NKG2A ligand, e.g., the extracellular region of HLA-E. In one embodiment, the HLA-E extracellular region 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: 42 or 43. In this embodiment, the immunoinhibitory molecule may further comprise the transmembrane region of the corresponding HLA-E, but generally does not also comprise the corresponding intracellular region. That is, the immunoinhibitory molecule is not a full-length HLA-E.

[0029] In one embodiment, the immunoinhibitory protein-binding domain of the present invention is an antibody targeting FasL. FasL is a type II cell membrane surface glycoprotein and a member of the tumor death factor receptor superfamily (TNFRSF). FasL is distributed on the surface of activated T lymphocytes, NK cells, monocytes, macrophages, etc., and its ligand is Fas. The Fas / FasL system has been found to be an important pathway of apoptosis. Specifically, binding of FasL to Fas forms a death-inducing complex, which further activates the caspase signaling pathway and ultimately induces apoptosis through phosphorylation of intracellular tyrosine and serine.

[0030] Any anti-FasL antibody known in the art can be used in the present invention. In one embodiment, an 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 the same as the CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO:66, and 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:65. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO:62, CDR2-L as shown in SEQ ID NO:63, and CDR3-L as shown in SEQ ID NO:64, while the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:59, CDR2-H as shown in SEQ ID NO:60, and CDR3-H as shown in SEQ ID NO:61.

[0031] 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 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:66, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:66, and wherein the heavy chain variable region 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:65, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:66. The anti-FasL antibody of the invention has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications compared to the amino acid sequence of SEQ ID NO:65. Preferably, the modifications are conservative modifications, such as conservative amino acid substitutions, additions, and deletions. In one preferred embodiment, the anti-FasL antibody of the invention comprises a heavy chain variable region shown in SEQ ID NO:65 and a light chain variable region shown in SEQ ID NO:66.

[0032] In one embodiment, the antibody targeting FasL 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: 67 or has one or more (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 67. Preferably, the antibody targeting FasL is as set forth in SEQ ID NO: 67.

[0033] In one embodiment, the immunoinhibitory protein binding domain of the present invention comprises the extracellular region of a FasL ligand, e.g., the extracellular region of Fas. In one embodiment, the Fas extracellular region 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:68. In this embodiment, the immunoinhibitory molecule may further comprise the corresponding transmembrane region of Fas, but generally does not also comprise the corresponding intracellular region. That is, the immunoinhibitory molecule is not full-length Fas. In one embodiment, the Fas transmembrane region 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:69.

[0034] In one embodiment, the immunoinhibitory protein-binding domain of the present invention is an antibody targeting CTLA4. CTLA4, also known as CD152, is primarily expressed on the surface of activated T cells. It shares a high degree of homology with the T cell surface costimulatory molecule receptor CD28, but its affinity for its co-ligands CD80 and CD86 is significantly higher than that of CD28. In contrast to the activating functions of CD28, which promote cytokine production, cell survival, and cell differentiation, CTLA4 transmits inhibitory signals to T cells.

[0035] Any anti-CTLA4 antibody known in the art can be used in the present invention. In one embodiment, an antibody targeting CTLA4 comprises a light chain variable region and a heavy chain variable region, wherein CDR1-L, CDR2-L, and CDR3-L contained in the light chain variable region are the same as CDR1-L, CDR2-L, and CDR3-L contained in SEQ ID NO:77, and wherein CDR1-H, CDR2-H, and CDR3-H contained in the heavy chain variable region are the same as CDR1-H, CDR2-H, and CDR3-H contained in SEQ ID NO:76. In one embodiment, the light chain variable region comprises CDR1-L as shown in SEQ ID NO:73, CDR2-L as shown in SEQ ID NO:74, and CDR3-L as shown in SEQ ID NO:75, while the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:70, CDR2-H as shown in SEQ ID NO:71, and CDR3-H as shown in SEQ ID NO:72.

[0036] 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 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:77, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:77, and wherein the heavy chain variable region 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:76, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:77. The anti-CTLA4 antibody of the invention has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:76. Preferably, the modifications are conservative modifications, such as conservative amino acid substitutions, additions, and deletions. In one preferred embodiment, the anti-CTLA4 antibody of the invention comprises a heavy chain variable region set forth in SEQ ID NO:76 and a light chain variable region set forth in SEQ ID NO:77.

[0037] In one embodiment, the CTLA4-targeting antibody 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: 78, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications relative to the amino acid sequence of SEQ ID NO: 78. Preferably, the CTLA4-targeting antibody is as set forth in SEQ ID NO:78.

[0038] In one embodiment, the immunoinhibitory protein binding domain of the invention comprises the extracellular region of a CTLA4 ligand, e.g., the extracellular region of CD80 or CD86. In one embodiment, the CD80 extracellular region 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:79, and the CD86 extracellular region 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:80. In this embodiment, the immunoinhibitory molecule may further comprise the corresponding transmembrane region of CD80 or CD86, but generally does not also comprise the corresponding intracellular region. That is, the immune inhibitory molecule is not full-length CD80 or CD86. In one embodiment, the CD80 transmembrane region 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:81, and the CD86 transmembrane region 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:82.

[0039] In one embodiment, the engineered cells express one exogenous immune inhibitory molecule, and the immune inhibitory molecule is an anti-PD1 antibody, an anti-NKG2A antibody, an anti-FasL antibody, an anti-CTLA4 antibody, a Fas extracellular region, a CD80 extracellular region, a CD86 extracellular region, a PDL1 extracellular region, a PDL2 extracellular region, an HLA-E extracellular region, or a combination thereof, for example, an anti-NKG2A antibody (or an HLA-E extracellular region) and an anti-PD1 antibody (or a PDL1 extracellular region, a PDL2 extracellular region), an anti-NKG2A antibody (or an HLA-E extracellular region) and an anti-FasL antibody (or a Fas extracellular region), an anti-NKG2 The combinations include combinations of an A antibody (or an HLA-E extracellular region) and an anti-CTLA4 antibody (or an extracellular region of CD80 or CD86), an anti-PD1 antibody (or an extracellular region of PDL1 or PDL2) and an anti-FasL antibody (or an extracellular region of Fas), an anti-PD1 antibody (or an extracellular region of PDL1 or PDL2) and an anti-CTLA4 antibody (or an extracellular region of CD80 or CD86), and an anti-FasL antibody (or an extracellular region of Fas) and an anti-CTLA4 antibody (or an extracellular region of CD80 or CD86) (the two binding domains in the combination may be linked via a linker).

[0040] In one embodiment, the engineered cells express two exogenous immune inhibitory molecules, each of which contains a binding domain that binds to a different immune inhibitory protein, e.g., the two exogenous immune inhibitory molecules bind to NKG2A and PD1, NKG2A and CTLA4, NKG2A and FasL, PD1 and CTLA4, PD1 and FasL, or CTLA4 and FasL, respectively. For example, in one embodiment, the engineered cells express two exogenous immune inhibitory molecules, each of which binds to an anti-NKG2A antibody (or HLA-E extracellular domain) and an anti-PD1 antibody (or a PDL1 extracellular domain, a PDL2 extracellular domain), an anti-NKG2A antibody (or an HLA-E extracellular domain) and an anti-FasL antibody (or a Fas extracellular domain), or an anti-NKG2A antibody (or an HLA-E extracellular domain) and an anti-CTLA4 antibody (or a CD4 domain). Examples of exogenous immune inhibitory molecules include an anti-PD1 antibody (or an anti-PD1 antibody, such as an anti-PD1 antibody), an anti-FasL antibody (or an anti-Fas extracellular region), an anti-PD1 antibody (or an anti-PD1 antibody, such as an anti-PD1 antibody), an anti-PD1 antibody (or ...

[0041] As used herein, the term "conservative modification" refers to an amino acid modification that does not affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. These conservative modifications are conservative amino acid substitutions, additions, and deletions. Modifications may be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications may be selected based on, for example, similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0042] As used herein, the term sequence "identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is generally expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with the exact same sequence have 100% identity. As known to those skilled in the art, several algorithms may be used to determine sequence identity, 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.

[0043] As used herein, the term "transmembrane domain" refers to a polypeptide structure that allows an immunoinhibitory molecule to be expressed on the cell surface and that can anchor an immunoinhibitory protein-binding domain on a cell membrane. The transmembrane domain may be natural or synthetic and may be derived from any membrane-associated or transmembrane protein. When the target-binding domain binds to a target, the transmembrane domain can transduce signals. Transmembrane domains that are particularly applicable to the present invention include, for example, TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD28, CD45, CD4, CD5, 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 β, IL-2R γ, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl 1d, ITGAE, CD103, ITGAL, CDl 1a, LFA-1, ITGAM, CDl 1b, ITGAX, CDl 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRT AM, Ly9, CD160, PSGL1, CDI00, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or NKG2C. In some embodiments, the transmembrane domain is derived from the molecules CD8a, CD4, CD28, 4-1BB, CD47, CD80, CD86, CD152, and PD1, or may be synthetic and comprise primarily 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.

[0044] In one embodiment, the immunoinhibitory molecule further comprises a hinge region located between the immunoinhibitory protein binding domain and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to an antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antibody. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a natural molecule, for example, derived in whole or in part from the extracellular region of CD8, CD4, or CD28, or derived in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In preferred embodiments, the hinge region comprises a portion of the hinge region of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge. In one embodiment, the hinge region is derived from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:26. In one embodiment, the hinge region is derived from CD8a, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO:27 or 28. In one embodiment, the hinge region is derived from IgG4, 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:29.

[0045] As used herein, a "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. It is an intracellular functional signaling domain from a costimulatory molecule, including the entire intracellular region of the costimulatory molecule or a functional fragment thereof. A "costimulatory molecule" refers to a homologous binding partner that mediates a costimulatory response (e.g., proliferation and survival) by specifically binding to a costimulatory ligand. The costimulatory signaling domain of any costimulatory molecule applies to the immune inhibitory molecules described herein. Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the invention include, but are not limited to, intracellular regions derived from the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, 4-1BB, CD270, CD272, B7-H3, ICOS, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof. Preferably, the costimulatory domain of the CAR of the invention is 4-1BB, CD28, or 4-1BB+CD28. In one embodiment, the costimulatory domain is derived 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 set forth in SEQ ID NO:18 or 19. In one embodiment, the costimulatory 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 to the amino acid sequence set forth in SEQ ID NO:17.

[0046] The immune inhibitory molecules of the present invention do not contain a primary signaling domain. As used herein, the term "primary signaling domain" refers to protein structures that act together to trigger primary signal transduction after antigen-receptor binding, which are typically intracellular sequences of T cell receptors and coreceptors. Primary signaling domains typically contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of primary signaling domains 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 immune inhibitory molecules of the present invention do not contain the CD3ζ intracellular region. For example, the CD3ζ intracellular region 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:21, 22, or 20.

[0047] In one embodiment, the immunoinhibitory molecule of the present invention further comprises a signal peptide, which, when expressed in cells, e.g., T cells, directs the nascent protein to the endoplasmic reticulum and then to the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment, which tends to form a single α-helix. The end of the signal peptide generally contains an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave the signal peptide during or after the shifting process 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 useful in the present invention is derived from B2M, 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: 23. In one embodiment, the signal peptide useful in the present invention 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: 24 or 25.

[0048] Functional exogenous receptors In one embodiment, the engineered cells of the present invention can also express a functional exogenous receptor, preferably 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.

[0049] As used herein, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from each component of a TCR, generally consisting of a TCR subunit and an antibody linked thereto, expressed on the cell surface, where the TCR subunit includes at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.

[0050] As used herein, the term "T cell antigen coupler" or "TAC" comprises three functional domains: 1. a tumor-targeting domain comprising a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting group; 2. an extracellular domain that is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor and the TCR receptor into close proximity; and 3. a transmembrane domain and an intracellular domain of the CD4 co-receptor, where the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as an early step in T cell activation.

[0051] As used herein, the term "T cell receptor" or "TCR" refers to a characteristic marker on the surface of T cells, which non-covalently binds to CD3 to form a complex. Antigen-presenting cells present antigen peptides to T cells via major histocompatibility complex molecules (MHC), which then bind to the TCR complex and induce a series of intracellular signal transduction events. TCRs consist of six peptide chains that form heterodimers, which are generally classified into αβ and γδ types. Each peptide chain contains a constant region and a variable region, where the variable region is responsible for binding to a specific antigen and MHC molecule. The term "recombinant TCR receptor" refers to an artificially constructed T cell receptor that further contains an antigen (e.g., tumor antigen) binding domain.

[0052] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide, which generally comprises an antigen (e.g., tumor antigen)-binding domain (e.g., an antibody or antigen ligand), a transmembrane domain, an optional costimulatory domain, and a primary signaling domain, each of which is connected via a linker. CARs can redirect the specificity and responsiveness 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, which comprises 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, etc.

[0053] In one embodiment, a 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 the antigen or a ligand of the antigen. In one embodiment, the antigen is 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, Claudin6, 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 LightChain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, for MSLN、MUC1、MUC16、MAGE-A1、MAGE3、MAD-CT-1、MelanA / MART1、ML-IAP、MYCN hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D catalyst, OY- TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostasis, PAP, PDGFR-β PCTA-1 / ガレン8, p53, p53 protein complex, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK. RAGE-1, ROR1, Ras activator, 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, Tie2, 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 telomere end reverse transcriptase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variants of tumor necrotic regions, 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 alpha, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from ROR1, CD19, Claudin18.2, MSLN, GPRC5D, CD7 and BCMA.

[0054] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD19, e.g., an antibody that targets CD19. In one embodiment, the antibody that targets CD19 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1-L as set forth in SEQ ID NO:8, CDR2-L as set forth in SEQ ID NO:9, and CDR3-L as set forth in SEQ ID NO:10, and the heavy chain variable region comprises CDR1-H as set forth in SEQ ID NO:5, CDR2-H as set forth in SEQ ID NO:6, and CDR3-H as set forth in SEQ ID NO:7.

[0055] 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 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:12, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:12, and wherein the heavy chain variable region 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:11, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:12. The antibody of the present invention has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:11. Preferably, the modifications are conservative modifications, such as conservative amino acid substitutions, additions, and deletions. In one preferred embodiment, the antibody of the present invention comprises a heavy chain variable region set forth in SEQ ID NO:11 and a light chain variable region set forth in SEQ ID NO:12.

[0056] In one embodiment, the CD19-targeting antibody 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: 13, or has one or more (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO: 13. Preferably, the CD19-targeting antibody is as set forth in SEQ ID NO: 13.

[0057] In one embodiment, the functional exogenous receptor comprises an extracellular domain that specifically recognizes Claudin18.2, such as an antibody that targets Claudin18.2. In one embodiment, the antibody that targets Claudin18.2 is a single domain antibody. Preferably, the single domain antibody comprises CDR1 as set forth in SEQ ID NO:1, CDR2 as set forth in SEQ ID NO:2, and CDR3 as set forth in SEQ ID NO:3.

[0058] 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, or has one or more (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) amino acid modifications compared to the amino acid sequence of SEQ ID NO:4. Preferably, the antibody targeting Claudin18.2 is as set forth in SEQ ID NO:4.

[0059] In one embodiment, the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises 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, which targets CD19, CD7, CD19, CD20, CD22, CD30, CD33, CD38, 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, more preferably 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. For definitions of the structures of the transmembrane domain, costimulatory domain, primary signaling domain, and optional hinge region, signal peptide, etc. of CARs usable in the present invention, see the section "Immune inhibitory molecules" above.

[0060] In one embodiment, the CAR of the present invention may further comprise a switch structure to control the expression time of the CAR. For example, the switch structure may be in the form of a dimerization domain, which undergoes a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain and activating a signaling pathway by binding to a targeted antigen. Alternatively, a switch domain may be used to link the binding domain and the signaling domain, respectively, and only when the switch domains bind to each other (e.g., in the presence of an inducing compound) can the binding domain and the signaling domain be linked via a dimer, thereby activating the signaling pathway. The switch structure may also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain and prevent binding to a targeted antigen, and after cleavage of the masking peptide by, for example, a protease, the extracellular binding domain can be exposed, resulting in a "normal" CAR structure. Any of a variety of switch structures known to those skilled in the art can be used in the present invention.

[0061] In one embodiment, the CAR of the present invention may further comprise a suicide gene, i.e., express a cell death signal that can be induced by an exogenous substance, allowing CAR cells to be eliminated when necessary (e.g., when severe toxic side effects occur). For example, the suicide gene may be in the form of an inserted epitope, such as the CD20 epitope or RQR8, and CAR cells can be eliminated when necessary using antibodies or reagents targeting these epitopes. The suicide gene may be herpes simplex virus thymidine kinase (HSV-TK), which can cause cell death when induced by ganciclovir treatment. The suicide gene may also be iCaspase-9, which can induce iCaspase-9 dimerization with chemical inducers such as AP1903 and AP20187, thereby activating downstream Caspase-3 molecules and causing apoptosis. Any of a variety of suicide genes known to those skilled in the art may be used in the present invention.

[0062] Engineered cells In one embodiment, the engineered cells of the present invention are engineered immune cells.

[0063] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). For example, an immune cell may be a B cell, a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell, or a NKT cell. Immune cells can be obtained from many sources, such as from a subject (e.g., isolated from a subject's peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor, etc.), from an in vitro cultured cell line (e.g., Jurkat, SupT1, NK92, etc.), or differentiated from a stem cell (e.g., derived from umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs, etc.). Preferably, the immune cells are T cells or NK cells, more preferably T cells. T cells may be enriched or purified. T cells may be at any stage of development, 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, and the like. In one preferred embodiment, the immune cells are human T cells. T cells can be obtained from a subject's blood using many techniques known to those skilled in the art, such as Ficoll apheresis.

[0064] In one embodiment, the engineered cells of the present invention are engineered stem cells or cells differentiated from such stem cells. Examples of stem cells include, but are not limited to, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs, etc.

[0065] In one embodiment, the endogenous HLA-I class gene and / or HLA-II class gene of the engineered cells of the invention are unmodified. In one embodiment, the endogenous HLA-I class gene of the engineered cells of the invention are unmodified. In one embodiment, the endogenous HLA-II class gene of the engineered cells of the invention are unmodified. In one embodiment, the endogenous HLA-I class and HLA-II class genes of the engineered cells of the invention are unmodified. In one embodiment, the expression of at least one endogenous HLA-I class gene and / or at least one HLA-II class gene of the engineered cells of the invention is downregulated. In one embodiment, the expression of at least one endogenous HLA-I class gene of the engineered cells of the invention is downregulated. In one embodiment, the expression of at least one endogenous HLA-II class gene of the engineered cells of the invention is downregulated. In one embodiment, the expression of at least one endogenous HLA-I class gene and at least one endogenous HLA-II class gene of the engineered cells of the invention is downregulated. In one embodiment, the expression of at least one endogenous TCR / CD3 gene in an engineered cell of the invention is downregulated. In one embodiment, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-I class gene in an engineered cell of the invention is downregulated. In one embodiment, the expression of at least one endogenous TCR / CD3 gene and at least one endogenous HLA-II class gene in an engineered cell of the invention is downregulated. In one embodiment, the expression of at least one endogenous TCR / CD3 gene, at least one endogenous HLA-I class gene and at least one endogenous HLA-II class gene in an engineered cell of the invention is downregulated. Preferably, the HLA-I class gene is selected from HLA-A, HLA-B, HLA-C and B2M.Preferably, the HLA-II class gene is selected from HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, and CIITA, and more preferably, RFX5, RFXAP, RFXANK, and CIITA. Preferably, the TCR / CD3 gene is selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, and CD3ζ.

[0066] 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 whom the material is introduced. Two or more individuals are considered allogeneic to one another if they differ in genes at one or more loci. In some cases, genetic differences in allogeneic material from individuals of the same species may be sufficient to allow antigenic interactions to occur.

[0067] Pharmaceutical Composition The present invention further provides a pharmaceutical composition, which comprises an engineered cell according to the present invention as an active agent and one or more pharmaceutically acceptable excipients, and thus the present invention further covers the use of said engineered cell in the manufacture of a pharmaceutical composition or medicament.

[0068] 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 active ingredient (i.e., capable of producing the required therapeutic effect without causing undesired local or systemic effects), and is known in the art (see, e.g., 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, coating agents, adsorbents, anti-adherents, fluidizers, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. As known to those skilled in the art, appropriate excipients are selected to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Generally, the selection of an appropriate excipient depends on the particular active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0069] The pharmaceutical composition according to the present invention can be administered by various routes. Generally, administration is completed parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

[0070] The pharmaceutical compositions according to the present invention may be prepared in various forms, for example, solid, liquid, gaseous or lyophilized, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form specifically adapted to the required method of administration. The processes for producing known drugs of the present invention may include, for example, conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. For example, pharmaceutical compositions comprising immune cells as described herein are generally provided in the form of a solution and preferably contain a pharmaceutically acceptable buffer.

[0071] The pharmaceutical composition according to the present invention can also be administered in combination with one or more other drugs (biological agents, e.g., antibody reagents, and / or small molecules) or therapeutic methods (e.g., surgery, chemotherapy, or radiation therapy) applied to treat and / or prevent the disease to be treated. Preferred examples of drugs applied in combination include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, isocyclophosphamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, and the like. E), well-known anticancer drugs such as 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; and 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. The pharmaceutical compositions of the present invention can also be used in combination with one or more other therapeutic methods, such as chemotherapy and radiation therapy.

[0072] In one embodiment, the pharmaceutical compositions of the invention are used to treat a subject suffering from cancer, an infection, or an autoimmune disease.

[0073] In one embodiment, the cancer is a cancer associated with target expression that binds to a functional exogenous receptor. For example, the cancer may be brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma (hospital gallbladder cancer), or the like. Cancers of the urinary system, including Hodgkin's lymphoma and non-Hodgkin's lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, cervical or endometrial cancer, malignancies of the urinary system, vaginal cancer, and other cancers and sarcomas, and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NH (including L, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, and bulky disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), and B-cell lymphoma. These include, but are not limited to, follicular prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, dispersed large B-cell lymphoma, follicular lymphoma, chronic myeloid leukemia (CML), malignant lymphoproliferative disorders, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablastic lymphoma, preleukemia, plasmacytoid dendritic cell neoplasm, and post-transplant lymphoproliferative disorder (PTLD), and other diseases associated with target expression. Preferably, diseases that can be treated using 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.

[0074] In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

[0075] In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus.

[0076] The present invention will be described in detail below in conjunction with examples, with reference to the drawings. It should be noted that those skilled in the art should understand that the drawings and the embodiments of the present invention are merely for illustrative purposes and do not limit the present invention in any way. Unless contradictory, the features of the embodiments and embodiments in this application can be combined with each other. [Brief explanation of the drawings]

[0077] [Figure 1] This shows the killing activity of Fas-knockout CAR-T cells against target cells. [Figure 2] Cytokine release levels after Fas-knockout CAR-T cells (A: CD19-targeting CAR-T cells; B: Claudin18.2-targeting CAR-T cells) were co-incubated with target cells. [Figure 3] The figure shows the inhibitory effect of Fas-knockout CAR-T cells (A: CAR-T cells targeting CD19, B: CAR-T cells targeting Claudin18.2) on the T cell killing activity. [Figure 4] 1 shows the in vivo tumor-inhibitory effect of Fas-knockout CAR-T cells. [Figure 5] Figure 1 shows the killing activity of CAR-T cells in which TGFBR1 or TGFBR2 (A: CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1, B: CAR-T cells containing immune inhibitory molecules targeting FasL) have been knocked out against target cells. [Figure 6]Cytokine release levels after TGFBR1 or TGFBR2 knockout CAR-T cells were co-incubated with target cells (A: IL2 and IFN-γ release levels of CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1; B: IL2 release levels of CAR-T cells containing immune inhibitory molecules targeting FasL; C: IFN-γ release levels of CAR-T cells containing immune inhibitory molecules targeting FasL). [Figure 7] Figure 1 shows the in vivo tumor-inhibitory effect of CAR-T cells in which TGFBR1 or TGFBR2 has been knocked out (A: CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1; B: CAR-T cells containing immune inhibitory molecules targeting FasL). DETAILED DESCRIPTION OF THE INVENTION

[0078] Exemplary CAR and immune inhibitory molecule structures used in the examples below: The CAR structure comprises, in order, 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). The extracellular domain contained in the CD19 CAR structure is an anti-CD19 scFv (SEQ ID NO:13). The extracellular domain contained in the Claudin18.2 CAR structure is an anti-Claudin18.2 vhh (SEQ ID NO:4).

[0079] The structure of the PD1-targeting immune inhibitory molecule IIM-PDL1 comprises, in order, a PDL1 signal peptide (SEQ ID NO: 53), a PDL1 extracellular domain (SEQ ID NO: 55), a PDL1 transmembrane domain (SEQ ID NO: 54), and a CD28 costimulatory domain (SEQ ID NO: 17).

[0080] The structure of the immune inhibitory molecule IIM-NKG2A, which targets NKG2A, comprises an anti-NKG2A scFv (SEQ ID NO:41), 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), which are linked in order.

[0081] The structure of IIM-FasL, an immune inhibitory molecule that targets FasL, contains, in order, a B2M signal peptide (SEQ ID NO: 23), an anti-FasL scFv (SEQ ID NO: 67), an IgG4 hinge region (SEQ ID NO: 29), a CD28 transmembrane domain (SEQ ID NO: 14), and a CD28 costimulatory domain (SEQ ID NO: 17).

[0082] Example 1. Construction of CAR-T immune cells of the present invention The coding sequence of CD19 CAR was synthesized and cloned into pGEM-T Easy vector (Promega) to obtain the CAR19 plasmid. The CAR19 plasmid further contained the coding sequence of IIM-PDL1, an immune inhibitory molecule that targets PD1 and is linked via T2A (SEQ ID NO: 30), to obtain the CAR19P plasmid.

[0083] The coding sequence of Claudin18.2 CAR was synthesized and cloned into pGEM-T Easy vector (Promega) to obtain the CAR18.2 plasmid. The CAR18.2 plasmid further contained the coding sequences of the PD1-targeting immune inhibitory molecules IIM-PDL1 and IIM-NKG2A, which are linked via T2A (SEQ ID NO: 30), to obtain the CAR18.2AP plasmid. The CAR18.2 plasmid further contained the coding sequences of the FasL-targeting immune inhibitory molecules IIM-FasL, which are linked via T2A (SEQ ID NO: 30), to obtain the CAR18.2FL plasmid.

[0084] The plasmid was diluted in 3 ml of Opti-MEM (Gibco) in a sterile tube, followed by the addition of the packaging vector psPAX2 (Addgene) and the envelope vector pMD2.G (Addgene) in a ratio of 4:2:1 (plasmid:viral packaging vector:viral envelope vector). 120 μl of X-treme GENE HP DNA Transfection Reagent (Roche) was then added, immediately mixed thoroughly, and incubated at room temperature for 15 minutes. The plasmid / vector / transfection reagent mixture was then added dropwise to a flask of 293T cells. Virus was collected at 24 and 48 hours, combined, and then ultracentrifuged at 25,000 g for 2.5 hours at 4°C to obtain concentrated lentivirus.

[0085] T cells were activated with DynaBeads CD3 / CD28 CTS™ (Gibco) and cultured for 1 day at 37°C and 5% CO. The next day, concentrated lentivirus was added, and after 3 days of continuous culture, T cells expressing CD19 CAR (CAR19), T cells expressing CD19 CAR and IIM-PDL1 combination (CAR19P), T cells expressing Claudin18.2 CAR and IIM-FasL (CAR18.2FL), and T cells expressing Claudin18.2 CAR and a combination of two immune inhibitory molecules (IIM-PDL1 + IIM-NKG2A) (CAR18.2AP) were obtained. In addition, the lentivirus was used to transfect T cells (CAR19, CAR19P, and CAR18.2AP) in which the Fas gene had been knocked out using the CRISPR / Cas9 system, to obtain CAR19-Fas KO T cells, CAR19P-Fas KO T cells, and CAR18.2AP-Fas KO T cells.

[0086] Example 2. Killing effect of CAR-T cells on target cells 1×10 4Target cells, Raji cells, were plated in a 96-well plate at a concentration of cells / well. NT cells, CAR19P T cells, CAR19-Fas KO T cells, and CAR19P-Fas KO T cells were plated in a 96-well plate at effector-target ratios of 4:1, 2:1, 1:1, or 0.5:1. After 18 hours of co-culture, fluorescence was measured using a microplate reader. Killing efficiency was calculated according to the formula: (mean target cell fluorescence - mean sample fluorescence) / mean target cell fluorescence × 100%. The results are shown in Figure 1.

[0087] As can be seen, CAR19P T cells, CAR19-Fas KO T cells, and CAR19P-Fas KO T cells all showed effective specific killing of target cells, and the killing activity of CAR19P-Fas KO T cells was significantly improved compared to the previous two, indicating that the combination of downregulation of the Fas gene and expression of immune inhibitory molecules produced a synergistic effect, maximizing the killing effect of CAR19T cells.

[0088] Example 3. Cytokine release levels after CAR-T cells are co-incubated with target cells 1×10 5Target cells (Raji cells) were plated in a 96-well plate at a concentration of 100 cells / well, and NT cells, CAR19P T cells, and CAR19P-Fas KO T cells were added at a 1:1 ratio. After 18–24 hours of co-culture, the cell co-culture supernatants were collected. IL2 and IFN-γ levels in the co-culture supernatants were measured using the Human IL-2 DuoSet ELISA Kit (R&D Systems) and the Human IFN-γ DuoSet ELISA Kit (R&D Systems), respectively. The results are shown in Figure 2A. Compared with NT cells, the release of both cytokines, IL2 and IFN-γ, was significantly higher after co-culture with the target cells, demonstrating the specificity of cytokine release. Furthermore, knockout of the Fas gene significantly enhanced IFN-γ release in CAR19 T cells.

[0089] Using a similar method, we detected the cytokine release levels after co-incubation of Claudin18.2-targeting CAR-T cells with target cells NUGC4-18.2, and the results are shown in Figure 2B. Similarly, knockout of the Fas gene significantly increased the IFNγ release levels of CAR18.2 T cells.

[0090] Example 4. Knockout of the Fas gene significantly enhances CAR-T cell survival To verify the killing-resistant ability of CAR-T cells with downregulated Fas gene expression, we first used 1 × 10 5 The CAR-T cells of the present invention were plated in a 96-well plate at a concentration of 100 cells / well, and effector CAR7 T cells (T cells expressing CD7 CAR, labeled with Far-red) were also plated in the 96-well plate at an effector-target ratio of 1:1. Co-culture was performed, and the survival rate of the CAR-T cells of the present invention was detected 24 hours later by flow cytometry. The results are shown in Figure 3A (targeting CD19) and Figure 3B (targeting Claudin18.2).

[0091] As can be seen, the survival rates of only CAR19P-T cells and CAR18.2AP-T cells expressing immunoinhibitory molecules were comparable to those of NT cells, and further knockout of the Fas gene significantly improved the survival of CAR-T cells.

[0092] Example 5. Inhibitory effect of CAR T cells on tumors in vivo Fifteen 6-8 week-old healthy female NPI mice were divided into three groups, each with five mice: NT cells, CAR19P-T cells, and CAR19P-Fas KO T cells. On day 0, each mouse received 5 × 10 5 Six days later, 2x10 Raji cells were injected into each mouse via the tail vein. 6 NT cells or the corresponding CAR-T cells were injected intravenously into the tail vein. The mouse condition was evaluated weekly. The mouse survival curve is shown in Figure 4.

[0093] As can be seen, the survival rate of mice treated with CAR19P-T cells was significantly higher than that of the control group, and the CAR19P-Fas KO T cell group, in which the Fas gene was knocked out, showed the longest survival period, indicating that knocking out the endogenous Fas gene can enhance the in vivo killing activity of CAR-T cells.

[0094] Example 6. Construction of CAR-T cells in which TGFBR1 or TGFBR2 has been knocked out and which express immune inhibitory molecules, and verification of their function CAR-T cells expressing immune inhibitory molecules and in which TGFBR1 or TGFBR2 has been knocked out were constructed according to the method of Example 1. Specifically, the CAR18.2AP and CAR18.2FL plasmids prepared in Example 1 were packaged into lentiviruses, respectively, and then transfected with TGFBR1-knockout T cells using the CRISPR / Cas9 system to obtain CAR18.2AP-TR1 KO T cells and CAR18.2FL-TR1 KO T cells, or with TGFBR2-knockout T cells using the CRISPR / Cas9 system to obtain CAR18.2AP-TR2 KO T cells and CAR18.2FL-TR2 KO T cells.

[0095] The killing activity of the CAR-T cells was detected using the method described in Example 2, and the results are shown in Figure 5A (CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1) and Figure 5B (CAR-T cells containing immune inhibitory molecules targeting FasL). As can be seen, compared to CAR18.2AP T cells, additional knockout of the TGFBR1 or TGFBR2 gene did not significantly affect the killing activity of CAR-T cells, whereas compared to CAR18.2FL T cells, additional knockout of the TGFBR1 or TGFBR2 gene enhanced the killing activity of CAR-T cells.

[0096] The cytokine release levels after co-incubation of the CAR-T cells with target cells were detected using the method described in Example 3. The results are shown in Figure 6A (IL2 and IFN-γ release levels of CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1), Figure 6B (IL2 release levels of CAR-T cells containing immune inhibitory molecules targeting FasL), and Figure 6C (IFN-γ release levels of CAR-T cells containing immune inhibitory molecules targeting FasL). As can be seen, additional knockout of the TGFBR1 or TGFBR2 gene did not significantly affect the cytokine release levels compared to CAR18.2AP T cells or CAR18.2FL T cells.

[0097] Twenty 6-8 week-old healthy female NPI mice were divided into four groups, with five mice per group: NT cells, CAR18.2AP T cells, CAR18.2AP-TR1 KO T cells, and CAR18.2AP-TR2 KO T cells. Twenty 6-8 week-old healthy female NPI mice were divided into four groups, with five mice per group: NT cells, CAR18.2FL T cells, CAR18.2FL-TR1 KO T cells, and CAR18.2FL-TR2 KO T cells. On day 0, each mouse received 4 × 10 6 NUGC4-18.2 cells were injected intravenously into the tail vein. After 14 days, 2x10 cells were injected into each mouse depending on the group. 6 NT cells or the corresponding CAR-T cells were injected intravenously into the tail vein. Mice were evaluated weekly. The progression of mouse tumor burden is shown in Figure 7A (CAR-T cells containing immune inhibitory molecules targeting NKG2A and PD1) and Figure 7B (CAR-T cells containing immune inhibitory molecules targeting FasL). As can be seen, compared to CAR18.2AP T cells, additional knockout of the TGFBR1 or TGFBR2 gene significantly reduced the tumor burden in mice and maintained it at a much lower level, with almost no tumor cells detected by the end of the experiment. Compared to CAR18.2FL T cells, additional knockout of the TGFBR1 or TGFBR2 gene significantly reduced the tumor burden in mice, and the tumor burden-reducing effect of CAR-T cells was even more pronounced after the TGFBR2 gene was knocked out.

[0098] In summary, knocking out endogenous genes such as Fas, TGFBR1, and TGFBR2 and inhibiting immunoinhibitory molecules does not adversely affect the killing activity or cytokine release characteristics of CAR-T cells (indeed, knocking out Fas and expressing immunoinhibitory molecules produces a synergistic effect, significantly improving the killing activity and IFNγ release levels), while significantly reducing the risk of immune rejection of CAR-T cells, improving their survival rate, and further enhancing the tumor-inhibitory effect of CAR-T cells compared to expressing immunoinhibitory molecules alone.

[0099] It should be noted that the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and those skilled in the art can make various modifications and changes to the present invention. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should all be included in the protection scope of the present invention.

Claims

1. 1. An engineered cell, comprising: (i) the expression of at least one endogenous gene selected from Fas, TNFR1, DR3, DR4, DR5, TGFBR1, and TGFBR2 is downregulated; (ii) expressing an exogenous immune inhibitory molecule comprising one or more immune inhibitory protein binding domains, a transmembrane domain and a costimulatory domain, but not a primary signaling domain, wherein the immune inhibitory protein binding domain binds to an immune inhibitory protein selected from PD1, NKG2A, FasL, and CLTA4.

2. 2. The engineered cell of claim 1, wherein the immune inhibitory protein binding domain comprises an anti-PD1 antibody, an anti-NKG2A antibody, an anti-FasL antibody, an anti-CTLA4 antibody, an extracellular region of PDL1, an extracellular region of PDL2, an extracellular region of HLA-E, an extracellular region of Fas, an extracellular region of CD80, an extracellular region of CD86, or a combination thereof.

3. The transmembrane domains include 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, and 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 3. The engineered cell of claim 1 or 2, wherein the transmembrane domain is selected from the group consisting of AM, Ly9, CD160, PSGL1, CD100, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C proteins.

4. 4. The engineered cell of any one of claims 1 to 3, wherein the costimulatory domain is selected from the intracellular regions 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.

5. The engineered cell of any one of claims 1 to 4, wherein the immune inhibitory molecule does not contain the CD3ζ intracellular domain.

6. The engineered cell of claim 1 , wherein the engineered cell expresses two exogenous immune inhibitory molecules.

7. The engineered cell of claim 6 , wherein the two exogenous immune inhibitory molecules contain binding domains that bind to different immune inhibitory proteins.

8. 8. The engineered cell of any one of claims 1 to 7, further expressing a functional exogenous receptor.

9. 9. The engineered cell of claim 8, wherein the functional exogenous receptor is selected from 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.

10. 10. The engineered cell of claim 9, wherein the functional exogenous receptor comprises an extracellular domain that specifically recognizes an antigen.

11. The engineered cell of claim 10 , wherein the extracellular domain comprises an antibody that specifically recognizes the antigen or a ligand of the antigen.

12. The antigens are 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, Claudin18.2, 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, LILRA, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, muthsp70-2, 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, 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 telomere end reverse transcriptase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tenascin, oncofetal variants of tumor necrotic regions, or any combination thereof.

13. 9. The engineered cell of claim 8, wherein the functional exogenous receptor is a chimeric antigen receptor, and the chimeric antigen receptor targets CD19, CD7, CD19, CD20, CD22, CD30, CD33, CD38, 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.

14. The engineered cell of any one of claims 1 to 13, wherein expression of at least one endogenous TCR / CD3 gene of the engineered cell is inhibited or silenced.

15. 15. The engineered cell of claim 14, wherein the TCR / CD3 genes are selected from TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, and combinations thereof.

16. The engineered cell of claim 1, wherein the HLA-I class gene and / or the HLA-II class gene of the engineered cell is unmodified.

17. 2. The engineered cell of claim 1, wherein expression of at least one endogenous HLA-I class gene and / or HLA-II class gene of the engineered cell is downregulated.

18. The engineered cell of any one of claims 1 to 17, 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.

19. 19. The engineered cell of claim 18, 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, an immature T cell, a γδ-T cell, or an αβ-T cell.

20. 20. The engineered cell of any one of claims 1 to 19, wherein the engineered cell is derived from umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSCs.

21. A pharmaceutical composition comprising the engineered cells of any one of claims 1 to 20 and one or more pharmaceutically acceptable excipients.

22. 22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is used to treat cancer, an infection, or an autoimmune disease.

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