CN111234033B

CN111234033B - Multi-chain chimeric antigen receptors and uses thereof

Info

Publication number: CN111234033B
Application number: CN202010072106.8A
Authority: CN
Inventors: 史其萍; 江雯; 贺小宏; 任江涛; 王延宾; 韩露
Original assignee: Nanjing Bioheng Biotech Co Ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2021-05-11
Anticipated expiration: 2040-01-21
Also published as: WO2021147929A1; CN111234033A

Abstract

The present invention relates to a multi-chain chimeric antigen receptor comprising: (a) a chimeric receptor comprising a first protein interaction domain, a transmembrane domain, and an intracellular signaling domain; and (b) an Fc fusion polypeptide comprising an antigen binding region, a second protein interaction domain, and an Fc region, wherein the first protein interaction domain is capable of specifically binding to the second protein interaction domain. The invention also relates to engineered immune cells comprising the multi-chain chimeric antigen receptor of the invention and pharmaceutical compositions thereof, and the use of the engineered immune cells/pharmaceutical compositions in the treatment of cancer.

Description

Multi-chain chimeric antigen receptors and uses thereof

Technical Field

The present invention relates to the field of immunotherapy, in particular, the invention relates to a multi-chain Chimeric Antigen Receptor (CAR) and its use, in particular in the treatment of cancer.

Background

In recent years, cancer immunotherapy technology has been rapidly developed, and particularly, chimeric antigen receptor T cell (CAR-T) -related immunotherapy has achieved excellent clinical effects in the treatment of hematological tumors. CAR-T cell immunotherapy is that T cells are genetically modified in vitro to enable the T cells to recognize tumor antigens, and the tumor antigens are amplified to a certain amount and then are infused back into a patient body to kill cancer cells, so that the purpose of treating tumors is achieved.

There are many cells involved in or associated with an immune response in humans, including T lymphocytes (also called T cells), B lymphocytes (also called B cells), natural killer cells (NK cells), macrophages, dendritic cells, mast cells, and the like. Among them, T cells are the main component of lymphocytes, and have various biological functions, such as direct killing of target cells, assistance or inhibition of antibody production by B cells, response to specific antigens, cytokine production, and the like. The immune response generated by T cells is cellular, and the effector forms of cellular immunity are two main types: one is to combine with the target cell specifically, destroy the target cell membrane, and kill the target cell directly; the other is the release of lymphokines, which ultimately amplifies and enhances the immune effect. NK cells are small in number but essential for innate immunity in humans. The recognition of foreign antigens by such immune cells does not require the mediation of antibodies and Major Histocompatibility Complex (MHC), and the immune killing response of NK cells is rapid. The wide and rapid immune killing capability of NK cells makes them an ideal immune cell in tumor immune cell therapy. Macrophages have multiple functions, and have phagocytosis effects on pathogens and the like, and can also play a role in presentation after being taken up. A large number of Tumor Associated Macrophages (TAMs) are also located throughout the Tumor microenvironment. They have a high degree of interaction with tumor cells, tumor stem cells, epidermal cells, fibroblasts, as well as T cells, B cells, NK cells, and the like. Dendritic Cells (DCs) are the most powerful professional Antigen Presenting Cells (APCs) of the body, and can efficiently take up, process and present Antigen. NK cells and macrophages have significant tumor infiltration superiority, and can efficiently present antigens to T cells. Moreover, NK cells also have the effect of activating DC cells.

Therefore, the activation of NK cells, macrophages, DC cells and the like while the CAR-T immunotherapy is carried out can help to solve the problems of the CAR-T cell therapy, such as immunosuppression of tumor microenvironment, tumor heterogeneity, difficult infiltration of T cells and the like, and obviously improve the overall therapeutic effect.

Summary of The Invention

In a first aspect, the present invention provides a multi-chain chimeric antigen receptor comprising: (a) an Fc fusion polypeptide comprising an antigen binding region, a first protein interaction domain, and an Fc region; and (b) a chimeric receptor polypeptide comprising a second protein interaction domain, a transmembrane domain, and an intracellular signaling domain, wherein the first protein interaction domain is capable of specifically binding to the second protein interaction domain.

In one embodiment, the antigen-binding region is selected from the group consisting of a sdAb, a nanobody, an antigen-binding ligand, a recombinant fibronectin domain, an anticalin, and a DARPIN.

In one embodiment, the antigen binding region is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a murine antibody, and a chimeric antibody.

In one embodiment, the target to which the antigen binding region binds is selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- β, SSEA-4, CD20, Folate receptor α, ERBB 20 (Her 20/neuu), MUC 20, EGFR, NCAM, Claudin18.2, Proudpase, PAP, ELF2 RB, ELBhrin B72, IGF-I receptor, CAGIBCI, CAGCADCOGAV 72, EPDG-72, EPGCHAV 72, EPDCHA-72, EPBCAA-72, EPDCASK-72, EPBCAA-72, EPOCHAV-72, EPTC 20, EPOCHAV-20, EPTC 20, EPC 20, EPOCHAV-20, EPTC 20, EPOCHAV-72, EPC 20, EPOCHAV-72, EPOCHAV-, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutants, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMSS 2 ETS fusion gene), NA17, PAX 84, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1, BORIS B, SART3, PAX5, OY-1, TES, SSAP-3748, SSLACE-3748, AK 5819-IRE 5, RU 5979, RU-599, RU-IRU 639, RU-5, RU-5979, RU-5, RU-599, RAG-related antigen 1, and its related genes, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D, and any combination thereof. Preferably, the target is selected from the group consisting of CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, claudin18.2, NKG2D, and any combination thereof.

In one embodiment, the chimeric receptor polypeptide comprises a transmembrane domain selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28 and CD 278.

In one embodiment, the chimeric receptor polypeptide comprises an intracellular signaling domain selected from the signaling domains of the following proteins: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular signaling domain is a signaling domain comprising CD3 ζ.

In one embodiment, the chimeric receptor polypeptide further comprises one or more co-stimulatory domains. Preferably, the co-stimulatory domain is a co-stimulatory signaling domain of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278(ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP 70. Preferably, the co-stimulatory domain is a co-stimulatory signaling domain of CD27, CD28, CD134, CD137, or CD 278.

In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain, preferably the CH2 and CH3 domains of IgG 1.

In one embodiment, the first protein interaction domain and the second protein interaction domain are a combination selected from the group consisting of: FKBP 506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB) of mTOR, FKBP and calcineurin a (cna), FKBP and cyclophilin (CyP), GAI and GID, Snap and Halo tags, Glucocorticoid Receptor (GR) and DHFR, PYL and ABI, dimerization docking domain of cAMP-dependent protein kinase a (pka) and anchoring domain of a-kinase anchoring protein (AKAP), avidin and biotin, leucine zipper domain and leucine zipper domain, and zinc finger domain and nucleotide tag. Preferably, the first protein interaction domain and the second protein interaction domain are both leucine zipper domains, e.g., AZip and BZip, respectively. Alternatively, preferably, the first protein interaction domain and the second protein interaction domain are a zinc finger domain and a nucleotide tag, respectively. In a specific embodiment, the multi-chain chimeric antigen receptor of the present invention may further comprise a second Fc fusion polypeptide comprising a second antigen-binding region, a third protein-interacting domain, and a second Fc region. For example, in a specific embodiment, the second protein interaction domain is a first portion of a nucleotide tag, the third protein interaction domain is a second portion of the nucleotide tag, and the complex specifically binds to the zinc finger domain as the first protein interaction domain only when the first portion of the nucleotide tag and the second portion of the nucleotide tag form a complex.

In a second aspect, the invention also provides a nucleic acid comprising a sequence encoding the multi-chain chimeric antigen receptor of the invention, a vector or vector system comprising said nucleic acid, and an immune cell comprising said nucleic acid or vector system.

In one embodiment, the invention provides a nucleic acid comprising a sequence encoding a chimeric receptor polypeptide of the invention and a sequence encoding an Fc fusion polypeptide of the invention. Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

In one embodiment, the invention provides a vector comprising the above-described nucleic acid. In particular, the vector is selected from the group consisting of a linear nucleic acid molecule, a plasmid, a retrovirus, lentivirus, adenovirus, vaccinia virus, Rous Sarcoma Virus (RSV), polyoma and adeno-associated virus (AAV), a bacteriophage, a phagemid, a cosmid, or an artificial chromosome. In some embodiments, the vector further comprises elements such as an origin of autonomous replication in immune cells, a selectable marker, a restriction enzyme cleavage site, a promoter, a poly a tail (polyA), a 3 'UTR, a 5' UTR, an enhancer, a terminator, an insulator, an operator, a selectable marker, a reporter, a targeting sequence, and/or a protein purification tag. In a specific embodiment, the vector is an in vitro transcription vector.

In one embodiment, the present invention provides a vector system comprising a first nucleic acid sequence encoding a chimeric receptor polypeptide of the present invention and a second nucleic acid sequence encoding an Fc fusion polypeptide of the present invention, the first and second nucleic acid sequences being located on different vectors. In another embodiment, the first nucleic acid sequence and the second nucleic acid sequence are located on the same vector.

In one embodiment, the invention provides an immune cell comprising a nucleic acid or vector system of the invention, which is capable of expressing a multi-chain chimeric antigen receptor of the invention. In a specific embodiment, the immune cell is selected from the group consisting of a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell. Preferably, the T cell is a CD4+/CD8+ double positive T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γ δ -T cell, or an α β -T cell.

In a third aspect, the present invention provides a pharmaceutical composition comprising a multi-chain chimeric antigen receptor of the invention as defined above or a nucleic acid encoding, vector or vector system thereof or an immune cell comprising the same, and one or more pharmaceutically acceptable excipients.

In a fourth aspect, the invention provides a method of treating a subject suffering from cancer, comprising administering to the subject an effective amount of a multi-chain chimeric antigen receptor, immune cell or pharmaceutical composition according to the invention.

In one embodiment, the multiple peptide chains comprised by the multi-chain chimeric antigen receptor of the present invention may be administered together or separately. For example, an immune cell or pharmaceutical composition comprising a first Fc fusion polypeptide, and an immune cell or pharmaceutical composition comprising a chimeric receptor polypeptide, respectively, can be administered to a subject. In another embodiment, the treatment may further comprise further administering to the subject an immune cell or pharmaceutical composition comprising a second Fc fusion polypeptide comprising a second antigen-binding region, a third protein-interacting domain, and a second Fc region.

In one embodiment, the cancer is selected from: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and CNS cancers, breast carcinoma, peritoneal carcinoma, cervical carcinoma, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial carcinoma, esophageal carcinoma, eye carcinoma, head and neck carcinoma, gastric carcinoma, Glioblastoma (GBM), liver carcinoma, hepatoma, intraepithelial tumors, kidney carcinoma, laryngeal carcinoma, leukemia, liver tumor, lung carcinoma, lymphoma, melanoma, myeloma, neuroblastoma, oral carcinoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, retinoblastoma, rhabdomyosarcoma, rectal carcinoma, cancer of the respiratory system, salivary gland carcinoma, skin carcinoma, squamous cell carcinoma, gastric carcinoma, testicular carcinoma, thyroid carcinoma, uterine or endometrial carcinoma, malignant tumors of the urinary system, vulval carcinoma and other carcinomas and sarcomas, as well as B-cell lymphoma, and also cancers of the head and neck, 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 prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disorder, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytoma-precursor lymphoma, leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD).

Detailed Description

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

Multi-chain chimeric antigen receptors

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide whose basic structure includes an antigen binding region (e.g., an antigen-binding portion of an antibody), a transmembrane domain, and an intracellular signaling domain. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner using the antigen-binding properties of monoclonal antibodies. non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigen independent of antigen processing, thus bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with the alpha and beta chains of the endogenous T Cell Receptor (TCR). Typically, the extracellular binding domain of a CAR consists of a single chain variable fragment (scFv) derived from a fusion of the variable and light chain regions of a monoclonal antibody of murine or human origin or chimeric. Alternatively, the scFv that can be used is derived from a Fab (rather than from an antibody, e.g., obtained from a Fab library). In various embodiments, such scFv is fused to a transmembrane domain, and subsequently fused to an intracellular signaling domain. Currently, with advances in technology, four different generations of CAR structures have emerged. The intracellular signaling domain of the first generation CARs contained only the primary signaling domain, e.g., CD3 ζ, and thus cells carrying the CARs (e.g., CAR-T cells) were poorly active and had short survival times in vivo. Second generation CARs incorporate a costimulatory domain, such as CD28 or 4-1BB, to enable cells to continue to proliferate, enhancing antitumor activity. Third generation CARs contain two costimulatory domains (e.g., CD28+4-1 BB), fourth generation CARs incorporate cytokines or costimulatory ligands to further enhance T cell responses, or suicide genes to self-destroy CAR cells when needed.

As used herein, the term "multi-chain chimeric antigen receptor" or "multi-chain CAR" refers to a CAR comprising at least two peptide chains, wherein each peptide chain comprises a protein interaction domain, and each peptide chain is capable of only one of target binding and signaling functions. Signaling while binding to the target can only occur when the at least two peptide chains bind to each other (e.g., via specific binding of a protein interaction domain). For example, when a multi-chain CAR comprises two peptide chains, one chain being an Fc fusion polypeptide responsible for target binding and the other chain being a chimeric receptor polypeptide responsible for signaling, the two peptide chains bind to each other through the respective protein interaction domains comprised. When the multi-chain CAR comprises three or more peptide chains, the protein-interacting domain comprised by the third chain may form a complex with the protein-interacting domain comprised by the second chain, thereby specifically binding to the protein-interacting domain comprised by the first chain and initiating a signaling pathway, or may compete with the protein-interacting domain comprised by the second chain for binding to the protein-interacting domain comprised by the first chain, e.g., by replacing the second chain with a stronger binding activity, thereby recognizing a new target and signaling.

In one embodiment, the multi-chain chimeric antigen receptor of the present invention comprises: (a) a chimeric receptor comprising a first protein interaction domain, a transmembrane domain, and an intracellular signaling domain; and (b) an Fc fusion polypeptide comprising an antigen binding region, a second protein interaction domain, and an Fc region, wherein the first protein interaction domain is capable of specifically binding to the second protein interaction domain.

As used herein, the term "protein interaction domain" refers to a domain that allows two separate polypeptides to specifically bind to each other. Provided herein are a number of exemplary protein interaction domain and their combinatorial pairs. In some embodiments, the first protein interaction domain in the multi-chain CAR can specifically bind to the second protein interaction domain. In some embodiments, specific binding occurs between two separate protein interaction domains. In some embodiments, specific binding occurs between three separate protein interaction domains. Exemplary protein interaction domains are known in the art and may be used in the embodiments described herein.

In one embodiment, the first protein interaction domain and the second protein interaction domain are a combination selected from the group consisting of: FKBP 506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB) of mTOR, FKBP and calcineurin a (cna), FKBP and cyclophilin (CyP), GAI and GID, Snap and Halo tags, Glucocorticoid Receptor (GR) and DHFR, PYL and ABI, dimerization docking domain of cAMP-dependent protein kinase a (pka) and anchoring domain of a-kinase anchoring protein (AKAP), avidin and biotin, leucine zipper domain and leucine zipper domain, and zinc finger domain and nucleotide tag.

In a preferred embodiment, the first protein interaction domain and the second protein interaction domain are both leucine zipper domains. As used herein, the term "leucine zipper domain" refers to a class of protein-protein interaction domains commonly found in transcription factors characterized by leucine residues evenly spaced by an alpha-helix. The leucine zipper may form a heterodimer or homodimer. In a specific embodiment, the first protein interacting domain and the second protein interacting domain are AZip and BZip, respectively. In one embodiment, AZip 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. 4 and BZip 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. 6. Other suitable leucine zipper domains may include SYNZIP 1-SYNZIP 48, as well as BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, and hepatad. Many leucine zipper domains are known in the art and can be used in the present invention.

In another preferred embodiment, the first protein interaction domain and the second protein interaction domain are a zinc finger domain and a nucleotide tag, respectively. The zinc finger domain consists of one alpha-helix and two antiparallel beta-sheets, and has the function of binding zinc ions. The zinc finger domain is capable of recognizing a specific base sequence, thereby regulating the activity of a gene comprising the specific base sequence. Preferably, the zinc finger domain binds DNA and the nucleotide tag is a DNA tag, more preferably a dsDNA tag. Examples of zinc finger domains and their associated nucleotide tags are well known to those skilled in the art.

In one embodiment, in the case of a zinc finger domain and a nucleotide tag, the nucleotide tag can be divided into multiple parts, e.g., two parts, three parts, and can only specifically bind to the zinc finger domain when the multiple parts are combined to form the complete nucleotide tag. Thus, in a particular embodiment, the multi-chain chimeric antigen receptor of the invention comprises: (a) a chimeric receptor polypeptide comprising a zinc finger domain, a transmembrane domain, and an intracellular signaling domain; (b) a first Fc fusion polypeptide comprising a first antigen binding region, a first portion of a nucleotide tag, and a first Fc region; and (c) a second Fc fusion polypeptide comprising a second antigen binding region, a second portion of a nucleotide tag, and a second Fc region, the complex being capable of specifically binding to the zinc finger domain only when the first portion of the nucleotide tag and the second portion of the nucleotide tag form a complex. The first portion may be ssDNA and the second portion is ssDNA complementary thereto, or the first portion is dsDNA having overhangs and the second portion is dsDNA having complementary overhangs, which first and second portions can hybridize under suitable conditions to form the complete dsDNA nucleotide tag required for binding to the zinc finger domain. In another specific embodiment, the dsDNA nucleotide tags are comprised of three parts that are present in each of the three Fc fusion polypeptides, e.g., the first part is ssDNA, the second part and the third part are ssDNA that are complementary to the first part, respectively, and do not overlap with each other, and the complex is capable of specifically binding to the zinc finger domain only when the nucleotide tags of the three parts form a complete nucleotide tag that is capable of being recognized by the zinc finger domain.

In one embodiment, the first protein interaction domain and the second protein interaction domain are chemically Induced protein interaction domains that bind specifically to form dimers only in the presence of specific chemicals, also known as the Chemically Induced Dimerization (CID) system. For example, exemplary chemically-induced protein-interaction domains include, but are not limited to, the following combinations: induction of bound FKBP and FRB by rapamycin and derivatives thereof such as photosensitive caged rapamycin; PYL and ABI bound by abscisic acid induction; GID and GAI induced by gibberellin; FKBP and calcineurin a (cna) that induce binding by FK 506; induction of bound FKBP and cyclophilin (CyP) by FKCsA; snap tags and Halo tags that bind induced by HaXS; the combined Glucocorticoid Receptor (GR) and dihydrofolate reductase (DHFR) was induced by dexamethasone-methotrexate (Dex-Mtx). Many CID systems are known to those skilled in the art, see, for example, Vo β S et al, Current Opinion in Chemical Biology, 2015, 28: 194-.

In one embodiment, the first protein interacting Domain and the second protein interacting Domain are the Dimerization Docking Domain (DDD) of cAMP-dependent protein kinase a (pka) and the Anchoring Domain (AD) of a-kinase Anchoring protein (AKAP), respectively. PKA has two types of R subunits (RI and RII), and each type has α and β isoforms, so there are four types of DDD: RI α, RI β, RII α, and RII β. AKAP is widely present in a variety of species and is localized to a variety of subcellular sites including the plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum. The AD used in AKAP to bind PKA is an amphipathic helix with 14-18 residues. The amino acid sequence of AD is quite different between AKAPs. The binding between AD and DDD is specific and very high in affinity. The sequences of various AD and DDD peptides and variants thereof are known to those of skill in the art, e.g., as described in Baillie et al, FEBS letters.2005, 579:3264. Wong; scott, nat. rev. mol.cell biol.2004,5: 959; PCT/US03/054842, incorporated herein by reference in its entirety.

In one embodiment, the first protein interaction domain and the second protein interaction domain are avidin and biotin, respectively. Avidin is a basic glycoprotein composed of 4 identical subunits, resistant to the action of a variety of proteolytic enzymes, and commonly used includes, for example, streptavidin. Biotin is widely found in various animal and plant tissues and comprises two cyclic structures, of which the imidazolone ring is the major part of the binding to avidin. The binding interaction between avidin and biotin has good stability and strong specificity, and is not influenced by organic solvents such as reagent concentration, PH environment, protein denaturant and the like.

Chimeric receptor polypeptides

As used herein, the term "chimeric receptor" refers to a polypeptide comprising a protein interaction domain located on a cell membrane that functions primarily to initiate a signaling pathway following specific binding of the protein interaction domain, thereby activating the activity of an immune cell comprising the chimeric receptor.

The chimeric receptor of the present invention comprises a first protein interaction domain, a transmembrane domain, and an intracellular signaling domain, wherein the protein interaction domain is defined as described above.

As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables expression of a chimeric receptor polypeptide on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and directs the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the multi-chain chimeric antigen receptor of the invention binds to a target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, the TCR α chain, the TCR β chain, the TCR γ chain, the TCR δ chain, the CD3 ζ subunit, the CD3 ε subunit, the CD3 γ subunit, the CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the human CD8 alpha chain and 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 of SEQ ID NO. 12.

In one embodiment, the chimeric receptor polypeptides of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an antigen binding region. In particular, the hinge region serves to provide greater flexibility and accessibility to the antigen binding region. The hinge region may comprise 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 from all or part of a naturally occurring molecule, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a portion of the hinge region of human CD8 a chain, Fc γ RIII a receptor, IgG4 or IgG1, more preferably the hinge of human CD8 a or 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 of SEQ ID No. 26 or 28.

As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specified function. The intracellular signaling domain is responsible for intracellular signaling after binding of the antigen at the antigen-binding region, resulting in activation of the immune cell and immune response. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.

In one embodiment, the intracellular signaling domain comprised by the chimeric antigen receptor of the present invention may be the cytoplasmic sequences of the T cell receptor and co-receptor that work together to trigger signaling upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same or similar function. The intracellular signaling domain comprises two different types of cytoplasmic signal sequences: those that elicit antigen-dependent primary activation, as well as those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. The primary cytoplasmic signal sequence may contain a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the invention include those derived from, but are not limited to, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d, alone. In a preferred embodiment, the signaling domain of the chimeric receptor polypeptides of the invention may comprise a CD3 zeta signaling domain having 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. 16.

In one embodiment, the chimeric antigen receptor of the present invention further comprises one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, which may comprise the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, co-stimulatory signaling domains derived from: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278(ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP 70. Preferably, the co-stimulatory domain of the CAR of the invention is a 4-1BB and/or CD28 fragment, more preferably having 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 of SEQ ID No. 14.

In a preferred embodiment, the chimeric antigen receptor of the invention comprises a CD8 a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. More preferably, the chimeric antigen receptor further comprises a CD28 co-stimulatory domain and a CD8 a hinge region or an IgG4 hinge region.

Fc fusion polypeptide

As used herein, the term "Fc fusion polypeptide" is a recombinant polypeptide comprising a protein interaction domain, an Fc region, and an antigen binding region, wherein the protein interaction domain is defined as described above. When normally expressed and secreted, the Fc fusion polypeptides of the present invention are capable of binding to Fc receptors on the surface of other immune cells, such as macrophages, NK cells, dendritic cells, etc., thereby recruiting these immune cells, performing additional killing on target cells or performing antigen presentation, and augmenting the killing effect of CART cells. In addition, the Fc fusion polypeptides of the invention can also provide additional antigen binding regions, i.e., provide individual target cell killing capabilities, as well as diverse antigen targeting.

As used herein, "antigen binding region" refers to any structure or functional variant thereof that can bind to an antigen. The antigen binding region can be an antibody structure including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and functional fragments thereof. For example, the antigen binding region includes, but is not limited to, Single Domain antibodies (sdabs), nanobodies (nbs), antigen binding ligands, recombinant fibronectin domains, anticalins, DARPINs, and the like, preferably selected from sdabs and nanobodies. In one embodiment, the Fc fusion polypeptide of the present invention comprises an antigen binding region that is not a Single chain antibody (scFv) in the present invention, the antigen binding region can be monovalent or bivalent, and can be monospecific, bispecific, or multispecific. In another embodiment, the antigen binding region may also be a specific binding polypeptide or receptor structure for a particular protein, such as PD1, PDL1, PDL2, TGF β, APRIL and NKG 2D.

"Single-chain antibody" or "scFv" is an antibody in which an antibody variable region (VH) and a light chain variable region (VL) are linked via a linker. The optimal length and/or amino acid composition of the linker may be selected. The length of the linker will significantly affect the variable region folding and interaction profiles of the scFv. In fact, if shorter linkers are used (e.g., between 5-10 amino acids), intra-strand folding may be prevented. For the choice of linker size and composition, see, e.g., Hollinger et al, 1993Proc Natl Acad. Sci. U.S.A.90: 6444-; U.S. patent application publication nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT publication nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference in their entirety.

"Single domain antibody" or "sdAb" refers to an antibody that naturally lacks a light chain, which comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also referred to as "heavy chain antibodies".

"Nanobody" or "Nb" refers to a VHH structure that is cloned and expressed individually, has structural stability comparable to that of an original heavy chain antibody and binding activity to an antigen, and is the smallest unit currently known to bind to a target antigen.

The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a parent, but contains at least one amino acid modification (i.e., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the Fc fusion polypeptide or chimeric receptor polypeptide of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including 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, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

Thus, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a parent amino acid sequence and retains the biological activity, e.g., binding activity, of the parent amino acid.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. One skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, 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.

In one embodiment, the antigen binding region of the invention binds to one or more targets selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD24, Folate receptor alpha, ERNYBB 24/neu, MUC 24, EGFR, NCAM, Prostase, ELF2 24, Ephrin B72, IGF-I receptor, CAIX, LMP 24, pOOOO-24, FuCOPAP-72, EPCTOC 72, EPTC-72, EPTC 24, EPT 5-5, EPTC-72, EPTC 24, EPTC-72, EPTC-24, EPT 5, EPT-72, EPT 5, EPTC-72, EPT 5, EPT-72, EPT 5, EPTC-72, EPT 5, EPT, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B, BORIS, SART3, PAX5, OY-TES, LCK, AK-4, SSX2, RAGE-1, human telomerase, reverse transcriptase 2, LRRU 2, SART 8672, GPC 2, CD2, CD2, GPC 2, CD2, CD2, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D, and any combination thereof. Preferably, the target is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D, and any combination thereof.

According to a particular embodiment, the antigen binding region binds to claudin18.2. According to another particular embodiment, the antigen binding region comprises a functional variant directed against the above sequences, such as a variant of SEQ ID NO:8 has the same CDR as SEQ ID NO:8 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The functional variant may be formed by substitution, addition or deletion of one or more (e.g., 1 to 10, 1 to 5 or1 to 3) amino acid residues. In particular, the functional variant is identical to SEQ ID NO:8 have the same or similar functions and activities.

In one embodiment, the multi-chain chimeric antigen receptor of the present invention may comprise two or more Fc fusion polypeptides, wherein each Fc fusion polypeptide comprises a protein interaction domain, an antigen binding region, and an Fc region, wherein the antigen binding regions may bind to the same or different targets, and wherein the Fc regions may be the same or different.

The term "Fc region" as used herein refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least part of the constant region. The Fc region has no antigen binding activity and is the site of interaction of an immunoglobulin with effector molecules or cells. The term includes native Fc regions and variant Fc regions. By "native Fc region" is meant a molecule or sequence comprising a non-antigen binding fragment, whether in monomeric or multimeric form, produced by digestion of an intact antibody. The immunoglobulin source that produces the native Fc region is preferably derived from a human. Native Fc fragments are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (e.g., disulfide) and non-covalent linkages. Natural Fc molecule monomer subunits have 1-4 intermolecular disulfide bonds between them, depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA 2). An example of a native Fc region is a disulfide-linked dimer produced by digestion of IgG with papain (see Ellison et al (1982), Nucleic Acids Res.10: 4071-9). The term "native Fc" as used herein generally refers to monomeric, dimeric and multimeric forms. A "variant Fc region" refers to an amino acid sequence that differs from the amino acid sequence of a "native" or "wild-type" Fc region due to at least one "amino acid modification" as defined herein, also referred to as an "Fc variant". Thus, "Fc region" also includes single chain Fc (scfc), i.e., a single chain Fc region composed of two Fc monomers linked by a polypeptide linker, which is capable of folding naturally into a functional dimeric Fc region. In one embodiment, the variant Fc region has at least about 80%, at least about 85%, at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or at least about 99% sequence identity to the native Fc region.

In a particular embodiment, the Fc fusion polypeptide of the invention comprises an Fc region preferably derived from IgG. Human IgG has four subtypes based on the antigenic difference of the r chain in IgG molecules: IgG1, IgG2, IgG3 and IgG4, wherein the IgG1 has the highest distribution abundance in serum. The constant region sequences of these four subtypes are highly homologous, but each subtype is specific for antigen binding, immune complex formation, complement activation, triggering effector cells, half-life, and placental transport properties. Generally, IgG1 and IgG3 have higher affinity for Fc receptors than IgG2 and IgG4 in structural and functional effects, and have a greater ability to activate antibody-dependent and complement-dependent cytotoxicity; the IgG2 and IgG4 subtypes have a blocking or inhibitory effector function. Thus, in a preferred embodiment, the Fc fusion polypeptide of the invention comprises an Fc region preferably derived from IgG1 to enhance the affinity of the Fc region for receptors and thereby increase the efficiency of recruitment of other immune cells.

In one embodiment, the Fc region of the present invention refers to a constant region that does not comprise CH 1. For example, in the case of IgA, IgD and IgG, the Fc region comprises the constant domains CH2 and CH 3; in the case of IgE and IgM, the Fc region comprises the constant domains CH2, CH3 and CH 4. In addition, for IgG, the Fc region may also comprise a lower hinge region between CH1 and CH 2. Thus, preferably, the Fc region of the invention comprises CH2 and CH3 of IgG1, more preferably further comprises a lower hinge region between CH1 and CH 2. In a particular embodiment, the Fc region has the same or similar receptor binding activity as the amino acid sequence shown in SEQ ID No. 10 and 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. 10.

Nucleic acids

The invention also provides a nucleic acid comprising a sequence encoding the chimeric receptor polypeptide of the invention and a sequence encoding the Fc fusion polypeptide of the invention.

As used herein, the term "nucleic acid" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form in single-and/or double-stranded form, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as found in Peptide Nucleic Acids (PNAs)). The nucleic acids of the invention may also contain one or more modified bases such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications are also contemplated, so long as the multi-stranded CARs of the invention can be expressed from the polynucleotide. The nucleic acid may be provided in an isolated form. In one embodiment, the nucleic acid may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcriptional termination signals), ribosome binding sites, introns, and the like.

The nucleic acid sequences of the invention may be codon optimized for optimal expression in a desired host cell (e.g., an immune cell); or for expression in bacterial, yeast or insect cells. Codon optimization refers to the replacement of codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, with the codons before and after the replacement encoding the same amino acid. Thus, the choice of optimal codons depends on the codon usage bias of the host genome.

Carrier

The invention also provides a vector comprising one or more nucleic acids according to the invention.

The present invention also provides a vector system comprising a first nucleic acid sequence encoding a chimeric receptor polypeptide and a second nucleic acid sequence encoding an Fc fusion polypeptide; the first nucleic acid sequence and the second nucleic acid sequence are located on the same vector or on different vectors.

As used herein, the term "vector" is a vector nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at a site specifically. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding the Fc fusion polypeptides or chimeric receptor polypeptides of the invention) in a suitable host cell and the translation of their mRNA. Suitable carriers for use in the present invention are known in the art and many are commercially available. In one embodiment, vectors of the invention include, but are not limited to, linear nucleic acid molecules (e.g., DNA or RNA), plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous sarcoma viruses (RSV, polyoma, and adeno-associated viruses (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC). the vectors themselves are typically nucleotide sequences, typically DNA sequences comprising inserts (transgenes) and larger sequences that serve as a "backbone" for the vector Selection markers, reporter genes, targeting sequences, and/or protein purification tags. In a specific embodiment, the vector is an in vitro transcription vector.

In one embodiment, the first nucleic acid sequence encoding the chimeric receptor polypeptide and the second nucleic acid sequence encoding the Fc fusion polypeptide are located on the same vector. For example, a chimeric receptor polypeptide of the invention and an Fc fusion polypeptide can be expressed independently without affecting each other by inserting a nucleic acid encoding a 2A peptide between the two nucleic acid sequences. As used herein, the term "2A peptide" is a cis-hydrolase acting element (CHYSEls) originally found in foot-and-mouth disease virus (FMDV). The 2A peptide has an average length of 18 to 22 amino acids. During protein translation, the 2A peptide can be cleaved from its last2 amino acids C-terminus by ribosome skipping. Specifically, the peptide chain binding group between glycine and proline is impaired at the 2A site, and initiates ribosome skipping to start translation from the 2 nd codon, thereby allowing independent expression of 2 proteins in1 transcription unit. This 2A peptide-mediated cleavage is widely present in eukaryotic animal cells. The expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.) can be improved by utilizing the higher cleavage efficiency of 2A peptide and the ability to promote balanced expression of upstream and downstream genes. Conventional 2A peptides comprise: P2A, T2A, E2A, F2A, and the like. In another embodiment, the first nucleic acid sequence encoding the chimeric receptor polypeptide and the second nucleic acid sequence encoding the Fc fusion polypeptide are located on different vectors.

Engineered immune cells and methods of making the same

The present invention provides engineered immune cells comprising a Chimeric Receptor polypeptide or a nucleic acid encoding thereof, and an Fc fusion polypeptide or a nucleic acid encoding thereof, also referred to herein as a gate CAR (Fc induced target cell engaging nucleic Antigen Receptor) cell. Thus, in one embodiment, an engineered immune cell of the invention comprises a first nucleic acid sequence encoding a chimeric receptor polypeptide of the invention and a second nucleic acid sequence encoding an Fc fusion polypeptide of the invention.

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, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cell may be a T cell, macrophage, dendritic cell, monocyte, NK cell, and/or NKT cell. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. The T cells may be any type of T cell and may be at any developmental stage, including, but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γ δ -T cells, α β -T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. 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 isolation. In the present invention, the immune cells are engineered to express a chimeric receptor polypeptide and an Fc fusion polypeptide.

Using conventional techniques known in the artMethods (e.g., by transduction, transfection, transformation, etc.) may introduce into an immune cell a first nucleic acid sequence encoding a chimeric receptor polypeptide and a second nucleic acid sequence encoding an Fc fusion polypeptide, such that they simultaneously express the chimeric receptor polypeptide and the Fc fusion polypeptide of the invention. "transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is used primarily for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection may be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their cargo into the interior. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. The term "transformation" is used to describe the non-viral transfer of a nucleic acid molecule or polynucleotide (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by direct uptake of the cell membrane from its surroundings and subsequent incorporation of foreign genetic material (nucleic acid molecules). The transformation may be achieved by artificial means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. Techniques for plant transformation include Agrobacterium-mediated transfer (such as by Agrobacterium tumefaciens: (A)A.tumefaciens) Rapid propelled tungsten or gold microprojectiles, electroporation, microinjection, and polyethylene glycol-mediated uptake.

In yet another embodiment, the immune cell of the invention further comprises at least one inactivated gene selected from the group consisting of: CD52, GR, TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε, CD247 ζ, HLA-I, HLA-II genes, immune checkpoint genes such as PD1 and CTLA-4. More particularly, the immune cell may comprise an inactivated gene of at least one selected TCR α or TCR β gene. This inactivation renders the TCR non-functional in the cell. This strategy is particularly useful for avoiding graft versus host disease (GvHD). Methods of inactivating a gene are known in the art, for example, by mediating DNA cleavage by meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, thereby inactivating the gene.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a multi-chain chimeric antigen receptor, nucleic acid, vector, system or engineered immune cell of the invention as an active agent, and one or more pharmaceutically acceptable excipients. Thus, the invention also encompasses the use of said multi-chain chimeric antigen receptor, nucleic acid, vector, system or engineered immune cell in the preparation of a pharmaceutical composition or medicament.

As used herein, the term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edied 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, anti-adherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, 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 extracts of fluid extracts, or in a form which is particularly suitable for the desired method of administration. Processes known in the art for the manufacture of medicaments may comprise, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells, multi-chain chimeric antigen receptors, or encoding nucleic acids or vectors thereof, such as described herein, are typically provided in solution form, and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the diseases to be treated. Preferred examples of the pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium ii (sorfimer Sodiumtofrin ii), temozolomide, topotecan, glucuronide (trimetrenate glucoside), oritavastin e (auristatin E), vincristine, and adriamycin; peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, and rnase; 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 IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides.

Method for preparing engineered immune cells

The invention also provides a method of making an engineered immune cell comprising introducing into an immune cell a nucleic acid sequence encoding a chimeric receptor polypeptide and an Fc fusion polypeptide of the invention or both, such that the immune cell expresses the chimeric receptor polypeptide and the Fc fusion polypeptide of the invention.

In one embodiment, the immune cell is a human immune cell, more preferably a human T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell.

Methods for introducing and expressing nucleic acids or vectors into immune cells are known in the art. For example, nucleic acids or vectors can be introduced into immune cells by physical methods, including calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Alternatively, chemical methods may be employed, such as the introduction of nucleic acids or vectors by colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In addition, nucleic acids or vectors can also be introduced using biological methods. For example, viral vectors, particularly retroviral vectors, have become the most common method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like.

After introducing the nucleic acid or vector into the immune cells, the resulting immune cells can be expanded and activated by one skilled in the art by conventional techniques.

Therapeutic applications

The invention also provides a method of treating a subject having cancer, comprising administering to the subject an effective amount of a multi-chain chimeric antigen receptor, immune cell, or pharmaceutical composition of the invention.

In one embodiment, an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.

In another embodiment, the treatment method of the invention is ex vivo treatment. Specifically, the method comprises the following steps: (a) providing a sample of a subject, said sample comprising immune cells; (b) introducing in vitro a multi-chain chimeric receptor of the invention or a nucleic acid or a vector system encoding it into said immune cells, obtaining modified immune cells, (c) administering said modified immune cells to a subject in need thereof. Preferably, the immune cells provided in step (a) are selected from T cells, NK cells and/or NKT cells; and the immune cells can be obtained from a sample (particularly a blood sample) of a subject by conventional methods known in the art. However, other immune cells capable of expressing the chimeric receptor polypeptides and Fc fusion polypeptides of the invention and performing the desired biological effector functions as described herein may also be used. Furthermore, the immune cells are typically selected to be compatible with the immune system of the subject, i.e. preferably the immune cells do not elicit an immunogenic response. For example, "universal recipient cells," i.e., universally compatible lymphocytes that can be grown and expanded in vitro to function as desired biological effects, can be used. The use of such cells would not require the obtaining and/or provision of subject autologous lymphocytes. The ex vivo introduction of step (c) may be carried out by introducing the nucleic acid or vector described herein into an immune cell via electroporation or by infecting an immune cell with a viral vector, which is a lentiviral, adenoviral, adeno-associated viral vector or retroviral vector as described previously. Other conceivable methods include the use of transfection reagents (such as liposomes) or transient RNA transfection.

In one embodiment, two or more peptide chains comprised by the multi-chain chimeric antigen receptor of the present invention may be administered together or separately. For example, an immune cell or pharmaceutical composition comprising the first Fc fusion polypeptide, and an immune cell or pharmaceutical composition comprising the chimeric receptor polypeptide, respectively, can be administered to a subject to activate a signaling pathway, as appropriate, to activate a killing function of the immune cell, as desired. In another embodiment, the treatment may further comprise further administering to the subject an immune cell or pharmaceutical composition comprising a second Fc fusion polypeptide comprising a second antigen-binding region, a third protein-interacting domain, and a second Fc region.

In one embodiment, when the protein-interacting domain is a chemically-induced dimerization domain, the method of treatment further comprises administering to the subject a chemical that can induce the protein-interacting domains to bind to each other. For example, when the protein-interacting domain is GIA and GID, respectively, the method of treatment further comprises administering gibberellin; when the protein interaction domain is a Snap tag and a Halo tag, respectively, the method of treatment further comprises administering HaXS; when the protein interaction domain is FRB and FKBP, respectively, the method of treatment further comprises administering rapamycin and derivatives thereof, such as photosensitive caged rapamycin; when the protein interaction domain is PYL and ABI, respectively, the method of treatment further comprises administering abscisic acid; when the protein interaction domain is FKBP and CyP, respectively, the method of treatment further comprises administering FKCsA; when the protein interaction domain is FKBP and CnA, respectively, the method of treatment further comprises administering FK 506; when the protein interaction domain is GR and DHFR, respectively, the method of treatment further comprises administering Dex-Mtx.

In one embodiment, the immune cell is an autologous or allogeneic cell, preferably a T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, more preferably a T cell, NK cell or NKT cell.

As used herein, the term "autologous" means that any material derived from an individual will be reintroduced into the same individual at a later time.

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. When the genes at one or more loci are different, two or more individuals are considered allogeneic to each other. In some cases, genetic differences in allogenic material from individuals of the same species may be sufficient for antigen interactions to occur.

As used herein, the term "subject" is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

In one embodiment, the disease is a cancer associated with expression of a target to which an antigen binding region binds. For example, the cancer includes, but is not limited to: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and CNS cancers, 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, hepatoma, intraepithelial tumors, kidney cancer, laryngeal cancer, leukemia, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenoid lung cancer, 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, cancer of the respiratory system, Salivary gland carcinoma, skin carcinoma, squamous cell carcinoma, gastric carcinoma, testicular carcinoma, thyroid carcinoma, uterine or endometrial carcinoma, malignant neoplasms of the urinary system, vulval carcinoma and other carcinomas and sarcomas, and B-cell lymphomas including low-grade/follicular non-Hodgkin's 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-cracked cellular NHL, large lump 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 prolymphocytic leukemia, T-cell lymphoma, T-cell lymphocytic leukemia, A blast-like plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmacytoma, pre-leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the disease that can be treated with the multi-chain chimeric antigen receptor, nucleic acid, vector, immune cell or pharmaceutical composition of the present invention is selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.

In one embodiment, the method further comprises administering to the subject one or more additional chemotherapeutic agents, biologies, drugs or treatments. In this embodiment, the chemotherapeutic agent, biological agent, drug or treatment is selected from the group consisting of radiation therapy, surgery, antibody agents and/or small molecules and any combination thereof.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: schematic design of a preferred embodiment of the present invention.

FIG. 2: levels of scFv expression are shown for Fite-CAR-1s and Fite-CAR-2s T cells.

FIG. 3: the killing effect of Fite-CAR-1s and Fite-CAR-2s T cells on target cells is shown.

FIG. 4: levels of secretion of scFv-Fc fusion polypeptides in Fite-CAR-1s and Fite-CAR-2s T cells are shown. Analysis was performed using Two-way ANOVA and statistical analysis was performed using T test. Indicates a P value of less than 0.05, indicates a P value of less than 0.01, all reached significant levels.

FIG. 5: the expression of the AZip and Fc fusion polypeptides in Fite-CAR-2s T cells is shown.

FIG. 6: levels of sdAb-Fc secretion in fix-CARX-1 s and fix-CARX-2 s T cells (a) and killing effect on target cells (B) are shown. Analysis was performed using Two-way ANOVA and statistical analysis was performed using T test. Indicates a P value of less than 0.05, indicates a P value of less than 0.01, all reached significant levels.

FIG. 7: the IFN- γ release levels of Fite-CARX-1s and Fite-CARX-2s T cells are shown.

FIG. 8: NK cell killing effects of Fite-CARX-1s and Fite-CARX-2s T cells are shown. Analysis was performed using Two-way ANOVA and statistical analysis was performed using T test. Indicates a P value of less than 0.05, indicates a P value of less than 0.01, all reached significant levels.

Detailed Description

The sequence summary used in the following examples is shown in table 1 below.

TABLE 1 sequences used in the present invention

SEQ ID NO	Description of the invention
		SEQ ID NO：1	Nucleotide sequence of Claudin18.2-scFv
SEQ ID NO：2	Amino acid sequence of Claudin18.2-scFv
		SEQ ID NO：3	Nucleotide sequence of AZip
SEQ ID NO：4	Nucleotide sequence of AZip
		SEQ ID NO：5	Nucleotide sequence of BZip
SEQ ID NO：6	Nucleotide sequence of BZip
		SEQ ID NO：7	Nucleotide sequence of Claudin18.2 sdAb
SEQ ID NO：8	Amino acid sequence of Claudin18.2 sdAb
		SEQ ID NO：9	Nucleotide sequence of Fc region
SEQ ID NO：10	Amino acid sequence of Fc region
		SEQ ID NO：11	Nucleotide sequence of transmembrane domain CD8 alpha
SEQ ID NO：12	Amino acid sequence of transmembrane domain CD8 alpha
		SEQ ID NO：13	Nucleotide sequence of co-activation domain 4-1BB
SEQ ID NO：14	Amino acid sequence of the coactivation domain 4-1BB
		SEQ ID NO：15	Nucleotide sequence of signaling domain CD3 zeta
SEQ ID NO：16	Amino acid sequence of signaling domain CD3 ζ
		SEQ ID NO：17	Nucleotide sequence of linker peptide IgG
SEQ ID NO：18	Amino acid sequence of linker peptide IgG
		SEQ ID NO：19	Nucleotide sequence of signal peptide CD8 alpha
SEQ ID NO：20	Amino acid sequence of signal peptide CD8 alpha
		SEQ ID NO：21	Nucleotide sequence of signal peptide GM-CSFR alpha
SEQ ID NO：22	Amino acid sequence of signal peptide GM-CSFR alpha
		SEQ ID NO：23	Nucleotide sequence of F2A peptide
SEQ ID NO：24	Amino acid sequence of the F2A peptide
		SEQ ID NO：25	Nucleotide sequence of CD8 alpha hinge region
SEQ ID NO：26	Amino acid sequence of CD8 alpha hinge region
		SEQ ID NO：27	Nucleotide sequence of IgG4 hinge region
SEQ ID NO：28	Amino acid sequence of IgG4 hinge region

The T cells used in all examples of the invention were primary human CD4+ CD8+ T cells isolated from healthy donors by leukapheresis using Ficoll-Paque (TM) PREMIUM (GE Healthcare, cat. No. 17-5442-02).

Example 1: construction of classical CAR T cells

The following coding sequences were synthesized and cloned sequentially into pGEM-T Easy vector (Promega, cat # A1360): CD8 alpha signal peptide (SEQ ID NO: 19), anti-Claudin18.2 scFv (SEQ ID NO: 1), CD8 alpha hinge region (SEQ ID NO: 25), CD8 alpha transmembrane region (SEQ ID NO: 11), 4-1BB costimulatory domain (SEQ ID NO: 13), CD3 zeta intracellular signaling domain (SEQ ID NO: 15), CAR plasmid was obtained and correct insertion of the target sequence was confirmed by sequencing.

After diluting the above plasmid by adding 3ml of Opti-MEM (Gibco, cat # 31985-: viral envelope vector =4:2:1 packaging vector psPAX2 (addge, cat # 12260) and envelope vector pmd2.g (addge, cat # 12259) were added. Then, 120ul of X-treme GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately, incubated at room temperature for 15min, and the plasmid/vector/transfection reagent mixture was added dropwise to the 293T cell culture flask. The viruses were collected at 24 hours and 48 hours, and after combining them, concentrated lentiviruses were obtained by ultracentrifugation (25000 g, 4 ℃, 2.5 hours).

T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, cat. No. 40203D) and cultured at 37 ℃ and 5% CO2 for 1 day. Then, concentrated lentivirus was added and after continued culturing for 3 days, CAR T (i.e., con-CAR T) cells targeted to claudin18.2 were obtained.

Example 2: construction of Fite-CAR T cells

Construction of the Fite-CAR plasmid: the coding sequence for the CD8 α signal peptide (SEQ ID NO: 19), Claudin18.2-scFv (SEQ ID NO: 1), BZip (SEQ ID NO: 5), IgG linker peptide (SEQ ID NO: 17), Fc region (SEQ ID NO: 9), F2A peptide (SEQ ID NO: 23), GM-CSFR α signal peptide (SEQ ID NO: 21), AZip (SEQ ID NO: 3), CD8 α hinge region (SEQ ID NO: 25), CD8 α transmembrane region (SEQ ID NO: 11), 4-1BB costimulatory domain (SEQ ID NO: 13), CD3 intracellular zeta signaling domain (SEQ ID NO: 15) was cloned into pGEM-T Easy vector (Promega, cat # A1360), a Fite-CAR-1s plasmid was obtained and correct insertion of the target sequence was confirmed by sequencing. The same procedure was used to obtain the Fite-CAR-2s plasmid, which contained the same elements as Fite-CAR-1s, the only difference being that the hinge region was derived from IgG4 (SEQ ID NO: 27).

After diluting the above plasmid by adding 3ml of Opti-MEM (Gibco, cat # 31985-: viral envelope vector =4:2:1 packaging vector psPAX2 (addge, cat # 12260) and envelope vector pmd2.g (addge, cat # 12259) were added. Then, 120ul of X-treme GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately, incubated at room temperature for 15min, and the plasmid/vector/transfection reagent mixture was added dropwise to the 293T cell culture flask. The virus was collected at 24 and 48 hours and after pooling, ultracentrifugation (25000 g, 4 ℃, 2.5 hours) yielded concentrated Fite-CAR lentivirus.

T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, cat. No. 40203D) and cultured at 37 ℃ and 5% CO2 for 1 day. Then, after adding concentrated filt-CAR lentivirus and continuing the culture for 3 days, filt-CAR T cells were obtained.

At 37 deg.CAnd 5% CO2 for 11 days, Biotin-SP (Long spacer) Affinipure Goat Anti-Mouse IgG, F (ab')₂As a primary antibody, Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) and APC Streptavidin (BD Pharmingen, cat # 554067) or PE Streptavidin (BD Pharmingen, cat # 554061) were used, and the expression level of scFv on Fite-CAR T cells was examined by flow cytometry, as shown in FIG. 2 (NT is an unmodified wild-type T cell).

It can be seen that both of the filt-CAR T cells of the invention are efficient in expressing scFv, indicating that the chimeric receptor polypeptide is capable of specific binding to the Fc fusion polypeptide and at a level comparable to con-CAR.

Example 3: functional validation and optimization of Fite-CAR T cells

3.1 detection of the killing Effect on target cells

When T cells kill target cells, the number of target cells is reduced. When T cells are co-cultured with target cells expressing luciferase, the number of target cells is reduced and the amount of secreted luciferase is reduced. Luciferase catalyses the conversion of luciferin to oxidative luciferin, and in this oxidation process, bioluminescence is produced, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cell. Thus, the detected fluorescence intensity can reflect the killing ability of T cells to target cells.

The 293T-Claudin18.2 target cells used in this example were Claudin18.2 positive monoclonal cells selected by flow cytometry after infection of 293T cells with a lentivirus expressing Claudin18.2.

To examine the killing ability of the Fite-CAR T cells on target cells, first 1X10⁴A293T-Claudin18.2 target cell carrying a fluorescein gene is plated in a 96-well plate, then a Fite-CAR T cell, a Con-CAR T cell (positive control) and an untransfected T cell (negative control) are plated in the 96-well plate at an effective target ratio (namely, the ratio of the effector T cell to the target cell) of 32:1 for co-culture, and a fluorescence value is measured by a microplate reader after 16-18 hours. According to calculationThe formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and the killing efficiency was calculated, and the results are shown in FIG. 3.

It can be seen that both of the filt-CAR T cells of the invention are able to kill target cells efficiently compared to NT, and their killing effect is comparable to Con-CAR T cells.

3.2 detection of secretion level of scFv-Fc

If the filt-CAR T cells are able to secrete the scFv-Fc region efficiently, they are able to be recognized by Fc receptor (FcR) -expressing immune effector cells, including NK cells, macrophages, dendritic cells, etc., to recruit these immune effector cells and further enhance the killing effect on target cells. Thus, the inventors used enzyme-linked immunosorbent assay (ELISA) to detect scFv-Fc secretion levels of Fite-CAR T cells.

Fite-CAR-1s T cells, Fite-CAR-2s T cells, Con-CAR T and NT cells were cultured in x-vivo 15 medium (Lonza, cat # 04-418Q) without IL-2 at 37 ℃ and 5% CO2, respectively. After 24 hours, the culture was collected and centrifuged at 1600rpm for 5 minutes at 4 ℃ to obtain a cell culture supernatant.

The 96-well plate was coated with the capture antibody Recombinant Human Claudin-18.2 (N-8His) (Novoprotein, Cat. CR 53) and incubated overnight at 4 deg.C, then the supernatant was removed and 250. mu.L of PBST (1 XPBS with 0.1% Tween) solution containing 2% BSA (sigma, Cat. V900933-1 kg) was added and incubated for 2 hours at 37 deg.C. After removing the supernatant, 250. mu.L of PBST (1 XPBS with 0.1% Tween) was added and washed 3 times. Then 50. mu.L of cell culture supernatant was added to each well and incubated at 37 ℃ for 1 hour. The supernatant was removed and then 250 μ L PBST (1 x PBS with 0.1% tween) was added and washed 3 times. Then 50. mu.L of the detection antibody HRP Goat anti-mouse IgG (Biolegend, cat # 405306) was added to each well and incubated at 37 ℃ for 30 minutes. Discard the supernatant, add 250 μ L PBST (1 x PBS with 0.1% tween) and wash 5 times.

To each well 50 μ L of TMB substrate solution was added. The reaction was allowed to occur at room temperature in the dark for 30 minutes, after which 50. mu.L of 1mol/L H was added to each well₂SO₄To stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450nm was measured using a microplate reader, and the relative expression level of the scFv-Fc fusion polypeptide in the supernatant was calculated by the ratio to the reading of the NT cell culture supernatant, and the results are shown in FIG. 4.

Unexpectedly, no significant expression of scFv-Fc was detected in the supernatant of both Fite-CAR-1s T cells and Fite-CAR-2s T cells compared to Con-CAR T and NT cells. This is probably due to the fact that the scFv structures comprised by the Fc fusion polypeptide are cohesive to each other and thus are immobilized to the cell membrane by specific binding between the protein interaction domains, ultimately affecting the normal secretion of scFv-Fc.

To test the above hypothesis, chimeric receptor expression on Fite-CAR T cells (i.e., to detect AZip) was detected by flow cytometry using a combination of antibodies anti-c-Fos (BOSTER, cat # PA 1318), Biotin-goat anti-rabbit IgG (BOSTER, cat # BA 1003), APC Streptavidin (BD Pharmingen, cat # 554067), and antibody PE anti-Human IgG Fc (Biolegend, cat # 409304) to detect Fc fusion polypeptide expression on Fite-CAR-2s T cells, with the results shown in FIG. 5.

It can be seen that the Fc positive cells in the filte-CAR-2 s T cell population were also essentially simultaneously azep negative (35.1%) compared to the results for NT and con-CAR, indicating that both detected Fc fusion polypeptides bind to the chimeric receptor polypeptide via the interaction between BZip and azep, resulting in the occupation of the binding site of azep and thus no binding to the detection antibody. Furthermore, little azep-positive and Fc-negative (0.31%) cells were present in the Fite-CAR-2s T cell population, also indicating that there was essentially no unbound chimeric receptor polypeptide alone in the Fite-CAR-2s T cell population.

The above results demonstrate that the inability of scFv-Fc fusion polypeptides to secrete results from specific binding between protein interaction domains and from the adhesion between scFv structures. Since Fite-CAR-1s T cells and Fite-CAR-2s T cells do not secrete scFv-Fc efficiently, no other immune cells could be recruited to enhance the killing effect of CAR T cells on target cells.

Example 4: construction of Fite-CARX T cells

Due to the unique VHH structure of the single domain antibody (sdAb), containing only the heavy chain region and no light chain region, it is expected to solve the problem found in example 3 that scFv-Fc cannot be secreted due to the potential mutual adhesion of VL and VH domains. Therefore, the inventors replaced scFv with single domain antibody (sdAb) to obtain Fite-CARX T cells.

The Fite-CARX-1s T cells and Fite-CARX-2s T cells used in this example contained the same elements and their order of attachment as Fite-CAR-1s T cells and Fite-CAR-2s T cells, respectively, with the only difference that Claudin18.2 scFv (SEQ ID NO: 1) was replaced with Claudin18.2 sdAb (SEQ ID NO: 7).

Example 5: functional validation of Fite-CARX T cells

The level of secretion of the sdAb-Fc fusion polypeptide in the fix-carm T cells was measured according to the method described in example 3.2 and the results are shown in figure 6A.

It can be seen that significantly secreted sdAb-Fc fusion polypeptides can be detected in both the gate-card T supernatants compared to Con-CAR T cells and NT cells, indicating that the single domain antibody structure can effectively avoid mutual attachment of scFv, thereby promoting secretion of Fc fusion polypeptides.

In addition, the killing effect of Fite-CARX T cells on 293T-Claudin18.2 target cells was tested according to the method described in example 3.1, and the results are shown in FIG. 6B.

It can be seen that both fix-CARX T cells were able to kill target cells efficiently compared to NT, and the killing effect was comparable to Con-CAR T cells.

Example 6: cytokine release from Fite-CARX T cells

When the T cells kill the target cells, the target cells decrease in number and release cytokines IL2, IFN-. gamma.and the like. The level of release of the cytokine IFN γ when the ite-carm T cells killed the target cells was determined using enzyme-linked immunosorbent assay (ELISA) according to the following procedure.

(1) Collecting cell co-culture supernatant

At 1x10⁵Per well target cells 293T-Claudin18.2 and non-target cells 293T were plated in 96-well plates, respectively, thenThen, Fite-CARX T, Con-CAR T (positive control) and NT cells (negative control) were co-cultured with target cells and non-target cells, respectively, at a ratio of 1:1, and cell co-culture supernatants were collected 18-24 hours later.

(2) ELISA detection of IFN gamma secretion in supernatant

The 96-well plate was coated with capture Antibody Purified anti-human IFN-. gamma.antibody (Biolegend, cat. 506502) and incubated overnight at 4 ℃ followed by removal of the Antibody solution and addition of 250. mu.L of PBST (1 XPBS with 0.1% Tween) solution containing 2% BSA (sigma, cat. V900933-1 kg) and incubation for 2 hours at 37 ℃. The plates were then washed 3 times with 250 μ L of PBST (1 XPBS with 0.1% Tween). mu.L of cell co-culture supernatant or standard was added to each well and incubated at 37 ℃ for 1 hour, after which the plates were washed 3 times with 250. mu.L of PBST (1 XPBS with 0.1% Tween). Then 50. mu.L of an Anti-Interferon gamma antibody [ MD-1 ] was added to each well](Biotin) (abcam, cat # ab25017), after 1 hour incubation at 37 ℃ the plates were washed 3 times with 250. mu.L PBST (1 XPBS with 0.1% Tween). HRP Streptavidin (Biolegend, cat # 405210) was added, and after incubation at 37 ℃ for 30 minutes, the supernatant was discarded, 250. mu.L of PBST (1 XPBS containing 0.1% Tween) was added, and washed 5 times. To each well 50 μ L of TMB substrate solution was added. The reaction was allowed to occur at room temperature in the dark for 30 minutes, after which 50. mu.L of 1mol/L H was added to each well₂SO₄To stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450nm was measured using a microplate reader, and the content of cytokine was calculated from a standard curve (plotted according to the reading and concentration of the standard), and the result is shown in FIG. 7.

It can be seen that no release of IFN γ was detected in non-target cells 293T, indicating that killing of both con-CAR T cells and ite-CARX T cells is specific. Also, upon killing of target cells, both filt-CARX T cells released the cytokine IFN- γ at levels comparable to Con-CAR T cells.

Example 7: killing effect of Fite-CARX T cell mediated NK cell on target cell

Since the filt-CARX T cells were able to kill target cells with high efficiency and secreted sdAb-Fc fusion polypeptides significantly, the inventors further tested whether they could mediate NK cells for tumor killing.

The NK cells used in this example were obtained by the following method: after the mouse spleen was ground, a mouse spleen lymphocyte isolate (TBD, cat. LTS1092 PK-200) was added thereto, and the mixture was centrifuged to obtain leucocyte layer cells. Then, PE Anti-mouse NK1.1 (Biolegend, cat # 108701) and Anti-PE Microbeads (Meitian whirlwind, cat # 130-.

The target cells of NUGC4-Claudin18.2 used in this example were Claudin18.2 positive monoclonal cells selected by flow cytometry after infection of NUGC4 cells with a lentivirus expressing Claudin18.2.

At 1x10⁴NuGC4-Claudin18.2 target cells carrying the fluorescein gene were plated in 96-well plates per well. Then, NK cells were resuspended using the supernatant of Fite-CARX T cells and fresh medium (media), respectively, and the resuspended NK cells were added to a 96-well plate at an effective target ratio of 4:1 (i.e., ratio of effective NK cells to target cells) for co-culture, and fluorescence was measured by a microplate reader after 16-18 hours. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and the killing efficiency was calculated, and the results are shown in fig. 8.

As can be seen, compared with a fresh culture medium, the two kinds of supernatant of the Fite-CARX T cells can effectively mediate the killing of NK cells to NUGC4-Claudin18.2 target cells, and the effect of the supernatant is obviously higher than that of a fresh culture medium control group.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

<110> Nanjing Beijing Heng Biotechnology Ltd

<120> multi-chain chimeric antigen receptor and use thereof

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

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caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcacc agctactgga taaactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggaaat atttatcctt ctgatagtta tactaactac 180

aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtac aagatcgtgg 300

aggggtaact cctttgacta ctggggccaa ggcaccactc tcacagtctc ctcaggtgga 360

ggcggttcag gcggaggtgg ctctggcggt ggcggatcgg acattgtgat gacacagtct 420

ccatcctccc tgactgtgac agcaggagag aaggtcacta tgagctgcaa gtccagtcag 480

agtctgttaa acagtggaaa tcaaaagaac tacttgacct ggtaccagca gaaaccaggg 540

cagcctccta aactgttgat ctactgggca tccactaggg aatctggggt ccctgatcgc 600

ttcacaggca gtggatctgg aacagatttc actctcacca tcagcagtgt gcaggctgaa 660

gacctggcag tttattactg tcagaatgat tatagttatc cattcacgtt cggctcgggg 720

acaaagttgg aaataaaa 738

<210> 2

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu

130 135 140

Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln

145 150 155 160

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

180 185 190

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val

210 215 220

Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys

245

<210> 3

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ggctccgagc tgacggacac tttgcaggcc gagaccgatc aacttgagga cgagaagagt 60

gcattgcaaa ccgaaatagc gaacttgctg aaggagaaag aaaagctcga atttatattg 120

gctgctcacc gc 132

<210> 4

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Gly Ser Glu Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu

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Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu

20 25 30

Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His Arg

35 40

<210> 5

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gaaagaataa gtcgcctcga agaaaaggtg aaaacgctga aaagccaaaa cacagagttg 60

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Glu Arg Ile Ser Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ser Gln

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Asn Thr Glu Leu Ala Ser Thr Ala Ser Leu Leu Arg Glu Gln Val Ala

20 25 30

Gln Leu Lys Gln Lys Val Leu Ser His Val

35 40

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caggtccaac ttgttgaatc aggcggtggc cttgtccagc cggggggttc cctcaggttg 60

agttgcgccg cttccggttc aattttctca ataaacgcta tgggttggta taggcaggcg 120

ccggggaagg gactggagct cgtagcagca attaccttcg gcggagggtc tacaaattat 180

gccgactcag tgaagggaag atttacgatt agcagagaca attcaaagaa tactctctac 240

ttgcaaatga atagccttag agccgaggac actgctgttt actattgtaa tgctgacttg 300

ctggttggtg gctttcctcg acgaaacgta tattggggcc agggcaccct cgtaacggtc 360

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<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ala Ile Thr Phe Gly Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Asp Leu Leu Val Gly Gly Phe Pro Arg Arg Asn Val Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 639

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 60

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 120

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 180

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 240

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 300

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 360

cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 420

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 480

cctcccgtgc tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag 540

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 600

cactacacgc agaagagcct ctccctgtct ccgggtaaa 639

<210> 10

<211> 232

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Glu Pro Lys Ser Gln Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 11

<211> 75

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gcaaa 75

<210> 12

<211> 25

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys Lys

20 25

<210> 13

<211> 120

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 60

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 120

<210> 14

<211> 40

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

1 5 10 15

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

20 25 30

Glu Glu Glu Glu Gly Gly Cys Glu

35 40

<210> 15

<211> 339

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300

acctacgacg cccttcacat gcaggccctg ccccctcgc 339

<210> 16

<211> 113

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 16

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 17

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

gagcccaaat ctcaggacaa aactcacaca tgcccaccgt gcccagcacc tgaactc 57

<210> 18

<211> 19

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Glu Pro Lys Ser Gln Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu

<210> 19

<211> 63

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 20

<211> 21

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 20

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 21

<211> 66

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

atgctgctgc tcgtgacaag cctgctgctg tgcgagctgc cccaccctgc ctttctgctg 60

atcccc 66

<210> 22

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 23

<211> 66

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

gtgaaacaga ctttgaattt tgaccttctc aagttggcgg gagacgtgga gtccaaccca 60

gggccg 66

<210> 24

<211> 22

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 25

<211> 135

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 26

<211> 45

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 27

<211> 39

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

gaatcaaaat atggtcctcc ttgcccgcca tgtccggat 39

<210> 28

<211> 13

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 28

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Asp

1 5 10

Claims

1. A multi-chain chimeric antigen receptor comprising:

(a) an Fc fusion polypeptide comprising, from N-terminus to C-terminus, a first antigen binding region, a first protein interaction domain, and an Fc region; and

(b) a chimeric receptor polypeptide comprising, from N-terminus to C-terminus, a second protein interaction domain, a transmembrane domain, and an intracellular signaling domain,

wherein the first protein interaction domain is capable of specifically binding to a second protein interaction domain, wherein the first protein interaction domain and second protein interaction domain are in a combination selected from the group consisting of: FKBP 506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB) of mTOR, FKBP and calcineurin a (cna), FKBP and cyclophilin (CyP), GAI and GID, Snap and Halo tags, Glucocorticoid Receptor (GR) and DHFR, PYL and ABI, dimerization docking domain of cAMP-dependent protein kinase a (pka) and anchoring domain of a-kinase anchoring protein (AKAP), avidin and biotin, leucine zipper domain and zinc finger domain and nucleotide tag;

wherein the first antigen binding region is a Claudin18.2-targeting single domain antibody comprising CDR sequences identical to those comprised by the antibody set forth in SEQ ID NO. 8.

2. The multi-chain chimeric antigen receptor of claim 1, wherein the first antigen-binding region is selected from a human antibody, a humanized antibody, and a murine antibody.

3. The multi-chain chimeric antigen receptor of claim 1, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154.

4. The multi-chain chimeric antigen receptor of claim 1, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66 d.

5. The multi-chain chimeric antigen receptor of claim 1, wherein the chimeric receptor polypeptide further comprises one or more co-stimulatory domains.

6. The multi-chain chimeric antigen receptor of claim 5, wherein the co-stimulatory domain is a co-stimulatory signaling domain of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD223(LAG3), CD270 (HVEM), CD272 (BTLA), CD273(PD-L2), CD274(PD-L1), CD276 (B7-H3), CD278(ICOS), CD357 (GITR), DAP10, LAT, NKG2C, NKG 76, LIGHT, slpm, and ZAP 70.

7. The multi-chain chimeric antigen receptor of any one of claims 1-6, wherein the Fc region comprises a CH2 domain and a CH3 domain.

8. The multi-chain chimeric antigen receptor of claim 1, wherein the first and second protein-interacting domains are AZip and BZip, respectively.

9. The multi-chain chimeric antigen receptor of claim 1, wherein the first and second protein interaction domains are a zinc finger domain and a nucleotide tag, respectively.

10. A nucleic acid comprising a first nucleic acid sequence encoding a chimeric receptor polypeptide and a second nucleic acid sequence encoding an Fc fusion polypeptide, said chimeric receptor polypeptide and said Fc fusion polypeptide comprising a multi-chain chimeric antigen receptor according to any one of claims 1-9.

11. A vector comprising the nucleic acid of claim 10.

12. A vector system comprising a first nucleic acid sequence encoding a chimeric receptor polypeptide and a second nucleic acid sequence encoding an Fc fusion polypeptide, said chimeric receptor polypeptide and said Fc fusion polypeptide comprising the multi-chain chimeric antigen receptor of any one of claims 1-9, said first and second nucleic acid sequences being on different vectors.

13. An immune cell comprising the multi-chain chimeric antigen receptor of any one of claims 1-9, the nucleic acid of claim 10, the vector of claim 11, or the vector system of claim 12.

14. The immune cell of claim 13, wherein the vector is a linear nucleic acid molecule, a plasmid, a retrovirus, a lentivirus, an adenovirus, a vaccinia virus, a Rous Sarcoma Virus (RSV), a polyoma virus, and an adeno-associated virus (AAV), a bacteriophage, a cosmid, or an artificial chromosome.

15. The immune cell of any one of claims 13-14, which is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

16. The immune cell of claim 15, wherein the immune cell is a T cell selected from the group consisting of: CD4+/CD8+ double positive T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, gamma delta-T cells and alpha beta-T cells.

17. A pharmaceutical composition comprising a multi-chain chimeric antigen receptor according to any one of claims 1-9, a nucleic acid according to claim 10, a vector according to claim 11, a vector system according to claim 12, or an immune cell according to any one of claims 13-16, and one or more pharmaceutically acceptable excipients.

18. Use of a multi-chain chimeric antigen receptor according to any one of claims 1 to 9, a nucleic acid according to claim 10, a vector according to claim 11, a vector system according to claim 12 or an immune cell according to any one of claims 13 to 16 or a pharmaceutical composition according to claim 17 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is selected from: sarcoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, gastric cancer, liver cancer, kidney cancer, larynx cancer, lung cancer, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, thyroid cancer.

19. The use of claim 18, wherein the brain and CNS cancer is Glioblastoma (GBM).