Immune effector cells co-expressing chemokine receptors

Abstract

Provided are an immune effector cell co-expressing a chemokine receptor, a pharmaceutical composition, a drug kit, and a method for treating a tumor, wherein the immune effector cell comprises a receptor that specifically recognizes claudin18.2 and a protein that recognizes SDF-1. Also provided are an expression construct, an expression vector, and a virus, wherein the expression construct comprises sequentially linked expressions of a receptor that binds to a tumor-associated antigen, and an expression of a protein that recognizes SDF-1.

Description

[0001] This case claims the priority of CN2019107353670, priority date: 2019 / 08 / 09. Technical Field

[0002] The present invention belongs to the field of cellular immunotherapy. More specifically, the present invention relates to immune effector cells that co-express chemokine receptors and receptors that bind to tumor-associated antigens. Background Art

[0003] Tumors, especially solid tumors, are complexes composed of tumor cells and their surrounding matrix cells and non-cellular components. The occurrence and development of tumors is a dynamic process of mutual promotion and co-evolution between tumor cells and their microenvironment. The tumor microenvironment plays a vital role in the growth and metastasis of tumors. Cancer associated fibroblasts (CAFs), as one of the most important components in the tumor microenvironment, mainly promote tumor development by secreting various extracellular matrix components (such as collagen, proteoglycans, proteases, growth factors, cytokines and chemokines).

[0004] In the field of immune cell therapy, the homing of immune effector cells to the tumor site is an important prerequisite for immune cells to play a tumor-killing role, and it is also a difficult problem in this field. For example, pancreatic cancer is one of the most deadly malignant tumors. Because pancreatic tumor cells can activate tumor-specific immune responses, they can also stimulate immunosuppressive responses at a higher intensity and establish an immunosuppressive microenvironment to escape immune surveillance, making traditional radiotherapy and chemotherapy regimens ineffective in the treatment of pancreatic cancer. Even the cell immunotherapy, which is currently developing well, has encountered great challenges in the treatment of pancreatic cancer due to the limited homing ability of therapeutic cells.

[0005] Therefore, further research is needed in this field to find ways to enable immune cells to better home to tumor sites and exert their greater tumor-killing effects, thereby improving the efficacy of immune effector cells in tumor immunotherapy. Summary of the invention

[0006] The first aspect of the present application relates to an immune effector cell co-expressing a chemokine receptor, characterized in that the cell comprises: a receptor that specifically recognizes claudin18.2; and a protein that recognizes SDF-1.

[0007] Specifically, the protein that recognizes SDF-1 is an antibody that recognizes SDF-1 or a receptor for SDF-1.

[0008] Specifically, the receptor of SDF-1 is CXCR4 or CXCR7.

[0009] Specifically, the amino acid sequence of the scFV that specifically recognizes the receptor of claudin18.2 has at least 90% identity with the amino acid sequence shown in SEQ ID NO:2.

[0010] Specifically, the immune effector cells are selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.

[0011] Specifically, the receptor of SDF-1 is CXCR4, and preferably, the amino acid sequence of CXCR4 has at least 90% identity with the sequence shown in SEQ ID NO: 19. Preferably, the amino acid sequence of CXCR4 is the sequence shown in SEQ ID NO: 19.

[0012] Specifically, the receptor that specifically recognizes claudin18.2 is a chimeric receptor; preferably, the chimeric receptor is selected from: a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0013] Specifically, the chimeric receptor is a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane region and an intracellular signal region connected in sequence; wherein the extracellular domain comprises an antibody or a ligand; preferably, the antibody is a single-chain antibody or a domain antibody. The transmembrane region comprises a transmembrane region sequence of CD8 or CD28. The intracellular signal region is selected from: intracellular signal region sequences of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, or a combination thereof.

[0014] Preferably, the extracellular domain of the chimeric antigen receptor is scFv, and more preferably, the scFv contains HDCR1, HCDR2, HCDR3 shown in SEQ ID NOs: 24, 25, 26, and LDCR1, LCDR2, LCDR3 shown in SEQ ID NOs: 27, 28, 29. More preferably, the extracellular domain of the chimeric antigen receptor contains the amino acid sequence shown in SEQ ID NO: 2.

[0015] In a specific embodiment, the chimeric antigen receptor comprises any one of the following (i), (ii) and (iii):

[0016] (i) the above-mentioned scFv, CD8 and CD3ζ;

[0017] (ii) the above-mentioned scFv, CD8, CD137 and CD3ζ; or

[0018] (iii) the above-mentioned scFv, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule and CD3ζ.

[0019] In a specific embodiment, the cell contains a nucleic acid having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 18. Preferably, the cell contains the nucleotide sequence shown in SEQ ID NO:18.

[0020] In a specific embodiment, the chimeric antigen receptor has at least 90% identity with the amino acid sequence shown in SEQ ID NO:21, 22 or 23.

[0021] The second aspect of the present application relates to an immune effector cell co-expressing a chemokine receptor, characterized in that the cell comprises: a receptor that specifically recognizes a pancreatic cancer antigen; and a protein that recognizes SDF-1.

[0022] Specifically, the protein that recognizes SDF-1 is an antibody that recognizes SDF-1, or a receptor for SDF-1;

[0023] Specifically, the receptor of SDF-1 is CXCR4 or CXCR7.

[0024] Specifically, the amino acid sequence of the scFV that specifically recognizes the receptor of claudin18.2 has at least 90% identity with the amino acid sequence shown in SEQ ID NO:2.

[0025] Specifically, the immune effector cells are selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.

[0026] Specifically, the receptor of SDF-1 is CXCR4, and preferably, the amino acid sequence of CXCR4 has at least 90% identity with the sequence shown in SEQ ID NO: 19. Preferably, the amino acid sequence of CXCR4 is the sequence shown in SEQ ID NO: 19.

[0027] Specifically, the receptor that specifically recognizes pancreatic cancer antigens is a chimeric receptor; preferably, the chimeric receptor is selected from: a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0028] Specifically, the chimeric receptor is a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane region and an intracellular signal region connected in sequence; wherein the extracellular domain comprises an antibody or a ligand; preferably, the antibody is a single-chain antibody or a domain antibody. The transmembrane region comprises a transmembrane region sequence of CD8 or CD28. The intracellular signal region is selected from: intracellular signal region sequences of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, or a combination thereof.

[0029] Preferably, the extracellular domain of the chimeric antigen receptor is scFv, and more preferably, the scFv contains HDCR1, HCDR2, HCDR3 shown in SEQ ID NOs: 24, 25, 26, and LDCR1, LCDR2, LCDR3 shown in SEQ ID NOs: 27, 28, 29. More preferably, the extracellular domain of the chimeric antigen receptor contains the amino acid sequence shown in SEQ ID NO: 2.

[0030] In a specific embodiment, the chimeric antigen receptor comprises any one of the following (i), (ii) and (iii):

[0031] (i) the above-mentioned scFv, CD8 and CD3ζ;

[0032] (ii) the above-mentioned scFv, CD8, CD137 and CD3ζ; or

[0033] (iii) the above-mentioned scFv, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule and CD3ζ.

[0034] In a specific embodiment, the cell contains a nucleic acid having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 18. Preferably, the cell contains the nucleotide sequence shown in SEQ ID NO:18.

[0035] In a specific embodiment, the chimeric antigen receptor has at least 90% identity with the amino acid sequence shown in SEQ ID NO:21, 22 or 23.

[0036] In the third aspect of the present application, an expression construct is provided, characterized in that the expression construct comprises sequentially connected: an expression of a receptor that binds to a tumor-associated antigen, and an expression of a protein that recognizes SDF-1; preferably, the two expressions are connected by a tandem fragment; more preferably, the tandem fragments include F2A, PA2, T2A, and / or E2A.

[0037] Specifically, the protein that recognizes SDF-1 is an antibody that recognizes SDF-1, or a receptor of SDF-1; more preferably, the receptor of SDF-1 is CXCR4 or CXCR7.

[0038] More specifically, the receptor of SDF-1 is CXCR4.

[0039] Specifically, the receptor that binds to the tumor-associated antigen has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 21, 22 or 23.

[0040] More specifically, the nucleic acid sequence of the CXCR4 expression product is a sequence that is at least 90% identical to the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:19.

[0041] In the fourth aspect of the present application, an expression vector is provided, which contains the expression construct encoding the above-mentioned third aspect of the present application.

[0042] In the fifth aspect of the present application, a virus is provided, characterized in that it comprises the vector described in the fourth aspect of the present application.

[0043] In the sixth aspect of the present application, a pharmaceutical composition is provided, characterized in that it comprises the immune effector cells described in the first aspect or the second aspect of the present application; and a pharmaceutically acceptable carrier.

[0044] In a seventh aspect of the present application, a medicine kit is provided, characterized in that it comprises:

[0045] A container, and the pharmaceutical composition of the sixth aspect of the present application in the container; or

[0046] A container, and the immune effector cells described in the first aspect or the second aspect of the application located in the container.

[0047] In the eighth aspect of the present application, a method for treating tumors is provided, characterized in that the immune effector cells described in the first aspect or the second aspect of the present application are administered to an individual suffering from a tumor, and lymphocyte clearance is performed on the individual or not; preferably, lymphocyte clearance is performed on the individual.

[0048] Beneficial effects of the present invention

[0049] The immune effector cells constructed by the present invention can interact with CXCL12 in the tumor microenvironment, so that the immune effector cells can chemotaxis and home to tumor cells under the action of CXCL12, and then the immune effector cells can better play the role of killing tumors. This is because CAR-T cells with high expression of CXCR4 can spread or transfer to tumor tissues with high expression of SDF1α / CXCL12 through chemotaxis. CXCR4 on the cell surface is activated after binding to its ligand. The activated CXCR4 can participate in cell proliferation, cell migration and other processes through multiple signal pathways, such as Wnt / β-catenin, NF-κB, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A The MSCV-CLDN18.2-BBZ plasmid map is shown; Figure 1B The MSCV-CLDN18.2-BBZ-F2A-CXCR4 plasmid map is shown;

[0051] Figure 2 The positive rate of CAR-T cell infection is shown;

[0052] Figure 3 The in vitro cytotoxicity of CLDN18.2-BBZ CAR T cells and CLDN18.2-BBZ-CXCR4 CAR T cells against tumor cells was shown;

[0053] Figure 4 shows the in vivo treatment experiment of CAR-T cells after lympholysis on the mouse subcutaneous transplant tumor model of PANC02-A2 pancreatic cancer cells: Figure 4A The results of transplanted tumor volume detection are shown. Figure 4B The results of mouse body weight test are shown. Figure 4C The results of transplanted tumor weight test are shown. Figure 4D The results of the inhibition of PANC02-A2 pancreatic cancer by CLDN18.2-BBZ and CLDN18.2-BBZ-CXCR4 CAR-T cells are shown. DETAILED DESCRIPTION

[0054] After in-depth research, the inventors have revealed for the first time a genetically engineered immune effector cell that co-expresses CXCR4. The immune effector cell can interact with CXCL12 in the tumor microenvironment, so that the immune effector cell chemotaxis homes to tumor cells under the action of CXCL12, and the immune effector cell can better play a role in killing tumors.

[0055] Unless specifically defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification, including definitions, shall prevail. In addition, unless otherwise specified, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0056] Unless otherwise indicated, the practice of the present invention employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are well described in the literature, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & S.J. Higginseds. 1984); Transcription And Translation (BD Hames & S.J. Higginseds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCaloseds., 1987, Cold Spring Harbor Laboratory); ImmunochemicalMethods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0057] In order to better understand the present invention, the relevant terms are defined as follows:

[0058] The term "genetically engineered cells" refers to cells transformed by genetic engineering. Specifically, the genetically engineered cells of the present invention refer to cells that co-express receptors that bind to antigens (such as tumor antigens) and exogenous open reading frames (for example, CXCR4 open reading frames), thereby being able to exert targeted killing effects; in particular, they refer to T cells that co-express chimeric antigen receptors that specifically bind to tumor antigens and CXCR4.

[0059] The term "CXCR4" is a specific receptor for the chemokine stromal cell-derived factor-1 (CXCL12). CXCR4 is expressed in some cells and organs. Its encoding gene is located on human chromosome 2q21, encoding 352 amino acid residues. The encoding sequence is highly conserved. Its α-helix spans the membrane seven times, with an extracellular N-terminus, three extracellular loops, three intracellular loops and an intracellular C-terminus, of which the N-terminus is the main binding site for the ligand. The C-terminus is located in the cytoplasm and has a Ser / Thr site. It is not related to receptor and ligand binding and is directly involved in signal transduction.

[0060] The term "CXCL12" (also known as SDF-1) belongs to the CXC chemokine family and is a chemotactic protein secreted by bone marrow stromal cells and other closely related mesothelial and epithelial cells. The gene is located on the long arm of chromosome 10. The gene coding region is 267bp in length, encoding an 89 amino acid polypeptide and producing six splice variants. CXCL12 is expressed in stromal fibroblasts of tissues and organs such as the brain, breast, liver, and lungs. It participates in cell migration and chemotaxis of leukocytes and plays an important role in tumor proliferation and migration. CXCL12 can bind to the tumor cell surface receptor CXCR4. The axis formed after the specific binding of CXCL12 / CXCR4 can not only promote tumor growth and tumor angiogenesis, but also promote tumor cell proliferation and migration.

[0061] The term "immune effector cell" refers to a cell that participates in an immune response, for example, a cell that promotes immune effects. Examples of immune effector cells include T cells, for example, α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. Preferably, the T cells include autologous T cells, xenogeneic T cells, allogeneic T cells, and the natural killer cells are allogeneic NK cells. As used herein, the term "immune effector function or immune effector response" refers to an immune effector cell, such as a function or reaction that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing of a target cell or inhibits growth or proliferation.

[0062] The term "chimeric antigen receptor" (CAR) includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and / or a co-stimulatory molecule, in one aspect, the stimulatory molecule is a ζ chain bound to a T cell receptor complex; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and / or CD28.

[0063] The term "extracellular binding domain" (also called extracellular antigen binding domain) includes an antibody or ligand that specifically recognizes an antigen (such as a tumor-associated antigen), and the antibody is preferably a single-chain antibody or a domain antibody. More preferably, the extracellular antigen binding region of the chimeric antigen receptor is connected to the transmembrane domain of CD8 or CD28 through the CD8 hinge region, and the intracellular signaling domain is immediately behind the transmembrane domain.

[0064] The term "transmembrane domain" (also referred to as a transmembrane domain) refers to a region in a protein sequence that spans a cell membrane and may include one or more additional amino acids adjacent to the transmembrane domain, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids in the intracellular region). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the chimeric receptor, for example, in one embodiment, the transmembrane domain may be from the same protein from which the signaling domain, the costimulatory domain, or the hinge domain is derived. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such a domain to the transmembrane domain of the same or different surface membrane protein, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another chimeric receptor on the surface of a cell expressing the chimeric receptor. The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In one aspect, the transmembrane domain is capable of conducting a signal to the intracellular domain whenever the chimeric receptor binds to the target. The transmembrane domain particularly used in the present invention can include at least the following transmembrane domains: for example, α, β or ζ chains of T-cell receptors, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.In certain embodiments, the transmembrane domain may include at least the following transmembrane domains: for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C.

[0065] In some cases, the transmembrane domain can be connected to the extracellular region of CAR via a hinge (e.g., a hinge from human protein), such as the antigen binding domain of CAR. Optionally, a short oligopeptide or polypeptide linker with a length between 2 and 10 amino acids can form a key between the transmembrane domain of CAR and the cytoplasmic region. Glycine-serine doublets provide a particularly suitable linker.

[0066] The term "intracellular domain" has the same meaning as "intracellular signaling domain", including intracellular signaling domains. The intracellular signaling domain refers to the part of the protein that transduces the immune effector function signal and guides the cell to perform a specific function, which can guide the activation of the immune effector function of the immune cell. The immune effector function of T cells can be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines. Although the entire intracellular signaling domain can usually be used, it is not necessary to use the entire chain in many cases. As long as the immune effector function signal can be transduced, a truncated portion can be used instead of the complete chain.

[0067] Those skilled in the art can also use technical means known in the art to promote the fusion expression of chemokine receptors and chimeric antigen receptors on the surface of immune effector cells, including but not limited to the use of self-cleavage sequences for the fusion expression of chemokine receptors and chimeric antigen receptors. In a specific embodiment, the cleavable protein can be selected from but not limited to 2A polypeptides and IRES. 2A polypeptides can be selected from F2A, PA2, T2A, E2A, etc.; the self-cleavage sequence is preferably F2A or P2A. Among them, F2A is a core sequence of 2A (or "self-cleavage polypeptide 2A") from foot-and-mouth disease virus, which has the "self-cleavage" function of 2A and can achieve co-expression of upstream and downstream genes. 2A provides an effective and feasible strategy for constructing gene therapy multicistronic vectors due to its high cleavage efficiency, high balance of upstream and downstream gene expression, and short self-sequence.

[0068] The term "tumor-associated antigen" refers to an antigen expressed in a tumor. The "tumor-associated antigen" may be selected from (but not limited to): EGFR, GPC3, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4 EGFR, CD19, CD33, IL13R, LMP1, PLAC1, NY-ESO-1, MAGE4, ​​MUC1, MUC16, LeY, CEA, CAIX (carbonic anhydrase IX), CD123.

[0069] In the present invention, various tumors known in the art can be included in the present invention, for example, the tumors include (but are not limited to): breast cancer, liver cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer.

[0070] The term "claudin 18.2" or "claudin 18A2" (CLD18.2, CLD18A2, CLDN18A2, or CLDN18.2) herein may also refer to homologs, orthologs, interspecies homologs, codon-optimized forms, truncated forms, fragmented forms, mutant forms, or any other known derivative forms, such as post-translationally modified variants, of a known claudin 18A2 sequence. In some embodiments, the claudin 18A2 or claudin 18A2 peptide is a peptide having GenBank accession number NP_001002026 (mRNA: NM_001002026)

[0071] The term "CAFs" is also called tumor-associated fibroblasts. They are the most abundant host cells in the microenvironment of solid tumors and acquire an activated phenotype under the influence of the microenvironment. Different from normal fibroblasts, CAFs are characterized by the expression of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP), and can secrete a large number of growth factors, such as VEGF, TGF-β, hepatocyte growth factor, etc., can synthesize and deposit ECM, produce various collagens, adhesion proteins, and mediate ECM remodeling. CAFs have been verified to be important in the occurrence, development, metastasis and recurrence of tumors, and it has been revealed that they promote tumor growth by dominating the tumor microenvironment.

[0072] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, complete immunoglobulins or antibody fragments, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0073] "Single-chain antibody (scFv)" herein refers to an antibody defined as follows, which is a recombinant protein comprising a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, and the linker associates these two domains to ultimately form an antigen binding site. The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. The single-chain antibody used in the present invention can be further modified using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods, alone or in combination. The method of introducing such a modification into its DNA sequence based on the amino acid sequence of an antibody is well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2002) NY. The modification referred to is preferably performed at the nucleic acid level. The above-mentioned single-chain antibody may also include its derivatives.

[0074] The term "single domain antibody" (sdAb), also known as nanobodies, consists of a single antibody variable domain. Single domain antibodies have a small molecular weight and strong stability. Although they have a simple structure, they can still achieve an affinity for binding to specific antigens that is comparable to or even higher than that of traditional antibodies. Therefore, single domain antibodies are widely used in bispecific antibodies and cell therapies (such as chimeric antigen receptor T cells).

[0075] The term "chimeric receptor" refers to a fusion molecule formed by connecting DNA fragments or corresponding cDNAs of proteins from different sources using genetic recombination technology, including an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to: chimeric antigen receptors (CARs), chimeric T cell receptors (TCRs), and T cell antigen couplers (TACs).

[0076] The term "T cell receptor (TCR)" mediates T cells to recognize specific major histocompatibility complex (MHC)-restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. The classical TCR receptor is composed of two peptide chains, α and β, each of which can be divided into a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region, etc. Its antigen specificity exists in the V region, and the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3. In one aspect, T cells expressing classical TCR can induce the specificity of T cell TCR to target antigens by using methods such as antigen stimulation on T cells.

[0077] The term "T cell antigen coupler (TAC)" includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (DARPin) or other targeting groups; 2. Extracellular domain, single-chain antibody that binds to CD3, thereby bringing the TAC receptor into close proximity with the TCR receptor; 3. Transmembrane region and intracellular region of CD4 co-receptor, wherein the intracellular region is connected to protein kinase LCK, catalyzing the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.

[0078] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides constituting TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells. The chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least a portion of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit and the antibody domain are operatively connected, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is integrated into the TCR expressed on the T cell.

[0079] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by producing a second messenger or by acting as an effector in response to such a messenger. An intracellular signaling domain may include the entire intracellular portion of a molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0080] The term "co-stimulatory molecule" refers to a signal that binds to a cell-stimulatory signaling molecule, such as TCR / CD3, and in combination results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0081] The terms "activation" and "activation" are used interchangeably and may refer to the process by which a cell changes from a quiescent state to an active state. The process may include a response to an antigen, migration, and / or a phenotypic or genetic change in a functionally active state. For example, the term "activation" may refer to the process by which a T cell is gradually activated. For example, a T cell may require at least one signal to be fully activated.

[0082] The genetically engineered cells of the present invention can be used to prepare pharmaceutical compositions or diagnostic reagents. In addition to an effective amount of the immune cells, the composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they will not produce adverse, allergic or other adverse reactions, such as cell cryoprotectants. The term "cell cryoprotectant" can be a composition, for example, it can contain isotonic salts, buffer salts, glycerol, DMSO, ethylene glycol, propylene glycol, acetamide, polyvinyl pyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, hydroxyethyl starch, serum, etc.

[0083] The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be injection or other treatment methods.

[0084] The term "lymphocyte clearance" or "lymphocyte clearance" means clearing the lymphocytes in the subject. It includes administering a lymphocyte clearance agent, whole body radiation therapy, or a combination thereof. For example, in order to increase the expansion or maintenance of immune effector cells in the subject, before, at the same time, after, or in any combination of administering a therapeutically effective amount of CAR-T cells, one or more agents capable of substantially clearing the subject's lymphocytes, whole body radiation therapy, or a combination thereof may be administered to the subject alone or in combination. Lymphocyte clearance therapy may be administered under conditions sufficient to achieve a lymphocyte clearance rate of 50%-100% in the subject.

[0085] The lymphocyte removal agent can be an anti-tumor chemotherapeutic agent, such as fludarabine, cyclophosphamide, or a combination thereof. The physician can select a specific lymphocyte removal agent and its appropriate dosage according to the subject to be treated, such as CAMPATH, anti-CD3 antibodies, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, melphalan, cyclophosphamide, fludarabine, and systemic radiation therapy.

[0086] The immune effector cells are administered before, during, and after the clearing therapy, and can also be administered in combination, i.e., before and during, before and after, during and after, or before, during, and after the clearing therapy. In certain embodiments, the clearing therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, before the immune effector cell therapy. In certain embodiments, the qinglin treatment is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 day, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, after the administration of the immune effector cell therapy.

[0087] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0088] Example 1: Genetically engineered cell construction

[0089] 1. Plasmid Construction

[0090] Using conventional molecular biological methods in the art, the scFv used in this embodiment is a Claudin 18.2-targeting antibody (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2), having HDCR1, HCDR2, HCDR3 as shown in SEQ ID NO: 24, 25, 26, and LDCR1, LCDR2, LCDR3 as shown in SEQ ID NO: 27, 28, 29, and the chimeric antigen receptor used is a second-generation chimeric antigen receptor, having a transmembrane domain of CD8, an intracellular domain of 4-1BB (CD137), and CD3ζ. Figure 1A The plasmid map shown was used to construct plasmid MSCV-CLDN18.2-BBZ.

[0091] In order to verify the results in mice, the transmembrane domain, intracellular domain, etc. selected for preparing CAR in this example are all derived from mice. If the present invention is used for clinical purposes, the transmembrane domain, intracellular domain, etc. for preparing CAR are preferably of human origin.

[0092] Using MSCV-IRES-GFP (purchased from Addgene) as a vector, a retroviral plasmid MSCV-CLDN18.2-BBZ expressing a second-generation chimeric antigen receptor was constructed. The CLDN18.2-BBZ sequence consists of a mouse CD8α signal peptide (nucleotide sequence as shown in SEQ ID NO:3, amino acid sequence as shown in SEQ ID NO:4), a scFv targeting Claudin 18.2 (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2), a mouse CD8 hinge and transmembrane domain (nucleotide sequence as shown in SEQ ID NO:5, amino acid sequence as shown in SEQ ID NO:6), a mouse 4-1BB intracellular signaling domain (nucleotide sequence as shown in SEQ ID NO:7, amino acid sequence as shown in SEQ ID NO:8), and the intracellular segment CD3ζ of mouse CD3 (nucleotide sequence as shown in SEQ ID NO:9, amino acid sequence as shown in SEQ ID NO:10).

[0093] The F2A-CXCR4 sequence was inserted into the MSCV-CLDN18.2-BBZ plasmid to construct the lentiviral plasmid MSCV-CLDN18.2-BBz-CXCR4 expressing the second-generation chimeric antigen receptor. Figure 1BF2A-CXCR4 is composed of F2A (nucleotide sequence as shown in SEQ ID NO: 11, amino acid sequence as shown in SEQ ID NO: 12) and mouse CXCR4 (nucleotide sequence as shown in SEQ ID NO: 13, amino acid sequence as shown in SEQ ID NO: 14).

[0094] 2. Using conventional viral packaging methods in the art, such as the method described in Di S, Zhou M, Pan Z, Sun R, Chen M, Jiang H, et al. Combined Adjuvant of Poly I:C Improves Antitumor Effects of CAR-TCells. (2019). Frontiers in oncology. 9: 241, MSCV-CLDN18.2-BBZ and MSCV-CLDN18.2-BBz-CXCR4 plasmids were transfected into 293T to package retroviruses, respectively, to obtain retroviruses.

[0095] 3. T cell activation: Grind the spleen of C57BL / 6 mice to obtain lymphocytes. After being treated with the CD3+ mouse T cell negative screening kit (Stemcell), the obtained mouse CD3+ T lymphocytes were added with Dynabeads Mouse T-activator CD3 / CD28 magnetic beads at a volume ratio of 1:1 for activation stimulation and placed in a cell culture incubator. The culture medium was RPMI 1640 complete culture medium (10% FBS + 50 μM β-mercaptoethanol + 100 U / mL of IL-2 + 1 ng / mL of IL-7).

[0096] Mouse spleen CD3+T lymphocytes activated for 24 hours were inoculated into a 24-well plate coated with Retronectin (5 μg / mL), and retrovirus was added for infection for 24 hours, and then fresh culture medium was replaced to obtain mouse CLDN18.2-BBZ cells and CLDN18.2-BBZ-CXCR4 cells. Flow cytometry was used to detect the positive rate of CAR-T cell infection. Figure 2 As shown, the positive rate of MSCV-CLDN18.2-BBZ cell infection was 43.3%, and the positive rate of MSCV-CLDN18.2-BBZ-CXCR4 cell infection was 33.2%.

[0097] Example 2: In vitro cytotoxicity experiment of CAR-T on mouse pancreatic cells PANC02-A2

[0098] 2.1 Construction of mouse pancreatic cancer cells PANC02-A2 stably overexpressing the huClaudin18.2 extracellular domain

[0099] In mouse pancreatic cancer cell line PANC02 (purchased from ATCC), the full-length sequence of CLDN18.2 (amino acid sequence as shown in SEQ ID NO: 20) was overexpressed using a lentiviral vector to obtain a stably expressing PANC02-A2 cell line; PANC02-A2 positive cell lines were screened by flow cytometry, and this cell line was used for subsequent research, and PANC02 cells were used as negative control cells for subsequent experiments.

[0100] 2.2 The untreated mouse T cells (UTD) in Example 1 were taken, and CLDN18.2-BBZ CAR T cells (the nucleotide sequence of CLDN18.2-BBZ is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16) and CLDN18.2-BBZ-CXCR4 CAR T cells (the nucleotide sequence of CLDN18.2-BBZ-CXCR4 is shown in SEQ ID NO: 17) were co-incubated with PANC02 cells and PANC02-A2 cells at 1: 3, 1: 1, and 3: 1, respectively. After 16 hours, the secretion of LDH in the supernatant was detected using Cytox 96 Non-Radioactive Cytotoxicity Assay, and the cytotoxicity of CLDN18.2-BBZ CAR T cells and CLDN18.2-BBZ-CXCR4 CAR T cells to tumor cells was calculated (as shown in Figure 1). Figure 3 For specific detection steps and calculation methods, see the Promaga Cytox 96 Non-Radioactive Cytotoxicity Assay instruction manual (Promaga, REF: G1782). Figure 3 It can be seen that when the effector-target ratio is 1:3 and 1:1, the tumor killing effect of CLDN18.2-BBZ-CXCR4 CAR T is better than that of CLDN18.2-BBZ CAR T cells.

[0101] Example 3: Anti-tumor treatment experiment of subcutaneous transplanted tumors

[0102] (1) Establishment and grouping of pancreatic cancer subcutaneous transplant tumor model in mice:

[0103] PANC02-A2 cells that were in the logarithmic growth phase and growing well were collected and subcutaneously inoculated into C57BL / 6 mice (mice with normal immune system) at a rate of 1×10 6 The tumor cell inoculation date is day 0 (ie, Day 0).

[0104] (2) On the 10th day after tumor inoculation (i.e., Day 10), mice were intraperitoneally injected with cyclophosphamide. Cyclophosphamide dosage: 100 mg / kg. 0.2 g of cyclophosphamide powder was fully dissolved in 20 ml of normal saline, and 200 μl was intraperitoneally injected into each mouse.

[0105] (3) On the 11th day after tumor inoculation (i.e., Day 11), CART cells (2×10 6 ). CLDN18.2-BBZ cells and CLDN18.2-BBZ-CXCR4 cells were constructed according to step 1 of this example.

[0106] The mice were divided into 3 groups, 5 mice in each group:

[0107] UTD group: 2×10 6 Mouse T cells;

[0108] CLDN18.2-BBZ group: 2×10 6 CLDN18.2-BBZ-CAR-T cells;

[0109] CLDN18.2-BBZ-CXCR4 group: 2×10 6 CLDN18.2-BBZ-CXCR4-CAR-T cells;

[0110] (4) Tumor volume detection. The changes in tumor volume of mice were continuously observed and measured, and the data were recorded three times a week. The formula for calculating tumor volume is: tumor volume = (tumor length * tumor width) 2 ) / 2.

[0111] The results of mouse tumor volume detection are as follows Figure 4A As shown, the results showed that CART co-expressing CXCR4 could significantly inhibit the tumor volume of mice. At the same time, it was detected that the weight changes of mice in each group were not obvious (such as Figure 4B This indicates that CART co-expressing CXCR4 did not cause significant toxic effects on mice.

[0112] (5) Tumor weight measurement. On Day 29, the mice were euthanized, the tumors were removed and the tumor weights were measured. The specific statistical results are as follows: Figure 4C As shown. This suggests that CAR-T cells co-expressing CXCR4 have a better anti-tumor effect on pancreatic cancer in mice.

[0113] (6) Calculation of tumor inhibition rate. The final tumor volume of mice on Day 29 was calculated using the following formula: tumor inhibition rate (%) = (the final tumor volume of mice in the UTD group - the final tumor volume of mice in the experimental group) / the final tumor volume of mice in the UTD group * 100. Figure 4DAs shown, the tumor inhibition rate of the CLDN18.2-BBZ group was 24.25%, which did not achieve a good effect in inhibiting tumor growth; the tumor inhibition rate of the CLDN18.2-BBZ-CXCR4 group was 90.18%, indicating that CAR-T cells co-expressing CXCR4 can inhibit the growth of pancreatic cancer in mice.

[0114] As an example, the antibodies selected in the above embodiments are humanized antibodies, but it should be known that the antibodies used can be mouse antibodies or humanized antibodies, and the transmembrane domains and intracellular domains used can also be of different species according to different purposes, such as human antibodies.

[0115] As an example, although the above embodiments use CAR-T cells, the T cells can also express other cytokines that enhance the function of CAR-T cells, such as CAR-T cells co-expressing CAR and type I interferon, CAR-T cells co-expressing CAR and PD1, etc.

[0116] As an example, although the above embodiment uses CAR-T cells, other immune cells may also be selected, such as NK cells, NK-T cells, and specific subtypes of immune cells may also be selected, such as γ / δT cells.

[0117] As an example, the above embodiment selects mouse CAR, but its signal peptide, hinge region, transmembrane region, etc. can be selected from other species according to different purposes, including but not limited to human signal peptide, hinge region, transmembrane domain, intracellular region, for example, the amino acid sequence of the CAR used can be the amino acid sequence shown in SEQ ID NO: 21, 22, 23. Antibodies can also select mouse antibodies or humanized antibodies or fully human antibodies against different targets according to different purposes.

[0118] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims of the present application. The sequences used herein are as follows:

[0119]

[0120]

[0121]

[0122]

Claims

1. An immune effector cell co-expressing a chemokine receptor, characterized in that: The cell comprises: a receptor that specifically recognizes claudin18.2; and a receptor that recognizes SDF-1; The receptor that specifically recognizes claudin18.2 is a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane region and an intracellular signaling region that are sequentially connected; The extracellular domain of the chimeric antigen receptor is scFv; The scFv contains HDCR1, HCDR2, HCDR3 shown in SEQ ID NOs: 24, 25, 26, and LDCR1, LCDR2, LCDR3 shown in SEQ ID NOs: 27, 28, 29; The receptor of SDF-1 is CXCR4, and the CXCR4 has an amino acid sequence as shown in SEQ ID NO: 19; the immune effector cells target pancreatic cancer.

2. The immune effector cell according to claim 1, characterized in that The immune effector cells are selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.

3. The cell according to claim 1, characterized in that The receptor that specifically recognizes claudin18.2 is a chimeric receptor.

4. The cell according to claim 3, characterized in that The chimeric receptor is selected from: a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

5. The immune effector cell according to claim 1, characterized in that The extracellular domain of the chimeric antigen receptor contains the amino acid sequence shown in SEQ ID NO:

2.

6. The immune effector cell according to claim 1, characterized in that The immune effector cell contains the nucleotide sequence shown in SEQ ID NO: 18; or contains the amino acid sequence encoded by SEQ ID NO:

18.

7. The immune effector cell according to claim 1, characterized in that The chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 21, 22 or 23.

8. An expression construct, characterized in that The expression construct comprises: an expression of a receptor that binds to a tumor-associated antigen, and a receptor CXCR4 that recognizes SDF-1, which are sequentially connected, wherein the CXCR4 has a nucleic acid sequence encoding an amino acid sequence shown in SEQ ID NO: 19; the tumor antigen is claudin18.2; the tumor is pancreatic cancer; the receptor that binds to the tumor-associated antigen is a chimeric antigen receptor, and the chimeric antigen receptor comprises an extracellular domain, a transmembrane region and an intracellular signal region that are sequentially connected; The extracellular domain of the chimeric antigen receptor is scFv; The scFv contains HDCR1, HCDR2, HCDR3 shown in SEQ ID NOs: 24, 25, 26, and LDCR1, LCDR2, LCDR3 shown in SEQ ID NOs: 27, 28, 29.

9. The expression construct according to claim 8, wherein The two expressions were linked by a tandem fragment.

10. The expression construct according to claim 9, wherein The tandem fragments include F2A, PA2, T2A, and / or E2A.

11. The expression construct according to claim 8, wherein The receptor binding to the tumor-associated antigen has the amino acids shown in SEQ ID NO: 21, 22 or 23.

12. An expression vector comprising an expression construct encoding any one of claims 8-11.

13. A virus, characterized in that It comprises the vector according to claim 12.

14. A pharmaceutical composition, characterized in that Comprising the immune effector cell according to any one of claims 1 to 7; and a pharmaceutically acceptable carrier.

15. A medicine box, characterized in that: include: A container, and the pharmaceutical composition of claim 14 located in the container; or A container, and the immune effector cell according to any one of claims 1 to 7 located in the container.

16. Use of the immune effector cells according to any one of claims 1 to 7 in the preparation of a drug for treating pancreatic cancer, and the individual is subjected to lymphocyte clearance or not.

17. The use according to claim 16, characterized in that The individual is subjected to lymphodepletion.

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