WO2023213280A1 - 靶向cldn18.2的嵌合抗原t细胞受体及其应用 - Google Patents

靶向cldn18.2的嵌合抗原t细胞受体及其应用 Download PDF

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WO2023213280A1
WO2023213280A1 PCT/CN2023/092137 CN2023092137W WO2023213280A1 WO 2023213280 A1 WO2023213280 A1 WO 2023213280A1 CN 2023092137 W CN2023092137 W CN 2023092137W WO 2023213280 A1 WO2023213280 A1 WO 2023213280A1
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cancer
cells
seq
chimeric antigen
car
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PCT/CN2023/092137
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French (fr)
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姜福伟
王超
王庆杨
杨翠青
曹卓晓
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上海先博生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present application relates generally to the fields of bioengineering and cell therapy, and specifically to T lymphocytes targeting CLDN18.2 and preparation methods and applications thereof.
  • Cancer as a malignant tumor, is a disease caused by an abnormality in the mechanism that controls cell division and proliferation.
  • the choice of cancer treatment depends on the location, malignancy, development and physical condition of the tumor, and new treatments are constantly being developed.
  • Immunotherapy uses the immune mechanism in the human body to fight against tumor cells. Among them, cancer vaccine therapy and monoclonal antibody therapy have made more progress, while immune cell therapy is the latest treatment technology developed in recent years.
  • Claudin 18 is a member of the cell surface protein claudin family (Claudin family). There are two splice variants: claudin 18.1 (Claudin18.1, CLDN18.1 for short) and claudin 18.2 (Claudin18. 2, referred to as CLDN18.2).
  • CLDN18 is a 4-transmembrane protein containing 2 extracellular domains and the N-terminus and C-terminus located in the cell. In the protein primary structure sequences of CLDN18.1 and CLDN18.2, only the amino acid residues at certain positions from the N-terminal signal peptide to the extracellular domain are different, especially in the extracellular domain 1 (extracellular loop 1). , only 8 amino acids differ.
  • CLDN18.1 NP_057453.1, NCBI database
  • SEQ ID NO: 1 The amino acid sequence of CLDN18.1 (NP_057453.1, NCBI database) is shown in SEQ ID NO: 1
  • nucleotide sequence NM_016369.4, Genbank database
  • SEQ ID NO: 2 The amino acid sequence of CLDN18.2 (NP_001002026.1, NCBI database) is shown in SEQ ID NO: 3
  • the nucleotide sequence NM_001002026.3, Genebank database
  • CLDN18.1 and CLDN18.2 are expressed in different tissues respectively.
  • Claudin18.1 is mainly expressed in lung tissue
  • Claudin18.2 is specifically expressed in normal gastric mucosa tissue, and is highly selectively and stably expressed in Specific tumor tissue is involved in the proliferation, differentiation and migration of cancer cells in gastric cancer, esophageal cancer, pancreatic cancer and other diseases, making it a potential effective molecular target for anti-tumor drugs.
  • Chimeric Antigen Receptor T cell therapy (Chimeric Antigen Receptor T cell, referred to as CAR-T cell) is a type of cellular immunotherapy: using Chimeric Antigen Receptor (Chimeric Antigen Receptor, referred to as CAR) in vitro Modify T cells isolated from the patient so that they can recognize specific cancer cells, and then expand the modified cells and infuse them back into the patient to achieve the effect of treating tumors.
  • This therapy uses the patient's own immune cells as a therapeutic tool to directly fight cancer cells, making it potentially effective for cancer patients with compromised or reduced immunity.
  • the present application provides a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising a signal peptide, Specifically recognizes the extracellular binding region, hinge region, transmembrane region, intracellular costimulatory domain and intracellular signaling domain of the CLDN18.2 protein, and the extracellular binding region includes an antibody that specifically recognizes CLDN18.2 or
  • the antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 13 LCDR1, LCDR2 and LCDR3 contained in the area (VL).
  • the present application provides an isolated nucleic acid molecule encoding the chimeric antigen receptor of the first aspect.
  • the present application provides an expression vector comprising the nucleic acid molecule described in the second aspect.
  • the present application provides cells that express the chimeric antigen receptor described in the first aspect, or contain the nucleic acid molecule described in the second aspect, or contain the expression vector described in the third aspect. .
  • the present application provides a method for preparing chimeric antigen receptor modified immune cells, the method comprising delivering the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect to the immune cells to be modified.
  • the present application provides a pharmaceutical composition, which comprises the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, and the chimeric antigen receptor described in the third aspect.
  • a pharmaceutical composition which comprises the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, and the chimeric antigen receptor described in the third aspect.
  • the present application provides the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the The cells, the product prepared by the method described in the fifth aspect, or the use of the pharmaceutical composition described in the sixth aspect in preparing a composition for preventing and/or treating cancer.
  • the present application provides a method for preventing and/or treating cancer, comprising administering to a patient in need an effective amount of the chimeric antigen receptor described in the first aspect, the second aspect
  • Figures 1A-1D show the cell affinity detection of antibodies Tab1, Tab2 and Tab3.
  • Figures 1A to 1D respectively show the relationship between the three antibodies and CHO-K1 cells expressing CLDN18.2, CHO-K1 cells expressing CLDN18.1, and tumors. Affinity detection results of NUGC4 cells and CHO-K1 cells.
  • Figure 2 shows a schematic structural diagram of each exemplary CAR-T plasmid of the present application.
  • Figures 3A-3C show the CAR expression of three CAR-T cells and the killing effect over time on the CHO-K1 cell line that highly expresses CLDN18.2.
  • Figure 3A shows the FACS detection results of CAR expression on the surface of CAR-T cells.
  • Figures 3B and 3C show the real-time killing results of target cells by CAR-T cells when the effect-to-target ratio is 1:1 and 1:5 respectively.
  • Figure 4A and Figure 4B respectively show the secretion levels of TNF- ⁇ (Figure 4A) and IFN- ⁇ ( Figure 4B) when three CAR-T cells kill CHO-K1 that highly expresses CLDN18.2. CAR-T cells and CHOK1-18.2 cells were co-incubated for 72 hours (effect-to-target ratio 1:5).
  • Figures 5A-5B show the evaluation results of multiple rounds of consecutive killing effects of three types of CAR-T cells on NUGC4-luc cells. After CAR-T cells were co-incubated with NUGC4-luc cells for 72 hours (effectiveness-to-target ratio 1:1), the T cells were resuspended and counted, and then co-incubated with the same number of fresh NUGC4-luc cells for the next round of killing experiments. By analogy, a total of 4 rounds of killing were completed.
  • Figure 5A It is the result of multi-round kill evaluation.
  • Figure 5B shows multiple rounds of CAR-T cell proliferation curves.
  • Figure 5C shows the expression level of PD-1 during the fourth round of killing experiment.
  • Figures 6A-6C show the anti-tumor efficacy test results of three CAR-T cells in the NUGC4-luc mouse tumor model.
  • Figure 6A shows the tumor growth curve of mice in each experimental group within 53 days of injection of CAR-T cells
  • Figure 6B shows the body weight change rate of mice in each experimental group within 53 days of injection of CAR-T cells
  • 6C shows the growth rate of mice in each experimental group within 53 days of injection of CAR-T cells Tumor growth inhibition rate after injection of CAR-T cells.
  • Figure 7A shows the growth curve from the in vitro expansion of four CLDN18.2 CAR-T cells
  • Figure 7B shows the CAR transfection efficiency of the four CLDN18.2 CAR-T cells detected by FACS.
  • Figures 8A and 8B respectively show the secretion levels of cytokines TNF- ⁇ (Figure 8A) and IFN- ⁇ (Figure 8B) when four CLDN18.2 CAR-T cells kill NUGC4-luc that highly expresses CLDN18.2. CAR-T cells and NUGC4-luc cells were co-incubated for 72 hours (effect-to-target ratio 1:5).
  • Figure 9A shows the results of multiple rounds of continuous killing of NUGC4-luc cells by four CLDN18.2 CAR-T cells;
  • Figure 9B shows the expression levels of the four CAR-T cell memory phenotypes after the third round of killing experiments;
  • Figure 9C shows the first The expression levels of TIM3 and LAG3 of four CAR-T cell exhaustion phenotypes after three rounds of killing experiments;
  • Figure 9D shows the expression levels of PD-1 of four CAR-T cell exhaustion phenotypes after the third round of killing experiments.
  • Figure 10 shows the anti-tumor efficacy test results of two CAR-T cells in the NUGC4-luc mouse tumor model.
  • Figure 10A shows the tumor growth inhibition rate in mice in the CAR-2 treatment group
  • Figure 10B shows the tumor growth inhibition rate in mice in the CLDN18.2-1 treatment group.
  • Figures 11A-11D show the anti-tumor efficacy test results of four high-dose CLDN18.2 CAR-T cells in the NUGC4-luc mouse tumor model.
  • Figure 11A shows the tumor growth curve of mice in each experimental group within 45 days of injection of CAR-T cells
  • Figure 11B shows the body weight change rate of mice in each experimental group within 45 days of injection of CAR-T cells
  • Figure 11C shows the rate of change in body weight of mice in each experimental group within 45 days of injection of CAR-T cells Tumor growth inhibition rate of CAR-T cells on day 34
  • Figure 11D shows the changes in serum IFN- ⁇ content of mice in each experimental group after injection of CAR-T cells.
  • Figures 12A-12C show the anti-tumor efficacy test results of high doses of three CLDN18.2 CAR-T cells in the patient-derived tumor xenograft model GA0006.
  • Figure 12A shows the tumor growth curve of mice in each experimental group within 35 days of injection of CAR-T cells
  • Figure 12B shows the body weight change rate of mice in each experimental group within 35 days of injection of CAR-T cells
  • Figure 12C shows the rate of change in body weight of mice in each experimental group within 35 days of injection of CAR-T cells Tumor growth inhibition rate calculated by CAR-T cells on days 21 and 35.
  • Figures 13A-13C show the anti-tumor efficacy test results of three CLDN18.2 CAR-T cells at different doses in the NUGC4-luc mouse tumor model.
  • Figure 13A shows the tumor growth curve of mice in each experimental group within 21 days of injection of CAR-T cells
  • Figure 13B shows the body weight change rate of mice in each experimental group within 21 days of injection of CAR-T cells
  • Figure 13C shows the rate of change in body weight of mice in each experimental group within 21 days of injection of CAR-T cells Tumor growth inhibition rate calculated by CAR-T cells on day 21.
  • Figure 14 shows the anti-tumor efficacy test results of two CLDN18.2 CAR-T cells at high and medium doses in the patient-derived tumor xenograft model GA0006.
  • Figure 14A shows the tumor growth curve of mice in each experimental group within 53 days of injection of CAR-T cells;
  • Figure 14B shows the body weight change rate of mice in each experimental group within 53 days of injection of CAR-T cells;
  • Figure 14C shows the rate of change in body weight of mice in each experimental group within 53 days of injection of CAR-T cells Tumor growth inhibition rate calculated by CAR-T cells for 25 days.
  • compositions including A and B
  • compositions composed of A and B as well as a composition containing other components in addition to A and B, all fall into the category Within the scope of the aforementioned "a composition”.
  • CLDN18 refers to claudin-18 and includes any variant (including CLDN18.1 and CLDN18.2), conformation, identity, or expression of CLDN18 naturally expressed by cells or cells transfected with a CLDN18 gene. Isoforms and species homologs.
  • CLDN18 refers to human CLDN18.
  • CLDN18.1 or Claudin18.1 includes any post-translationally modified variant, isoform, and interspecies expression of human CLDN18.1 that is naturally expressed by a cell or that is expressed by a cell transfected with a CLDN18.1 gene. homologues.
  • CLDN18.2 or “Claudin18.2” includes any post-translationally modified variant, isoform, and Interspecific homologs.
  • antibody refers to an immunoglobulin molecule that specifically binds or is immunoreactive to a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugated antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab') 2 fragments which lack The Fc fragment of the intact antibody (clears more quickly from the animal circulation) and therefore lacks Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the content of which is incorporated by reference This article).
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , scFv, and VHH.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers include GSTGSSGKPGSGEGSTKG and consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • antibodies may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, Llama, ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • humanized antibody means an antibody obtained by grafting CDR sequences derived from another mammalian species, such as mouse germline, onto human framework sequences. In order to retain binding affinity, some residues of the segment of the backbone (termed FR) can be modified. Humanized antibodies or fragments thereof according to the present application can be prepared by techniques known to those skilled in the art.
  • variable region herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen.
  • Heavy chain variable region is used interchangeably with “VH” and “HCVR”
  • light chain variable region is used interchangeably.
  • VL can be used interchangeably with “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • LCDR light chain variable region
  • frame region or "FR region” is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody.
  • FR region usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR CDR
  • Kabat et al. J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. . Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P.
  • CDR in this article can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, and the tool website used includes but is not limited to abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) ).
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • chimeric antigen receptor refers to an artificial immune effector cell surface receptor engineered to be expressed on immune effector cells and specifically bind an antigen, which contains at least (1) an extracellular antigen-binding structure domains, such as variable heavy or light chains of antibodies, (2) transmembrane domains that anchor the CAR into immune effector cells, and (3) intracellular signaling structures area. CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • the extracellular domain of the chimeric antigen receptor may also include a signal peptide and/or hinge region.
  • the intracellular domain of the chimeric antigen receptor may also include a costimulatory domain.
  • extracellular binding domain in the context of a chimeric antigen receptor refers to the portion of a protein that is located outside the cell membrane and is capable of binding to an antigen, target, or ligand.
  • the term "hinge region" in the context of a chimeric antigen receptor refers to the portion of a protein that connects two adjacent domains of the protein (eg, the extracellular domain and the transmembrane domain). Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region.
  • the hinge region may be derived in whole or in part from a natural molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.
  • signal peptide in the context of chimeric antigen receptors refers to the leader sequence at the amino terminus (N-terminus) of the nascent protein, which co- or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequently Make surface expressions.
  • a non-limiting example of a signal peptide is the CD8 ⁇ signal peptide.
  • transmembrane region in the context of chimeric antigen receptors is used interchangeably with “transmembrane domain” to refer to a thermodynamically stable region of a protein anchored within the cell membrane.
  • the transmembrane region can be obtained from natural proteins and can be selected from CD8 ⁇ molecules or CD28 molecules.
  • intracellular signaling domain in the context of chimeric antigen receptors refers to the structural region of a protein that is capable of transducing signals for a cell's effector function and directing the cell to perform a specific function.
  • the intracellular signaling domain is responsible for primary intracellular signal transmission after the antigen-binding domain binds the antigen, leading to the activation of immune cells and immune responses.
  • 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.
  • Exemplary intracellular signaling domains include CD3 ⁇ .
  • costimulatory signal in the context of chimeric antigen receptors refers to signals that bind to primary signals such as TCR/CD3 ligation resulting in T cell proliferation and/or up- or down-regulation of key molecules.
  • costimulatory signal binding domain in the context of chimeric antigen receptors includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to T cells cognate costimulatory molecules on the peptide, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, in addition to the primary signal provided by, for example, the TCR/CD3 complex binding to peptide-loaded MHC molecules. , activation, differentiation, etc.
  • antigen-presenting cells e.g., aAPCs, dendritic cells, B cells, etc.
  • Costimulatory signal binding domains may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L ), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, binding Toll ligand receptor and Agonists or antibodies that specifically bind to ligands of B7-H3.
  • CD7, B7-1 CD80
  • B7-2 CD86
  • PD-L1, PD-L2, 4-1BBL OX40L
  • IX40L inducible costimulatory ligand
  • IAM intercellular adhesion molecule
  • CD30L CD40, CD70, CD83, HLA-G, MICA, MICB
  • HVEM lympho
  • Costimulatory signal binding domains may also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytes Functionally related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.
  • T cells such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytes Functionally related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.
  • immune cell may refer to cells that participate in an immune response, such as promoting an immune effector response.
  • immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • lentivirus refers to the genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can enough to infect non-dividing cells. They can deliver large amounts of genetic information into the host cell's DNA, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant levels of gene transfer in vivo.
  • a "vector,” generally referred to as an "expression vector,” is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.
  • Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • telomere binding refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample.
  • an antibody that specifically binds to an antigen of one species may also bind to an antigen of one or more species. However, this interspecies reactivity does not in itself alter the specificity of the antibody.
  • an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, this cross-reactivity does not by itself change the specificity of the antibody.
  • the terms "specific recognition” or “specific binding” may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species to indicate that the interaction depends on the presence of a specific structure.
  • a specific structure e.g., an antigenic determinant or epitope
  • an antibody recognizes and binds to a specific protein structure rather than to proteins in general. If the antibody is specific for epitope "A”, then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing label "A” and the antibody will reduce the amount of label A bound to the antibody.
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the alignment of sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and nonhomologous sequences may be ignored for comparison purposes) followed by the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity.
  • the length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.
  • the terms "subject,” “subject,” and “patient” refer to an organism undergoing treatment for a particular disease or condition (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
  • treatment refers to surgical or pharmacological treatment with the purpose of preventing, slowing (reducing) undesirable physiological changes or pathologies in the subject treated, such as cell proliferative disorders (such as cancer or infectious diseases) progress.
  • have Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • terms such as slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • the term "effective amount” refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of the disease. "Effective amount” also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • compositions as used herein means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that is combined together to achieve a specific purpose.
  • the pharmaceutical compositions include combinations that are separated in time and/or space, so long as they can act together to achieve the purposes of the present application.
  • the components contained in the pharmaceutical composition eg, CAR-T cells according to the present application
  • the ingredients contained in the pharmaceutical composition can be administered to the individual as a whole, or separately.
  • the ingredients contained in the pharmaceutical composition are administered to the individual separately, the ingredients may be administered to the individual simultaneously or sequentially.
  • Pharmaceutical compositions according to the present application may include conventional components of cell culture to maintain the activity of CAR-T cells.
  • Pharmaceutically acceptable carriers may also include water, aqueous buffer solutions, isotonic saline solutions such as PBS (phosphate buffer saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc.
  • the compositions according to the present application may contain wetting agents, emulsifiers or buffer substances as additives.
  • the inventor of this application has developed a new chimeric antigen receptor targeting CLDN18.2 and its application after extensive research and development and testing, and provided a chimeric antigen receptor targeting CLDN18.2 and corresponding nucleic acid molecules, Vectors, immune effector cells, preparation methods and products thereof, pharmaceutical compositions, therapeutic uses, pharmaceutical uses and tumor or cancer treatment methods.
  • Each invention of the present application achieves at least one of the following beneficial effects: (1) having a high CAR expression positive rate or cell proliferation multiple; (2) being highly specific for specific antigenic epitopes; (3) It has excellent tumor cell killing and inhibition capabilities, especially at medium and high doses, showing better tumor killing effect; (4) lower side effects, strong specificity, and safer.
  • the present application provides a chimeric antigen receptor (CAR), which includes a signal peptide, an extracellular binding region that specifically recognizes CLDN18.2 protein, a hinge region, and a transmembrane region, an intracellular co-stimulatory domain and an intracellular signaling domain, the extracellular binding region includes an antibody or an antigen-binding fragment thereof that specifically recognizes CLDN18.2, and the antibody or an antigen-binding fragment thereof includes SEQ ID NO: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 13, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region (VL) shown in SEQ ID NO: 13.
  • CAR chimeric antigen receptor
  • the HCDR1, HCDR2 and HCDR3 have the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively, and the LCDR1, LCDR2 and LCDR3 have the sequences of SEQ ID NO: 14, SEQ ID NO: 14 and SEQ ID NO: 12 respectively.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 13.
  • the antibody or antigen-binding fragment is in the form of a scFv having the structure of VH-linker-VL or VL-linker-VH.
  • the linker has the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.
  • the signal peptide is a CD8 signal peptide.
  • the CD8 signal peptide has the amino acid sequence shown in SEQ ID NO: 29.
  • the hinge region is selected from the CD28 hinge region or the CD8 alpha hinge region. In some embodiments, the hinge region includes the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the transmembrane region is selected from the group consisting of CD4, CD8 ⁇ , CD28, PD1 and/or 4-1BB transmembrane region. In some embodiments, the transmembrane region has the amino acid sequence shown in SEQ ID NO: 22 or SEQ ID NO: 23.
  • the intracellular costimulatory signaling domain is selected from the group consisting of CD28 or 4-1BB intracellular costimulatory domains.
  • the CD28 intracellular costimulatory signaling domain has the amino acid sequence shown in SEQ ID NO: 24.
  • the 4-1BB intracellular costimulatory domain has the amino acid sequence shown in SEQ ID NO: 25.
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain.
  • the CD3 ⁇ intracellular signaling domain has the sequence set forth in SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
  • the chimeric antigen receptor includes sequentially linked CD8 signal peptide, extracellular binding domain, CD28 or CD8 ⁇ hinge region, CD28 or CD8 ⁇ transmembrane domain, CD28 or 4-1BB intracellular costimulatory binding domain and the CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID
  • the present application provides an isolated nucleic acid molecule encoding the chimeric antigen receptor described in the first aspect.
  • the nucleic acid molecule is DNA or RNA.
  • the RNA is mRNA.
  • the nucleic acid molecule is operably linked to a regulatory sequence that is recognized by a host cell transformed with the vector.
  • the present application provides an expression vector comprising the nucleic acid molecule described in the second aspect.
  • the present application provides cells that express the chimeric antigen receptor described in the first aspect, or contain the nucleic acid molecule described in the second aspect, or contain the expression vector described in the third aspect. .
  • the cells are immune cells.
  • the immune cells are selected from immune cells cultured and differentiated from T lymphocytes, NK cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.
  • the present application provides a method for preparing chimeric antigen receptor modified immune cells, the method comprising delivering the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect to the immune cells to be modified step.
  • the method of delivery includes viral transfection or delivery to the immune cells via cationic liposomes (eg, LNP).
  • cationic liposomes eg, LNP
  • the method further includes isolating and activating the immune cells prior to delivery.
  • the method further includes culturing the immune cells introduced into the nucleic acid molecule or expression vector.
  • the immune cells are selected from immune cells cultured and differentiated from T lymphocytes, NK cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.
  • the present application provides a pharmaceutical composition, said pharmaceutical composition comprising the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, The expression vector, the cell described in the fourth aspect, or the product prepared by the method described in the fifth aspect, and a pharmaceutically acceptable carrier.
  • compositions are used to prevent and/or treat cancer.
  • the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, primary peritoneal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, glioma, head and neck cancer , kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, prostate cancer, rectal cancer, skin cancer, testicular cancer, Tracheal and vulvar cancer.
  • the cancers are those in which CLDN18.2 is expressed on the surface of the cancer cells.
  • the cancer is a cancer that highly expresses CLDN18.2 (CLDN18.2+).
  • a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 60% of the cancer cells in the cancer cell population express CLDN18.2.
  • a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 70% of the cancer cells in the cancer cell population express CLDN18.2.
  • a cancer that highly expresses CLDN18.2 means that at least 80% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 90% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 95% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 98% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) means that at least 99% of the cancer cells in the cancer cell population express CLDN18.2.
  • the present application provides the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the The cells, the product prepared by the method described in the fifth aspect, or the use of the pharmaceutical composition described in the sixth aspect in preparing a composition for preventing and/or treating cancer.
  • the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, primary peritoneal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, glioma, head and neck cancer , kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, prostate cancer, rectal cancer, skin cancer, testicular cancer, Tracheal and vulvar cancer.
  • gastric cancer gastric cancer
  • esophageal cancer pancreatic cancer
  • ovarian cancer primary peritoneal cancer
  • bladder cancer bone cancer
  • brain cancer breast cancer
  • cervical cancer colon cancer
  • glioma head and neck cancer
  • kidney cancer leukemia, acute myeloid leukemia (AML), multiple myeloma
  • liver cancer lung cancer
  • lymphoma
  • the present application provides a method for preventing and/or treating cancer, comprising administering to a patient in need an effective amount of the chimeric antigen receptor described in the first aspect, the second aspect
  • the cancer is selected from gastric cancer, esophageal cancer, pancreatic cancer, ovarian cancer, primary Peritoneal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, glioma, head and neck cancer, kidney cancer, leukemia, acute myeloid leukemia (AML), multiple myeloma, liver cancer, lung cancer , lymphoma, melanoma, mesothelioma, medulloblastoma, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.
  • gastric cancer gastric cancer
  • esophageal cancer pancreatic cancer
  • ovarian cancer primary Peritoneal cancer
  • bladder cancer bone cancer
  • brain cancer breast cancer
  • cervical cancer colon cancer
  • glioma head and neck cancer
  • kidney cancer leukemia
  • AML acute myeloid leukemia
  • multiple myeloma liver cancer
  • lung cancer lymphoma
  • a CHO-K1 stably transduced cell line expressing human CLDN18.1, a CHO-K1 stably transduced cell line expressing human CLDN18.2, and tumor cells stably expressing both human CLDN18.2 and bacterial luciferase were constructed.
  • NUGC4 gastric cancer cell line
  • Plasmid construction Synthesize polynucleotides encoding recombinant proteins, and add EcoRI and SalI restriction sites and corresponding vector homologous sequences to both ends.
  • the plasmid pCDH-CMV-MCS-EF1-Puro was digested using restriction endonucleases EcoRI (Thermo, Cat# FD0274) and SalI (Thermo, Cat# FD0644), and the linear plasmid was recovered and purified by agarose gel electrophoresis.
  • the polynucleotide synthesized in the above steps was connected to the linearized vector using recombinase 5 ⁇ In-FusionHD enzyme (TaKaRa, Cat#ST0344).
  • the reaction system was as follows: 2 ⁇ l polynucleotide fragment (50 ng/ ⁇ l ), 1 ⁇ l linearized plasmid (50ng/ ⁇ l), 2 ⁇ l 5 ⁇ HD In-Fusion enzyme, 5 ⁇ l ddH 2 O. Use a pipette to mix gently, centrifuge briefly, and place at 50°C for reaction for 15 minutes. Add 10 ⁇ l of the recombinant reaction product to 100 ⁇ l of bacterial competent cells, place on ice for 5 minutes, spread the transformed bacterial solution evenly on an LB plate containing 100 ⁇ g/ml ampicillin, and incubate upside down in a constant temperature incubator for 12-16 hours. Randomly pick 3-5 clones from each plate for sequencing and identification.
  • Lentivirus preparation 293T cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat#GNHu17) were inoculated into 100mm culture dishes, and DMEM medium (Gibco, Cat) added with 10% FBS (Gibco, Cat#10099141) was used. #10566016) for cultivation.
  • CHO-K1 cells Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat#SCSP-507) or NUGC-4 cells (Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP74135) in a 10cm culture dish. 5 ⁇ 10 5 cells per dish.
  • 10 ⁇ g/ml polybrene Shanghai Yisheng Biotechnology Co., Ltd., Cat#40804ES76
  • Construct cell lines that stably express CLDN18 named CHOK1-18.1, CHOK1-18.2 and NUGC4-luc cells respectively.
  • the amino acid sequence of human CLDN18.1 used is shown in SEQ ID NO:1
  • the amino acid sequence of CLDN18.2 used is shown in SEQ ID NO. :3.
  • the amino acid sequence of bacterial luciferase used is shown in SEQ ID NO:5.
  • the corresponding amino acid sequences are shown in Table 1.
  • the amino acid sequence of detection antibody Tab1 is shown in SEQ ID NO: 6, the amino acid sequence of Tab2 is shown in SEQ ID NO: 7, and the amino acid sequence of Tab3 is shown in SEQ ID NO: 8 (refer to patent WO 2018/006882) (Tab1, 2, 3 Corresponding to the single-chain antibodies targeting CLDN18.2 in CAR1, CAR2 and CAR3 in Examples 3-6 below respectively, obtained by conventional preparation methods). Centrifuge and wash twice with FACS buffer (PBS buffer with 10% FBS added), and add 100 ⁇ l of fluorescently labeled secondary antibody Alexa to each well.
  • FACS buffer PBS buffer with 10% FBS added
  • the single chain antibody (Single Chain Antibody Fragment, referred to as scFv) used in this example is an antibody targeting Claudin 18.2.
  • the lentiviral vector pCDH-CMV -MCS-EF1-Puro was used as a template to construct lentiviral plasmids CAR18.2-1, CAR18.2-2, CAR18.2-3, Claudin 18.2-1, Claudin expressing second-generation chimeric antigen receptors (CAR). 18.2-2, Claudin 18.2-3 and Claudin 18.2-4.
  • the corresponding amino acid sequences are shown in Table 2 (the CDR sequences are divided according to Kabat), and the sequences corresponding to each plasmid expression element are shown in Table 3.
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VL1, linker, VH1, and CD8 ⁇ hinge. region, CD8 ⁇ transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 32;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VH1, linker, VL1, and CD8 ⁇ hinge. region, CD8 ⁇ transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 33;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VL2, linker, VH2, and CD8 ⁇ hinge. region, CD8 ⁇ transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 34;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VH1, linker, VL1, CD8 ⁇ hinge region, CD28 transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 35;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VH1, linker, VL1, CD8 ⁇ hinge region, CD8 transmembrane domain, 4-1BB intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 36;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VL2, linker, VH2, CD8 ⁇ hinge region, CD28 transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 37;
  • the CAR from the 5′ end to the 3′ end includes: CD8 ⁇ signal peptide, VH1, linker, VL1, The CD28 hinge region, CD28 transmembrane domain, CD28 intracellular costimulatory domain, and CD3 ⁇ signal transduction domain, the amino acid sequence is shown in SEQ ID NO: 38.
  • Example 4 Construction of chimeric antigen receptor T cells (CAR-T cells) expressing CAR-1, CAR-2 and CAR-3
  • T cells expressing multiple second-generation chimeric antigen receptors were constructed and named respectively CAR-1, CAR-2 and CAR-3. Specific steps are as follows:
  • Example 3 Take the chimeric antigen receptor lentiviral plasmids CAR18.2-1, CAR18.2-2, CAR18.2-3 and packaging plasmids (pRRE, pRSV-Rev and pVSV-G) prepared in Example 3, according to the Example Prepare lentivirus by the method described in 1. Collect the 293T cell supernatant (virus) after 48 hours, filter it with a 0.45 ⁇ m filter membrane, and concentrate it for later use.
  • CAR-T cells chimeric antigen receptor T cells
  • T cell proliferation Use Stemcell Easy Sep Kit (Stemcell, Cat#19055) according to the instructions to isolate T cells from peripheral blood mononuclear cells (PBMC) of different healthy donors.
  • PBMC peripheral blood mononuclear cells
  • the isolated T cells were cultured in culture dishes pre-embedded with 1 ⁇ g/ml CD3/CD28 antibody (Thermo, Cat#11131D).
  • the medium components are X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100U/ml penicillin-streptomycin (Gibco, Cat#15140-122) , 200IU/ml human IL2 factor (Beijing Shuanglu, Cat#S19991007).
  • Cell counting was performed twice a week, and when the cell density reached 2.5 ⁇ 10 6 cells/ml, the cells were subcultured and expanded.
  • CAR-T cell preparation Take the above-mentioned T cells in good condition and inoculate them into a 12-well cell culture plate pre-coated with 5 ⁇ g/ml recombinant human fibrin (Takara, Cat#T100B) at a rate of 1 ⁇ 10 6 cells/well. Add 50 ⁇ l concentrated lentivirus and 10 ⁇ g/ml Polybrene (Sigma-Aldrich, Cat#TR-1003) and centrifuged at 1000 g for 1 h at 4°C. The centrifuged culture plate was moved to a constant temperature incubator (37°C, 5% CO 2 ) and cultured. The cells were counted twice a week. When the cell density reached 2.5 ⁇ 10 6 cells/ml, they were subcultured and expanded.
  • Flow cytometry experiment was used to detect the expression and infection efficiency of CAR: wash the cells twice with PBS buffer, count and dilute the cells with PBS buffer to 2 ⁇ 10 6 cells/ml, and add 50 ⁇ l Fc receptor blocker ( BioLegend, Cat#422302) was incubated at room temperature for 10 minutes, and then 100 ⁇ l per well was added to the 96-well FACS reaction plate. Add 100 ⁇ l of CLDN18.2 protein containing His-tagged protein (Acro, Cat# CL2-H5546) at a concentration of 2 ⁇ g/ ⁇ l, and incubate on ice for 20 minutes.
  • FACS Flow cytometry experiment
  • Example 5 In vitro killing activity experiment of CAR-T cells CAR-1, CAR-2 and CAR-3
  • ELISA Enzyme-linked immunosorbent assay
  • the killing efficiency of CAR-1, CAR-2 and CAR-3 in each round was 100% or close to 100%; as shown in Figure 5B, the proliferation ability of CAR-2 cells after the third round of killing experiment significantly higher than the other two groups of CAR-T cells; Figure 5C shows that after the fourth round of killing experiments, CAR-2 cells The expression level of PD-1 on the cell surface was significantly lower than that of the other two groups of CAR-T cells.
  • Example 6 Anti-tumor efficacy test of cells expressing chimeric antigen receptors (CAR-T cells) in the NUGC4-luc mouse tumor model
  • NUGC4-luc cells in the logarithmic growth phase and in good growth status were collected, and 5 ⁇ 10 6 cells were subcutaneously inoculated into NPG mice (combined immunodeficient mice).
  • V (mm 3 ) 1/2 ⁇ (a ⁇ b 2 )
  • CAR-T cells (1 ⁇ 10 7 cells/animal) were injected into the tail vein with an injection volume of 200 ⁇ l/animal. The CAR-T cell injection diary was Day 0.
  • TGI (%) (tumor volume of mice in the PBS group - tumor volume of mice in the experimental group) /PBS group mouse tumor volume ⁇ 100%.
  • the in vitro and in vivo experimental activity verification according to Examples 4 to 6 shows that the in vitro and in vivo activity of CAR-2 is significantly better than that of CAR-1 and CAR-3.
  • the connection sequence (VH-VL) of the CAR-2 antibody plays a major advantageous role in activity verification, so the next step will be Antibodies with VH-VL connection sequences are mainly selected to construct the main CAR molecules.
  • Example 7 Construction of T cells expressing chimeric antigen receptors (CAR-T cells) CLDN18.2-1, CLDN18.2-2, CLDN18.2-3 and CLDN18.2-4
  • Claudin 18.2-1, Claudin 18.2-2, Claudin 18.2-3 and Claudin 18.2-4 prepared in Example 3 were used to construct T cells expressing second-generation chimeric antigen receptors, which were named CLDN18.2-1 and CLDN18 respectively. 2-2, CLDN18.2-3 and CLDN18.2-4. Specific steps are as follows:
  • CAR-T cells CLDN18.2-1, CLDN18.2-2, CLDN18.2-3 and CLDN18.2-4 were constructed, and flow cytometry (FACS) was used to detect the relationship between CAR expression and Infection efficiency: Wash the cells twice with PBS buffer. After counting, dilute the cells with PBS buffer to 2 ⁇ 10 6 cells/ml. Add 50 ⁇ l Fc receptor blocker (BioLegend, Cat#422302) and incubate at room temperature for 10 min, then press Add 100 ⁇ l per well to a 96-well FACS reaction plate.
  • Fc receptor blocker BioLegend, Cat#422302
  • Figure 7A shows that compared with uninfected T cells, the proliferation ability of CAR-T cells is not affected;
  • Figure 7B shows that the positive rates of CAR transfection are 60%-80%, and CLDN18.2-1 cells have the highest positive rate.
  • Example 8 In vitro killing activity experiment of T cells expressing chimeric antigen receptor (CAR-T) CLDN18.2-1, CLDN18.2-2, CLDN18.2-3 and CLDN18.2-4
  • CAR-T chimeric antigen receptor
  • FIG. 8A-8B Collect the cells in the well plate for tumor cell killing experiments. Specifically, add the firefly luciferase substrate D-Luciferin, and use a multifunctional microplate reader to read the bioluminescence value. The first round of killing efficiency is higher than 50%, and the second round of killing experiment is carried out.
  • Central Memory T cells are cells with long-term memory produced by naive T cells (Naive T Cells) after antigen activation, and can return to lymph nodes to receive antigen restimulation. T cells. Activated TCM cells can continue to produce a large number of cloned effector memory T cells (Effective Memory T Cell, TEM, CD62L-CD45RO+) cells carrying alloantigens under re-stimulation by antigens. TCM cells can pass through lymphatic shielding and return to lymph nodes while being activated by antigens.
  • Stem cell-like memory T cell (TSCM, CD62L+CD45RO-) is an important component of memory T cells and has stem cell-like self-renewal, multi-differentiation potential and immune reconstitution properties. Different from central memory T cell (TCM) or effector memory T cell (TEM) subsets, TSCM has a naive T cell phenotype and is less differentiated. Effector T cells (TE, CD62L-CD45RO-) are cells that proliferate and differentiate after T cells receive antigen stimulation; effector T cells describe a group of cells that actively respond to stimuli (such as Co- stimulation), including regulatory T cells, helper T cells, and cytotoxic T cells. Effector T cells have the function of releasing lymphokines.
  • TCM central memory T cell
  • TEM effector memory T cell
  • the immune checkpoint molecule PD-1 (programmed cell death protein 1, programmed death receptor 1) has an important immunosuppressive function. It is up-regulated on the surface of T cells in tumor tissues and prevents T cell activation by transmitting inhibitory signals. Flow cytometry was used to detect the expression of TCM, TEM, TSCM and TE, and the expression of PD-1 on the surface of T cells.
  • Figure 9B shows that after the third round of killing experiments, there is no significant difference in the memory phenotype of CAR-T cells in each group;
  • Figure 9C shows that after the third round of killing experiments, the expression levels of TIM3 and LAG3 in each group's exhaustion phenotype Comparable;
  • Figure 9D shows that after the third round of killing experiments, there was no significant difference in the expression of PD-1 on the cell surface of each group.
  • Example 9 Anti-tumor efficacy test of cells expressing chimeric antigen receptors (CAR-T cells) in the NUGC4-luc mouse tumor model
  • NUGC4-luc cells in the logarithmic growth phase and in good growth status were collected and inoculated subcutaneously into NPG mice (combined immunodeficient mice).
  • the inoculation amounts were 7 ⁇ 10 6 and 5 ⁇ 10 6 cells respectively.
  • V the formula is the same as Example 6
  • CAR-T cells (1 ⁇ 10 7 cells/animal) were injected into the tail vein with an injection volume of 200 ⁇ l/animal.
  • the CAR-T cell injection diary was Day0.
  • the grouping of mice and the injection of CAR-T cells are shown in Table 5.
  • the calculation formula is the same as in Example 6.
  • FIGS 10A and 10B The results of mouse tumor volume detection are shown in Figures 10A and 10B.
  • D3 and D4 in the figures represent the 3rd and 4th days after injection of CAR-T cells, respectively.
  • the results showed that after injection through the tail vein, CLDN18.2-1 cells could completely inhibit the growth of tumors in mice in the G3 group, and the tumors eventually disappeared completely, showing good anti-tumor effects.
  • Example 10 Anti-tumor efficacy test of cells expressing chimeric antigen receptors (CAR-T cells) in the NUGC4-luc mouse tumor model
  • NUGC4-luc cells in the logarithmic growth phase and in good growth status were collected, and 5 ⁇ 10 6 cells were subcutaneously inoculated into NPG mice (combined immunodeficient mice).
  • V the formula is the same as Example 6
  • mice with a tumor volume of about 50 mm3 the random number principle for random grouping.
  • CAR-T cells (1 ⁇ 10 7 cells/animal) were injected into the tail vein with an injection volume of 200 ⁇ l/animal.
  • the CAR-T cell injection diary was Day 0.
  • the grouping of mice and the injection of CAR-T cells are shown in Table 6. Continuously observe and measure the changes in tumor volume and body weight of the mice, and detect the IFN- ⁇ content in the serum. The measurements are recorded twice a week.
  • mice in Figure 11A The results show that on the 45th day after injection of CAR-T cells, CLDN18.2-1 and CLDN18.2-3 can completely inhibit the growth of mouse tumors and the tumors completely disappear; CLDN18.2-4 was able to inhibit the tumor growth of 4 mice in the group until the tumors completely disappeared. CLDN18.2-2 did not show the efficacy of inhibiting tumor growth.
  • the mice in the group were euthanized on the 34th day because the tumor volume reached the euthanasia standard.
  • the PBS treatment group was treated on day 34 due to tumor volume reaching euthanasia criteria.
  • the IFN- ⁇ content in the serum of mice in each group reached the highest on the 5th day after the start of treatment, and then gradually decreased. Except for the PBS treatment group, the IFN- ⁇ expression of mice in other groups was still detected on the 42nd day.
  • Example 11 Anti-tumor efficacy test of cells expressing chimeric antigen receptors (CAR-T cells) in patient-derived tumor xenograft model (PDX)
  • mice and the injection of CAR-T cells are shown in Table 7. Continuously observe and measure the changes in tumor volume and body weight of the mice, and record the measurements twice a week.
  • mice in Figure 12A The results of mouse tumor volume detection are shown in Figure 12A.
  • the results show that on the 35th day after injection of CAR-T cells, CLDN18.2-1 can inhibit the growth of mouse tumors, and the tumors of 2 of them disappeared completely; CLDN18.
  • the tumors of 4 and 5 mice in the 2-3 and CLDN18.2-4 groups were inhibited respectively, and the tumor of 1 mouse in both groups disappeared completely.
  • mice in each treatment group experienced weight loss, and the weight of the PBS, CLDN18.2-1 and CLDN18.2-3 treatment groups dropped to the lowest point on the 7th day after injection of CAR-T cells.
  • mice in the CLDN18.2-4 treatment group fell on the 14th day
  • the weight of the animals dropped significantly.
  • the weight of 3 animals dropped by more than 20% and they were euthanized.
  • the weight of the remaining mice gradually recovered.
  • the mouse tumor volume was measured on days 21 and 35, and the tumor inhibition rate TGI% was calculated (the formula is the same as in Example 6).
  • the CLDN18.2-1 treatment group and CLDN18.2-4 treatment group had significant tumor inhibition effects, with the tumor inhibition rates reaching 89% and 83% respectively.
  • the tumor inhibition rate of the CLDN18.2-3 treatment group It was 27%; on the 35th day, the tumor inhibition rate of the CLDN18.2-1 treatment group reached 90%, the tumor inhibition rate of the CLDN18.2-3 treatment group reached 73%, and the tumor inhibition rate of the CLDN18.2-4 treatment group reached 83%.
  • Example 12 Anti-tumor efficacy test of different doses of chimeric antigen receptor cells (CAR-T cells) in the NUGC4-luc mouse tumor model
  • NUGC4 cells that were in the logarithmic growth phase and grew well were collected, and 6 ⁇ 10 6 cells were subcutaneously inoculated into the right anterior scapula of NPG mice (combined immunodeficient mice).
  • V the formula is the same as Example 6
  • mice with tumor volumes of about 60 mm3 according to the random number principle for random grouping.
  • CAR-T cells (6 ⁇ 10 6 cells/animal, 3 ⁇ 10 6 cells/animal or 1.5 ⁇ 10 6 cells/animal) were injected through the tail vein for treatment. The injection volume was 200ul.
  • the CAR-T cell injection diary is day 0 (i.e. Day0).
  • the grouping of mice and the injection of CAR-T cells are shown in Table 8. Continuously observe and measure the changes in tumor volume and body weight of the mice, and record the measurements twice a week.
  • the results of mouse tumor volume detection are shown in Figure 13A.
  • the results show that on the 21st day after the injection of CAR-T cells, 6 ⁇ 10 6 CLDN18.2-4 cells can significantly inhibit the growth of mouse tumors, including 1 animal. The tumors completely disappeared; except for the CLDN18.2-4 (1.5 ⁇ 10 6 /mouse) treatment group, the other five groups also showed a tendency to inhibit tumor growth in mice after treatment.
  • the weight of the CLDN18.2-4 (6 ⁇ 10 6 /mouse) treatment group dropped to the lowest point on the 11th day, and then rebounded rapidly, while the weight of mice in the other groups showed a slow growth trend (as shown in Figure 13B).
  • the mouse tumor volume was measured and the tumor inhibition rate was calculated.
  • the calculation formula was the tumor inhibition rate TGI% (the formula is the same as Example 6).
  • the tumor inhibition rate of the CLDN18.2-4 (6 ⁇ 10 6 /animal) treatment group was 84%, showing good inhibitory effect on tumor growth.
  • the tumor inhibition rate of the CLDN18.2-1 (3 ⁇ 10 6 /bird), CLDN18.2-1 (6 ⁇ 10 6 /bird) and CLDN18.2-4 (3 ⁇ 10 6 /bird) treatment groups also exceeded 20% .
  • Example 13 Anti-tumor efficacy test of different doses of cells expressing chimeric antigen receptors (CAR-T cells) in patient-derived tumor xenograft models
  • the tumor tissue of tumor-bearing mice in the GA0006 xenograft model was cut into tumor pieces with a diameter of 2-3 mm, and then inoculated subcutaneously in the right front scapula of NCG mice (combined immunodeficient mice).
  • the mouse tumor volume V the formula is the same as in Example 6
  • the day of grouping was recorded as day 0 (i.e. Day 0), and CAR-T cells (5 ⁇ 10 6 /animal or 1 ⁇ 10 7 /animal) were injected through the tail vein for treatment, and the injection volume was 200 ⁇ l/animal.
  • mice were divided into 5 groups, with 6 mice in each group.
  • the grouping of mice and the injection of CAR-T cells are shown in Table 9. Continuously observe and measure the changes in tumor volume and body weight of the mice, and record the measurements twice a week.
  • the results of mouse tumor volume detection are shown in Figure 14A.
  • the results show that on the 53rd day after the injection of CAR-T cells, 1 ⁇ 10 7 CLDN18.2-1 cells were able to inhibit the tumor growth of 5 mice in the group, and Can inhibit the rate of tumor recurrence; 1 ⁇ 10 7 CLDN18.2-4 cells can inhibit the tumor growth of 4 mice in the group, and the tumors of 2 animals disappeared completely; CLDN18.2-1 (5 ⁇ 10 6 /mouse) and CLDN18.2-4 (5 ⁇ 10 6 /mouse) were able to inhibit the tumor growth of 1 or 2 mice in the group respectively.
  • mice in each treatment group showed weight loss: mice in the PBS, CLDN18.2-1 (5 ⁇ 10 6 /mouse) and CLDN18.2-4 (5 ⁇ 10 6 /mouse) treatment groups showed weight loss after The fluctuating trend of rising and then continuing to fall, because the average tumor volume exceeded 2000mm 3 in the late treatment period, euthanasia was performed on the 25th or 32nd day respectively; while CLDN18.2-1 (1 ⁇ 10 7 /bird) and CLDN18.2-4 (1 ⁇ 10 7 /mouse) The weight of the mice did not change significantly and showed a slow growth trend (as shown in Figure 14B).
  • the tumor inhibition rate of the CLDN18.2-4 (1 ⁇ 10 7 /bird) treatment group reached 89%, and the tumor inhibition rate of the CLDN18.2-1 (1 ⁇ 10 7 /bird) treatment group reached 85%.
  • the tumor inhibition rate of the CLDN18.2-4 (5 ⁇ 10 6 /bird) treatment group reached 46%, and the tumor inhibition rate of the CLDN18.2-1 (5 ⁇ 10 6 /bird) group reached 26%.
  • Each treatment group can well inhibit tumor growth in mice, indicating that CLDN18.2-1 and CLDN18.2-4 have good anti-tumor activity at medium and high doses.

Abstract

本申请涉及靶向CLDN18.2的嵌合抗原受体及其应用，并提供了靶向CLDN18.2的嵌合抗原受体以及相应的核酸分子、载体、免疫细胞、制备方法及其产品、药物组合物、治疗用途、制药用途和肿瘤或癌症治疗方法。

Description

靶向CLDN18.2的嵌合抗原T细胞受体及其应用
相关申请的引用
本申请要求2022年5月6日提交的中国专利申请202210497885.5号的优先权，通过援引加入的方式将该申请的内容全部并入本文，用于所有目的。
技术领域
本申请大体涉及生物工程和细胞治疗领域，具体而言，涉及靶向CLDN18.2的T淋巴细胞及其制备方法和应用。
背景技术
癌症(Cancer)，作为一种恶性肿瘤(Malignant tumor)，为由控制细胞分裂增殖机制失常而引起的疾病。癌症治疗方式的选择取决于肿瘤的位置、恶性程度、发展程度以及病人身体状态，新的治疗方法不断推陈出新。免疫疗法是利用人体内的免疫机制来对抗肿瘤细胞，其中癌症疫苗疗法和单克隆抗体疗法取得进展较多，而免疫细胞疗法是近几年最新发展的治疗技术。
密蛋白18(Claudin18，简称CLDN18)是细胞表面蛋白密蛋白家族(Claudin family)的成员，有两种剪接变体：密蛋白18.1(Claudin18.1，简称CLDN18.1)和密蛋白18.2(Claudin18.2，简称CLDN18.2)。CLDN18为4次跨膜蛋白，含有2个胞外域和位于细胞内的N端和C端。在CLDN18.1和CLDN18.2的蛋白一级结构序列中，仅N端信号肽至胞外域的某些位置的氨基酸残基上有所区别，尤其是在胞外域1(细胞外环1)上，仅有8个氨基酸不同。CLDN18.1的氨基酸序列(NP_057453.1，NCBI数据库)见SEQ ID NO：1，核苷酸序列(NM_016369.4，Genbank数据库)见SEQ ID NO：2。CLDN18.2的氨基酸序列(NP_001002026.1，NCBI数据库)见SEQ ID NO：3，核苷酸序列(NM_001002026.3，Genebank数据库)见SEQ ID NO：4。CLDN18.1和CLDN18.2分别在不同的组织中表达，其中，Claudin18.1主要表达在肺组织，而Claudin18.2则特异性表达于正常胃粘膜组织中，以及高度选择性、稳定地表达于特定肿瘤组织，参与胃癌、食管癌、胰腺癌等疾病中癌细胞的增殖分化和迁移，这使其成为潜在的抗肿瘤药物的有效分子靶点。
与传统的药物不同，嵌合抗原受体T细胞疗法(Chimeric Antigen Receptor T cell，简称CAR-T细胞)是一种细胞免疫疗法：在体外利用嵌合抗原受体(Chimeric Antigen Receptor，简称CAR)修饰从患者体内分离出的T细胞，使其能够识别特定的癌细胞，然后将改造后的细胞扩增并回输至患者体内，从而达到治疗肿瘤的效果。该疗法以患者自身的免疫细胞为治疗工具，直接用于对抗癌细胞，因而对免疫能力受损或下降的癌症患者具有潜在的功效。
因此，开发针对CLDN18.2的高效抗肿瘤的CAR-T细胞具有重要意义。
发明概述
在第一个方面，本申请提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含信号肽、 特异性识别CLDN18.2蛋白的胞外结合区、铰链区、跨膜区、胞内共刺激结构域和胞内信号传导结构域，所述胞外结合区包含特异性识别CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含SEQ ID NO：9所示重链可变区(VH)中包含的HCDR1、HCDR2和HCDR3，和SEQ ID NO：13所示轻链可变区(VL)中包含的LCDR1、LCDR2和LCDR3。
在第二个方面，本申请提供一种分离的核酸分子，所述分离的核酸分子编码第一个方面所述的嵌合抗原受体。
在第三个方面，本申请提供了一种包含第二个方面所述核酸分子的表达载体。
在第四个方面，本申请提供了表达上述第一个方面所述的嵌合抗原受体，或包含第二个方面所述的核酸分子，或包含第三个方面所述的表达载体的细胞。
在第五个方面，本申请提供了一种制备嵌合抗原受体修饰的免疫细胞的方法，所述方法包括将第二个方面所述的核酸分子或第三个方面所述的表达载体递送至待修饰的免疫细胞的步骤。
在第六个方面，本申请提供了一种药物组合物，所述药物组合物包含第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞，或通过第五个方面所述的方法制备的产品，以及药学可接受的载体。
在第七个方面，本申请提供了上述第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞、第五个方面所述的方法制备的产品，或第六个方面所述的药物组合物在制备预防和/或治疗癌症的组合物中的用途。
在第八个方面，本申请提供了一种预防和/或治疗癌症的方法，包含向有此需要的患者施用有效量的上述第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞、第五个方面所述的方法制备的产品，或第六个方面所述的药物组合物。
附图简要说明
图1A-1D显示了抗体Tab1、Tab2和Tab3的细胞亲和力检测，其中图1A至图1D分别显示三株抗体与表达CLDN18.2的CHO-K1细胞、表达CLDN18.1的CHO-K1细胞、肿瘤细胞NUGC4与CHO-K1细胞的亲和力检测结果。
图2显示了本申请各个示例性CAR-T质粒结构示意图。
图3A-3C显示了三种CAR-T细胞的CAR表达情况以及对高表达CLDN18.2的CHO-K1细胞系的随时间杀伤作用。图3A为CAR-T细胞表面CAR表达情况的FACS检测结果，图3B和3C分别是效靶比1：1和1：5时，CAR-T细胞对靶细胞的实时杀伤结果。
图4A和图4B分别显示了三种CAR-T细胞杀伤高表达CLDN18.2的CHO-K1时的TNF-α(图4A)和IFN-γ(图4B)的分泌水平。CAR-T细胞与CHOK1-18.2细胞共孵育72h(效靶比1：5)。
图5A-5B显示了三种CAR-T细胞对NUGC4-luc细胞的多轮连续杀伤作用评价结果。CAR-T细胞与NUGC4-luc细胞共孵育72h(效靶比1：1)后，将T细胞重悬计数，再与相同数量新鲜NUGC4-luc细胞共孵育进行下一轮杀伤实验。以此类推，共完成4轮杀伤。图5A 为多轮杀伤评价结果。图5B为多轮CAR-T细胞增殖曲线。图5C为第四轮杀伤实验过程中PD-1的表达水平。
图6A-6C显示了三种CAR-T细胞在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验结果。图6A表示各实验组小鼠在注射CAR-T细胞53天内的肿瘤生长曲线；图6B表示各实验组小鼠在注射CAR-T细胞53天内的体重变化率；6C表示各实验组小鼠在注射CAR-T细胞后的肿瘤生长抑制率。
图7A显示了来自四种CLDN18.2CAR-T细胞体外扩增的生长曲线，图7B为FACS检测的四种CLDN18.2CAR-T细胞的CAR转染效率。
图8A和图8B分别显示了四种CLDN18.2CAR-T细胞杀伤高表达CLDN18.2的NUGC4-luc时的细胞因子TNF-α(图8A)和IFN-γ(图8B)分泌水平。CAR-T细胞与NUGC4-luc细胞共孵育72h(效靶比1：5)。
图9A显示了四种CLDN18.2CAR-T细胞对NUGC4-luc细胞的多轮连续杀伤结果；图9B为第三轮杀伤实验后四种CAR-T细胞记忆表型的表达水平；图9C为第三轮杀伤实验后四种CAR-T细胞耗竭表型TIM3和LAG3的表达水平；图9D为第三轮杀伤实验后四种CAR-T细胞耗竭表型PD-1的表达水平。
图10显示了两种CAR-T细胞在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验结果。图10A为CAR-2治疗组的小鼠体内肿瘤生长抑制率，图10B为CLDN18.2-1治疗组的小鼠体内肿瘤生长抑制率。
图11A-11D显示了高剂量的四种CLDN18.2CAR-T细胞在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验结果。图11A表示各实验组小鼠在注射CAR-T细胞45天内的肿瘤生长曲线；图11B表示各实验组小鼠在注射CAR-T细胞45天内的体重变化率；图11C表示各实验组在注射CAR-T细胞第34天肿瘤生长抑制率；图11D表示各实验组小鼠在注射CAR-T细胞后血清内IFN-γ含量的变化。
图12A-12C显示了高剂量的三种CLDN18.2CAR-T细胞在病人来源肿瘤异种移植模型GA0006中的抗肿瘤药效试验结果。图12A表示各实验组小鼠在注射CAR-T细胞35天内的肿瘤生长曲线；图12B表示各实验组小鼠在注射CAR-T细胞35天内的体重变化率；图12C表示各实验组在注射CAR-T细胞第21天和35天所计算的肿瘤生长抑制率。
图13A-13C显示了不同剂量的三种CLDN18.2CAR-T细胞在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验结果。图13A表示各实验组小鼠在注射CAR-T细胞21天内的肿瘤生长曲线；图13B表示各实验组小鼠在注射CAR-T细胞21天内的体重变化率；图13C表示各实验组在注射CAR-T细胞第21天所计算的肿瘤生长抑制率。
图14显示了高、中两种剂量的两种CLDN18.2CAR-T细胞在病人来源肿瘤异种移植模型GA0006中的抗肿瘤药效试验结果。图14A表示各实验组小鼠在注射CAR-T细胞53天内的肿瘤生长曲线；图14B表示各实验组小鼠在注射CAR-T细胞53天内的体重变化率；图14C表示各实验组在注射CAR-T细胞25天所计算的肿瘤生长抑制率。
发明详细描述
术语定义和说明
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
尽管本申请的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
如本文所用，术语“CLDN18”指密蛋白-18，并包括细胞天然表达的或转染了CLDN18基因的细胞所表达的任何CLDN18的变体(包括CLDN18.1和CLDN18.2)、构象、同工型(isoform)和种间同源物(specieshomologs)。优选地，“CLDN18”指人CLDN18。
如本文所用，术语“CLDN18.1”或Claudin18.1包括细胞天然表达的或转染了CLDN18.1基因的细胞所表达的任何人CLDN18.1的翻译后修饰变体、同工型和种间同源物。
如本文所用，术语“CLDN18.2”或“Claudin18.2”包括细胞天然表达的或转染了CLDN18.2基因的细胞所表达的任何人CLDN18.2的翻译后修饰变体、同工型和种间同源物。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316，1983；其内容援引加入本文)。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、scFv和VHH。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头包括GSTSGSGKPGSGEGSTKG和由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。
在本文中，“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
术语“人源化抗体”意指将源自另一哺乳动物物种，诸如小鼠种系的CDR序列嫁接到人框架序列上获得的抗体。为了保留结合亲和力，可以修饰骨架(称为FR)区段的一些残基。通过本领域技术人员已知的技术可以制备根据本申请的人源化抗体或其片段。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构，每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统，使用的工具网站包括但不限于abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi))。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
如本文所用，“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工免疫效应细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构 域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。嵌合抗原受体的胞外结构域还可以包括信号肽和/或铰链区。嵌合抗原受体的胞内结构域还可以包括共刺激结构域。
如本文所用，嵌合抗原受体语境下的术语“胞外结合区”是指位于细胞膜之外且能够结合到抗原、靶标或配体的蛋白质的部分。
如本文所用，嵌合抗原受体语境下的术语“铰链区”是指连接蛋白质的两个相邻结构域(例如，胞外结构域与跨膜结构域)的蛋白质的部分。具体地，铰链区用来为抗原结合区提供更大的灵活性和可及性。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。
如本文所用，嵌合抗原受体语境下的术语“信号肽”是指在初生蛋白的氨基端(N端)处的前导序列，其将初生蛋白共翻译或翻译后导向内质网且随后进行表面表达。信号肽的非限制性实例为CD8α信号肽。
如本文所用，嵌合抗原受体语境下的术语“跨膜区”(简称TM)可以与“跨膜结构域”互换使用，指的是锚定在细胞膜内具有热力学稳定的蛋白质结构区域。跨膜区可以从天然蛋白质中获得，可选自CD8α分子或CD28分子。
如本文所用，嵌合抗原受体语境下的术语“胞内信号结构域”指的是能够传导细胞效应功能信号并指导细胞执行特定功能的蛋白质结构区域。胞内信号传导结构域负责在抗原结合结构域结合抗原以后的细胞内初级信号传递，从而导致免疫细胞和免疫反应的活化。换言之，胞内信号传导结构域负责活化其中表达CAR的免疫细胞的正常的效应功能的至少一种。示例性的胞内信号传导结构域包括CD3ζ。
如本文所用，嵌合抗原受体语境下的术语“共刺激信号”是指与诸如TCR/CD3连接之类的主要信号结合导致T细胞增殖和/或关键分子上调或下调的信号。
如本文所用，嵌合抗原受体语境下的术语“共刺激信号结合域”包括在抗原呈递细胞(例如，aAPC，树突状细胞，B细胞等)上的分子，其特异性结合T细胞上的同源共刺激分子，从而提供一种信号，该信号除了通过例如TCR/CD3复合物与载有肽的MHC分子结合提供的主要信号外，还介导T细胞反应，包括但不限于增殖、激活、分化等。共刺激信号结合域可包括但不限于CD7、B7-1(CD80)、B7-2(CD86)、PD-L1、PD-L2、4-1BBL、OX40L、诱导型共刺激配体(ICOS-L)、细胞间粘附分子(ICAM)、CD30L、CD40、CD70、CD83、HLA-G、MICA、MICB、HVEM、淋巴毒素β受体、3/TR6、ILT3、ILT4、结合Toll配体受体和特异性结合B7-H3的配体的激动剂或抗体。共刺激信号结合域还可以包括与T细胞上存在的共刺激分子特异性结合的抗体，例如但不限于，CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3，以及与CD83特异性结合的配体。
如本文所用的术语“免疫细胞”、“免疫效应细胞”或“效应细胞”可以指参与免疫应答例如促进免疫效应子应答的细胞。免疫效应细胞的例子包括T细胞，例如α/βT细胞和γ/δT细胞，B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和髓系来源的吞噬细胞。
如本文所用，“慢病毒”是指逆转录病毒科的属。慢病毒在逆转录病毒中是独特的，能 够感染非分裂细胞。它们可以将大量遗传信息传递到宿主细胞的DNA中，因此它们是基因传递载体中最有效的方法之一。HIV、SIV和FIV都是慢病毒的例子。源自慢病毒的载体提供了在体内实现显着水平的基因转移的手段。
如本文所用，“载体”，通常指“表达载体”，是包含分离的核酸并且可以用于将分离的核酸递送至细胞内部的物质组成。许多载体是本领域已知的，包括但不限于线性多核苷酸，与离子或两亲性化合物相关的多核苷酸，质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语还应解释为包括有助于核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物，脂质体等。病毒载体的实例包括但不限于腺病毒载体，腺相关病毒载体，逆转录病毒载体等。
如本文所用，“特异性识别”是指识别特异性抗原但基本上不识别或结合样品中其他分子的抗体。例如，与一种物种的抗原特异性结合的抗体也可以与一种或多种物种的抗原结合。但是，这种种间反应性本身并不改变抗体的特异性。在另一个实例中，特异性结合抗原的抗体也可以结合抗原的不同等位基因形式。但是，这种交叉反应性本身并不改变抗体的特异性。在一些情况下，术语“特异性识别”或“特异性结合”可以用于指抗体，蛋白质或肽与第二化学物种的相互作用，以表示该相互作用取决于特定结构的存在。化学物种上的特定结构(例如，抗原决定簇或表位)；例如，抗体识别并结合特定的蛋白质结构，而不是一般的蛋白质。如果抗体对表位“A”具有特异性，则在包含标记“A”和抗体的反应中存在包含表位A(或游离，未标记A)的分子将减少与抗体结合的标记A的量。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
如本文所用，术语“治疗”是指外科手术或药物处理，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有 益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
如本文所用，术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文使用的术语“药物组合物”其表示组合在一起以实现某种特定目的的至少一种药物以及任选地药学可接受的载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本申请的目的。例如，所述药物组合物中所含的成分(例如根据本申请的CAR-T细胞)可以以整体施用于个体，或者分开施用于个体。当所述药物组合物中所含的成分分开地施用于个体时，所述成分可以同时或依次施用于个体。根据本申请的药物组合物可以包括细胞培养的常规组分，以保持CAR-T细胞的活性。药学可接受的载体还可以包括水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。根据本申请的药物组合物或者药物制剂可通过任何适宜的途径施用，例如静脉内施用、皮内、皮下、肌内注射等。根据本申请的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
本申请的发明人经过大量研发和测试，开发出新的靶向CLDN18.2的嵌合抗原受体及其应用，并提供了靶向CLDN18.2的嵌合抗原受体以及相应的核酸分子、载体、免疫效应细胞、制备方法及其产品、药物组合物、治疗用途、制药用途和肿瘤或癌症治疗方法。本申请的各项发明实现了以下至少一种有益效果：(1)具有较高的CAR表达阳性率或细胞增殖倍数；(2)针对特定的抗原表位，具有高度的特异性；(3)具有优异的肿瘤细胞杀伤与抑制能力，尤其是在中高剂量时，显示更优异的肿瘤杀伤作用；(4)副作用较低，特异性强，更加安全。
在第一个方面，本申请提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含信号肽、特异性识别CLDN18.2蛋白的胞外结合区、铰链区、跨膜区、胞内共刺激结构域和胞内信号传导结构域，所述胞外结合区包含特异性识别CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含SEQ ID NO：9所示重链可变区(VH)中包含的HCDR1、HCDR2和HCDR3，和SEQ ID NO：13所示轻链可变区(VL)中包含的LCDR1、LCDR2和LCDR3。
在一些实施方式中，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示序列，LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示序列。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：9所示的重链可变区和SEQ ID NO:13所示的轻链可变区。
在一些实施方式中，所述抗体或抗原结合片段为scFv的形式，所述scFv具有VH-连接子-VL或VL-连接子-VH的结构。在一些实施方式中，所述连接子具有SEQ ID NO：17或SEQ ID NO：18所示的序列。
在一些实施方式中，信号肽为CD8信号肽。在一些实施方式中，CD8信号肽具有SEQ ID NO：29所示的氨基酸序列。
在一些实施方式中，铰链区选自CD28铰链区或CD8α铰链区。在一些实施方式中，铰链区包含SEQ ID NO：19、SEQ ID NO：20或SEQ ID NO：21所示的氨基酸序列。
在一些实施方式中，跨膜区选自CD4、CD8α、CD28、PD1和/或4-1BB跨膜区。在一些实施方式中，跨膜区具有SEQ ID NO：22或SEQ ID NO：23所示的氨基酸序列。
在一些实施方式中，胞内共刺激信号结构域选自CD28或4-1BB胞内共刺激结构域。在一些实施方式中，CD28胞内共刺激信号结构域具有如SEQ ID NO：24所示的氨基酸序列。在一些实施方式中，4-1BB胞内共刺激结构域具有如SEQ ID NO：25所示的氨基酸序列。
在一些实施方式中，胞内信号传导结构域为CD3ζ胞内信号传导结构域。在一些实施方式中，CD3ζ胞内信号传导结构域具有SEQ ID NO:26、SEQ ID NO：27或SEQ ID NO：28所示的序列。
在一些实施方式中，嵌合抗原受体包括顺序连接的CD8信号肽、胞外结合区、CD28或CD8α铰链区、CD28或CD8α跨膜结构域、CD28或4-1BB胞内共刺激结合结构域以及CD3ζ胞内信号传导结构域。
在一些实施方式中，嵌合抗原受体包含SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37或SEQ ID NO：38所述的氨基酸序列，或者包含与上述任一氨基酸序列相比具有至少85％(例如至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％)序列一致性的氨基酸序列。
在第二个方面，本申请提供一种分离的核酸分子，所述的分离的核酸分子编码第一个方面所述的嵌合抗原受体。
在一些实施方式中，所述核酸分子为DNA或RNA。
在一些实施方式中，所述RNA为mRNA。
在一些实施方案中，核酸分子可操作地连接到调控序列，调控序列可以被用载体转化过的宿主细胞识别。
在第三个方面，本申请提供了一种包含第二个方面所述核酸分子的表达载体。
在第四个方面，本申请提供了表达上述第一个方面所述的嵌合抗原受体，或包含第二个方面所述的核酸分子，或包含第三个方面所述的表达载体的细胞。
在一些实施方式中，细胞为免疫细胞。
在一些实施方式中，免疫细胞选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
在第五个方面，本申请提供了一种制备嵌合抗原受体修饰的免疫细胞的方法，所述方法包括将第二个方面所述的核酸分子或第三个方面所述的表达载体递送至待修饰的免疫细胞的 步骤。
在一些实施方式中，递送的方式包括病毒转染或通过阳离子脂质体(例如LNP)递送至所述免疫细胞。
在一些实施方式中，所述方法还包括在递送前，分离和激活所述免疫细胞。
在一些实施方式中，所述方法还包括培养导入所述核酸分子或表达载体后的免疫细胞。
在一些实施方式中，免疫细胞选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
在第六个方面，本申请提供了一种药物组合物，所述的药物组合物包含第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞，或通过第五个方面所述的方法制备的产品，以及药学可接受的载体。
在一些实施方式中，药物组合物用于预防和/或治疗癌症。在一些实施方式中，癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
在一些实施方案中，癌症是癌细胞表面表达CLDN18.2的那些癌症。在一些实施方案中，癌症是高表达CLDN18.2(CLDN18.2+)的癌症。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少60％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少70％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少80％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少90％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少95％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少98％的癌细胞表达CLDN18.2。在一些实施方案中，高表达CLDN18.2(CLDN18.2+)的癌症是指癌细胞群体中至少99％的癌细胞表达CLDN18.2。
在第七个方面，本申请提供了上述第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞、第五个方面所述的方法制备的产品，或第六个方面所述的药物组合物在制备预防和/或治疗癌症的组合物中的用途。在一些实施方式中，癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
在第八个方面，本申请提供了一种预防和/或治疗癌症的方法，包含向有此需要的患者施用有效量的上述第一个方面所述的嵌合抗原受体、第二个方面所述的核酸分子、第三个方面所述的表达载体、第四个方面所述的细胞、第五个方面所述的方法制备的产品，或第六个方面所述的药物组合物。在一些实施方式中，癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发 性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1人CLDN18.1和人CLDN18.2高表达细胞株构建
分别构建了表达人CLDN18.1的CHO-K1稳转细胞株，表达人CLDN18.2的CHO-K1稳转细胞株，与同时稳定表达人CLDN18.2和细菌荧光素酶(Luciferase)的肿瘤细胞NUGC4(胃癌细胞系)稳转株。具体步骤如下：
质粒构建：分别合成编码重组蛋白的多聚核苷酸，并分别在两端加上EcoRI和SalI酶切位点以及相应的载体同源序列。使用限制性内切酶EcoRI(Thermo，Cat#FD0274)和SalI(Thermo，Cat#FD0644)对质粒pCDH-CMV-MCS-EF1-Puro进行酶切，琼脂糖凝胶电泳回收纯化线性质粒。将以上步骤中合成的多聚核苷酸分别和线性化载体通过重组酶5×In-FusionHD酶(TaKaRa，Cat#ST0344)进行连接，反应体系如下：2μl多聚核苷酸片段(50ng/μl)，1μl线性化质粒(50ng/μl)，2μl 5×HD In-Fusion酶，5μl ddH2O。用移液器轻轻吹打混匀，短暂离心，置于50℃反应15min。将10μl重组反应产物加入到100μl细菌感受态细胞中，在冰上放置5min，将转化菌液均匀涂布在含有100μg/ml氨苄青霉素的LB平板上，在恒温培养箱中倒置培养12-16h。随机从每个平板上挑取3-5个克隆进行测序鉴定。将测序正确的菌液转接于100ml含100μg/ml氨苄青霉素的LB液体培养基中，37℃培养过夜，用MN无内毒素质粒中抽试剂盒(MN，Cat#740420.50)进行质粒抽提，定量后用无内毒超纯水稀释至1000ng/μl。
慢病毒制备：将293T细胞(中国科学院典型培养物保藏委员会细胞库，Cat#GNHu17)分别接种于100mm培养皿中，使用加入10％FBS(Gibco，Cat#10099141)的DMEM培养基(Gibco，Cat#10566016)进行培养。待293T细胞覆盖培养皿表面约70％时进行质粒转染：分别取表达重组蛋白的慢病毒质粒，与包装质粒(pRRE，pRSV-Rev和pVSV-G)混合后加入1.2ml Opti-MEM培养基(Thermofisher Scientific，Cat#31985070)中；加入35μl Fugene HD(Promega，Cat#04709691001)，混匀后室温孵育15min。最后将混合物加入293T细胞培养基，移入恒温培养箱(37℃，5％CO2)内培养。2天后收集293T细胞上清(病毒)，用0.45μm滤膜过滤，并浓缩备用。
重组细胞制备：将CHO-K1细胞(中国科学院典型培养物保藏委员会细胞库，Cat#SCSP-507)或NUGC-4细胞(南京科佰生物科技有限公司，Cat#CBP74135)接种于10cm培养皿，每皿各5×105个细胞。将已制备好的病毒加入状态良好的CHO-K1细胞或NUGC4 细胞，同时加入10μg/ml聚凝胺(上海翊圣生物科技有限公司，Cat#40804ES76)，混匀后放置培养箱，3～4h后更换培养基。第3天向培养基中添加入10μg/ml嘌呤霉素进行筛选(Gibco，Cat#A1113803)。2-3天后细胞大量死亡，更换新鲜培养基继续培养，筛选单克隆细胞株，扩培并冻存保种。
构建稳定表达CLDN18的细胞株，分别命名为CHOK1-18.1、CHOK1-18.2和NUGC4-luc细胞，所用的人CLDN18.1氨基酸序列见SEQ ID NO:1，所用的CLDN18.2氨基酸序列见SEQ ID NO:3，所用的细菌荧光素酶氨基酸序列见SEQ ID NO:5。相应氨基酸序列见表1。
表1.构建CLDN18细胞株中所用到的氨基酸序列详情

实施例2流式细胞技术(FACS)检测抗CLDN18.2抗体与CHOK1-18.1、CHOK1-18.2和肿瘤细胞NUGC-4结合实验
用PBS缓冲液(Gibco，Cat#10010-023)洗涤细胞2次，计数后将细胞用PBS缓冲液稀释至1×106个/ml，然后按每孔100μl加入96孔FACS反应板中，离心后添加100μl单链抗体Fc融合蛋白(2μg/ml)，冰上孵育20min。其中，检测抗体Tab1的氨基酸序列见SEQ ID NO：6，Tab2的氨基酸序列见SEQ ID NO：7，Tab3的氨基酸序列见SEQ ID NO：8(参考专利WO 2018/006882)(Tab1、2、3分别对应于下述实施例3-6中CAR1、CAR2和CAR3中靶向CLDN18.2的单链抗体，由常规制备方法获得)。用FACS缓冲液(加入10％FBS的PBS缓冲液)离心洗涤2次，加入每孔100μl荧光标记的二抗Alexa647-AffiniPure山羊抗 人IgG(Jackson Immuno，Cat#309-605-008)，冰上孵育20min。用FACS缓冲液离心洗涤3次，用100μl FACS缓冲液悬浮细胞，用FACS(CANTOII，购自BD公司)检测和分析结果，结果如图1A-1D表明，Tab1、Tab2和Tab3对CLDN18.2有特异结合活性，而对CLDN18.1无结合活性。
实施例3表达二代嵌合抗原受体载体构建
采用本领域常规分子生物学方法，本实施例采用的单链抗体(Single Chain Antibody Fragment，简称scFv)为靶向Claudin 18.2的抗体，参照图2所示的质粒图，以慢病毒载体pCDH-CMV-MCS-EF1-Puro为模板，构建了表达二代嵌合抗原受体(CAR)的慢病毒质粒CAR18.2-1、CAR18.2-2、CAR18.2-3、Claudin 18.2-1、Claudin 18.2-2、Claudin 18.2-3和Claudin 18.2-4。相应氨基酸序列见表2(其中CDR序列根据Kabat划分)，各质粒表达元件对应的序列见表3。
CAR18.2-1质粒(对应的CAR-T细胞下文中称为CAR-1)中CAR从5`端到3`端依次包括：CD8α信号肽、VL1、连接子(linker)、VH1、CD8α铰链区、CD8α跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:32；
CAR18.2-2质粒(对应的CAR-T细胞下文中称为CAR-2)中CAR从5`端到3`端依次包括：CD8α信号肽、VH1、连接子(linker)、VL1、CD8α铰链区、CD8α跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:33；
CAR18.2-3质粒(对应的CAR-T细胞下文中称为CAR-3)中CAR从5`端到3`端依次包括：CD8α信号肽、VL2、连接子(linker)、VH2、CD8α铰链区、CD8α跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:34；
Claudin18.2-1质粒(对应的CAR-T细胞下文中称为CLDN18.2-1)中CAR从5`端到3`端依次包括：CD8α信号肽、VH1、连接子(linker)、VL1、CD8α铰链区、CD28跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:35；
Claudin18.2-2质粒(对应的CAR-T细胞下文中称为CLDN18.2-2)中CAR从5`端到3`端依次包括：CD8α信号肽、VH1、连接子(linker)、VL1、CD8α铰链区、CD8跨膜结构域、4-1BB胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:36；
Claudin18.2-3质粒(对应的CAR-T细胞下文中称为CLDN18.2-3)中CAR从5`端到3`端依次包括：CD8α信号肽、VL2、连接子(linker)、VH2、CD8α铰链区、CD28跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:37；
Claudin18.2-4质粒(对应的CAR-T细胞下文中称为CLDN18.2-4)中CAR从5`端到3`端依次包括：CD8α信号肽、VH1、连接子(linker)、VL1、CD28铰链区、CD28跨膜结构域、CD28胞内共刺激域、及CD3ζ信号转导结构域，氨基酸序列见SEQ ID NO:38。
表2.嵌合抗原受体中各元件的氨基酸序列



表3.嵌合抗原受体中各质粒表达元件及对应的序列
实施例4：表达嵌合抗原受体T细胞(CAR-T细胞)CAR-1、CAR-2和CAR-3的构建
利用实施例3中制备的CAR18.2-1、CAR18.2-2、CAR18.2-3，构建多表达二代嵌合抗原受体的T细胞，分别命名为CAR-1、CAR-2和CAR-3。具体步骤如下：
4.1慢病毒制备
取实施例3制备的嵌合抗原受体的慢病毒质粒CAR18.2-1、CAR18.2-2、CAR18.2-3与包装质粒(pRRE，pRSV-Rev和pVSV-G)，按照实施例1所述方法制备慢病毒，48h后收集293T细胞上清(病毒)，用0.45μm滤膜过滤，并浓缩备用。
4.2嵌合抗原受体T细胞(CAR-T细胞)CAR-1、CAR-2和CAR-3的制备
T细胞增殖：按照说明书，使用Stemcell Easy Sep Kit试剂盒(Stemcell，Cat#19055)从不同健康捐献者的外周血单个核细胞(Peripheral blood mononuclear cell，简称PBMC)中分离T细胞。将分离后的T细胞置于1μg/ml CD3/CD28抗体(Thermo，Cat#11131D)预先包埋的培养皿中培养。培养基组分为X-VIVO15(Lonza，Cat#BEBP02-054Q)，5％人AB血清(Gemini，Cat#100-512)，100U/ml青霉素-链霉素(Gibco，Cat#15140-122)，200IU/ml人IL2因子(北京双鹭，Cat#S19991007)。每周进行2次细胞计数，当细胞密度到2.5×106个/ml时传代扩培。
CAR-T细胞制备：取上述状态良好的T细胞，按1×106个/孔接种到预先用5μg/ml重组人纤维蛋白(Takara，Cat#T100B)包被的12孔细胞培养板，添加50μl浓缩慢病毒和10μg/ml 聚凝胺(Sigma-Aldrich，Cat#TR-1003)，并在4℃下1000g离心1h。将离心后的培养板移入恒温培养箱(37℃，5％CO2)内培养，每周计数2次，当细胞密度达到2.5×106个/ml时传代扩培。
流式细胞实验(FACS)检测CAR的表达与感染效率：用PBS缓冲液洗涤细胞2次，计数后用PBS缓冲液稀释细胞至2×106个/ml，加入50μl Fc受体阻断剂(BioLegend，Cat#422302)室温孵育10min，然后按每孔100μl加入到96孔FACS反应板中。加入100μl浓度为2μg/μl的含His标签蛋白的CLDN18.2蛋白(Acro，Cat#CL2-H5546)，冰上孵育20min。用FACS缓冲液离心洗涤2次，加入每孔100μl二抗THETM His Tag Antibody(Genscript，Cat#A00612)，冰上孵育20min。用FACS缓冲液离心洗涤3次，用100μl FACS缓冲液悬浮细胞，用FACS(BD，CANTOII)检测和分析结果。结果如图3A所示，CAR的阳性率均在50％-70％，CAR-2细胞的阳性率最高，CAR-1和CAR-3细胞次之。
实施例5：CAR-T细胞CAR-1、CAR-2和CAR-3的体外杀伤活性实验
为了验证CAR-T细胞CAR-1、CAR-2和CAR-3的体外杀伤活性，使用实施例1内构建好的CHOK1-18.2作为靶细胞，CAR-T细胞作为效应细胞，进行验证。
5.1实时细胞杀伤分析实验
用PBS缓冲液清洗靶细胞CHOK1-18.2，加入胰蛋白酶溶液(Gibco，Cat#25200056)消化靶细胞，1000rpm离心5min，移除上清液并加入新鲜培养基重悬细胞，调整细胞浓度至4×105cells/ml。向E-plate 96孔板每孔中加入20000个细胞。将E-Plate 96孔板放置于实时无标记细胞分析仪(Agilent，RTCA MP)进行培养。第二天，加入相应效靶比(1：1或者1：5)的CAR-T细胞，继续培养2天或以上。培养过程中，每隔15min检测细胞指数(Cell Index，简称CI)，记录靶细胞生长情况。结果如图3B-3C所示，CAR-T细胞对高表达抗原CLDN18.2的CHOK1-18.2有较好杀伤活性，当效靶比为1：5时，CAR-2显示出良好的杀伤活性，而CAR-1和CAR-3的杀伤活性次之。
5.2酶联免疫吸附实验(ELISA)检测细胞因子分泌实验：取杀伤实验中的细胞培养基(72h)检测人肿瘤坏死因子α(TNF-α)和人干扰素γ(IFN-γ)，按照检测试剂盒(BD OptEIATM Human IFN-γELISA Set，Cat No#555142；BD OptEIATMHuman TNF ELISA Set，Cat No#555212)提供的手册进行操作。实验结果如图4A-4B所示：E：T＝1：5时，CAR-2的TNF-α分泌量最高，3个CAR-T细胞的IFN-γ分泌量相近。
5.3多轮长期杀伤实验：
第1轮杀伤实验，按照E：T为1：1，将CAR-T细胞和3×105个NUGC4-luc细胞共同加入24孔板中，在细胞培养箱中(37℃，5％CO2)共孵育3天，收集孔板中细胞进行杀伤肿瘤细胞实验。具体的，加入萤火虫萤光素酶底物D-Luciferin，使用多功能酶标仪读取生物荧光值。第一轮杀伤效率高于50％，进行第2轮杀伤实验。CAR-T细胞与NUGC4-luc细胞共孵育72h(效靶比1：1)后，将T细胞重悬计数，再与3×105个NUGC4-luc细胞共孵育进行下一轮杀伤实验(共孵育时间72h，效靶比1：1)。以此类推，共完成4轮杀伤。FACS检测CAR-T细胞的增殖情况和细胞表面PD-1的表达情况。如5A图所示，CAR-1、CAR-2和CAR-3在各轮的杀伤效率为100％或接近100％；如图5B所示，第三轮杀伤实验后CAR-2细胞的增殖能力明显高于其他两组CAR-T细胞；如图5C表明第四轮杀伤实验后，CAR-2细 胞表面的PD-1表达水平明显低于其他两组CAR-T细胞。
实施例6：表达嵌合抗原受体细胞(CAR-T细胞)在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验
为了评估CAR-T细胞CAR-1、CAR-2和CAR-3的抗肿瘤药效，使用小鼠胃癌皮下移植瘤模型进行抗肿瘤药效试验。具体如下：
收集处于对数生长期且生长状态良好的NUGC4-luc细胞，于NPG小鼠(联合免疫缺陷小鼠)皮下接种5×106个细胞。接种肿瘤后第7天测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(mm3)＝1/2×(a×b2)，根据随机数原则选择肿瘤体积在50mm3左右的小鼠进行随机分组。接种肿瘤后第8天，尾静脉注射CAR-T细胞(1×107个/只)，注射体积为200μl/只，CAR-T细胞注射日记为Day0。小鼠分组与CAR-T细胞注射情况见表4。持续观察并测量小鼠肿瘤体积及体重变化，每周测量记录2次并计算肿瘤抑制率，计算公式为抑瘤率TGI(％)＝(PBS组小鼠肿瘤体积-实验组小鼠肿瘤体积)/PBS组小鼠肿瘤体积×100％。
表4.体内抗肿瘤实验分组情况
小鼠肿瘤体积检测结果如图6A所示，结果表明：通过尾静脉注射后，CAR-2细胞能够完全抑制G3组小鼠体内肿瘤生长，组内有4只动物肿瘤最终完全消失，表现出良好的抑瘤效果(如图6A和6C所示)；G2、G3组小鼠体重变化不明显，呈缓慢增长趋势，同时检测到G4组在注射CAR-3细胞后的第11天体重开始持续下降(如图6B所示)，并且该组动物在实验过程中的第26天，第31天分别发生1只，3只动物死亡，最终在第38天全部死亡。
综上，根据实施例4-实施例6的体内外实验活性验证表明，CAR-2的体内外活性效果明显优于CAR-1和CAR-3，在CAR-T细胞其他元件(信号肽、铰链区、跨膜区、共刺激结构域及信号传导结构域)一致的情况下，推测是CAR-2抗体的连接顺序(VH-VL)在活性验证中起了主要的优势作用，因此接下来将主要选择VH-VL连接顺序的抗体来构建主要的CAR分子。
实施例7：表达嵌合抗原受体T细胞(CAR-T细胞)CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4的构建
利用实施例3制备的Claudin 18.2-1、Claudin 18.2-2、Claudin 18.2-3和Claudin 18.2-4，构建表达二代嵌合抗原受体的T细胞，分别命名为CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4。具体步骤如下：
参照实施例4的构建过程，构建CAR-T细胞CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4，并通过流式细胞实验(FACS)检测CAR表达与感染效率：用PBS缓冲液洗涤细胞2次，计数后用PBS缓冲液稀释细胞至2×106个/ml，加入50μl Fc受体阻断剂(BioLegend，Cat#422302)室温孵育10min，然后按每孔100μl加入到96孔FACS反应板中。 加入100μl浓度为2μg/μl的含His标签蛋白的CLDN18.2蛋白(Acro，Cat#CL2-H5546)，冰上孵育20min。用FACS缓冲液离心洗涤2次，加入每孔100μl二抗THETM His Tag Antibody(Genscript，Cat#A00612)，冰上孵育20min。用FACS缓冲液离心洗涤3次，用100μl FACS缓冲液悬浮细胞，用FACS(BD，CANTOII)检测和分析结果，结果如图7。图7A表明相较未感染的T细胞，CAR-T细胞的增殖能力未受到影响；图7B表明CAR转染的阳性率均在60％-80％，CLDN18.2-1细胞的阳性率最高，CLDN18.2-2和CLDN18.2-4细胞次之。
实施例8：表达嵌合抗原受体T细胞(CAR-T)CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4体外杀伤活性实验
为了验证CAR-T细胞CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4的体外杀伤毒性，使用实施例1内构建好的NUGC4-luc作为靶细胞，CAR-T细胞作为效应细胞，进行多轮长期杀伤实验验证。
第1轮杀伤实验，按照E：T为1：5，将CAR-T细胞和3×105个NUGC4-luc细胞共同加入24孔板中，在细胞培养箱中(37℃，5％CO2)共孵育3天。取出200μl培养基上清用以酶联免疫吸附实验(ELISA)检测细胞因子分泌：取第1轮杀伤实验中的细胞培养基检测人肿瘤坏死因子α(TNFα)和人干扰素γ(IFN-γ)，按照检测试剂盒(BD OptEIATMHuman IFN-γELISA Set，Cat No#555142；BD OptEIATMHuman TNF ELISA Set，Cat No#555212)提供的手册进行操作。实验结果如图8A-8B所示。收集孔板中细胞进行杀伤肿瘤细胞实验，具体的，加入萤火虫萤光素酶底物D-Luciferin，使用多功能酶标仪读取生物荧光值。第一轮杀伤效率高于50％，进行第2轮杀伤实验。CAR-T细胞与NUGC4-luc细胞共孵育72h(效靶比1：5)后，将T细胞重悬计数，再与一定数量新鲜NUGC4-luc细胞共孵育进行下一轮杀伤实验(共培养时间72h，效靶比1：5)。相同的，共完成4轮杀伤。
中央记忆型T细胞(Central Memory T cell，TCM，CD62L+CD45RO+)是初始T细胞(Naive T Cell)经过抗原激活后，产生的具有长期记忆性的，并能够归巢到淋巴结接受抗原再刺激的T细胞。被激活的TCM细胞，在抗原的再次刺激之下可继续产生大量的携带同种抗原的克隆化的效应记忆型T细胞(Effective Memory T Cell，TEM，CD62L-CD45RO+)细胞。TCM细胞能够通过淋巴屏蔽，回归淋巴结，同时处于被抗原激活的状态。干细胞样记忆性T细胞(stem cell-like memory T cell，TSCM，CD62L+CD45RO-)是记忆T细胞的重要组成，具有干细胞样自我更新、多分化潜能和免疫重构特性。有别于中枢记忆T细胞(central memory T cell，TCM)或效应记忆T细胞(effective memory T cell，TEM)亚群，TSCM具有初始T细胞表型且分化程度较低。效应T细胞(Effector T cells，TE，CD62L-CD45RO-)是T细胞接受抗原刺激后，经过增殖，分化形成的细胞；效应T细胞描述了一组细胞，该细胞包括主动响应刺激(例如Co-stimulation)的几种T细胞类型，包括调节性T细胞、辅助T细胞、细胞毒性T细胞，效应T细胞具有释放淋巴因子的功能。免疫检查点分子PD-1(programmed cell death protein 1，程序性死亡受体1)具有重要的免疫抑制功能，在肿瘤组织中的T细胞表面表达上调，并通过传递抑制信号阻止T细胞活化。流式检测TCM、TEM、TSCM和TE的表达情况，并检测T细胞表面PD-1的表达量。
如图8A-8B所示，E：T＝1：5时，CLDN18.2-1的TNF-α分泌量最高，CLDN18.2-2与CLDN18.2-3的TNF-α分泌量次之。4种CAR-T细胞的IFN-γ分泌量相当。如图9A所示， CLDN18.2-4在第4和5轮的杀伤效率为64％和25％，明显高于其他CAR-T细胞在第4轮(44％-50％)和第5轮(15％-19％)的杀伤效率；9B图表明第三轮杀伤实验后，各组CAR-T细胞记忆表型均无明显差异；9C图表明第三轮杀伤实验后，各组耗竭表型TIM3和LAG3的表达水平相当；9D图表明第三轮杀伤实验后，各组细胞表面PD-1表达无明显差异。
实施例9：表达嵌合抗原受体细胞(CAR-T细胞)在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验
为了评估CAR-T细胞CAR-2和CLDN18.2-1(治疗2组来自于两批实验)的抗肿瘤药效，使用小鼠胃癌皮下移植瘤模型进行抗肿瘤药效试验。具体如下：
收集处于对数生长期且生长状态良好的NUGC4-luc细胞，于NPG小鼠(联合免疫缺陷小鼠)皮下接种，接种量分别为7×106和5×106个细胞。接种肿瘤后第6天和第7天测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(公式同实施例6)，根据随机数原则选择肿瘤体积在60mm3左右的小鼠进行随机分组。接种肿瘤后第7天和第8天，尾静脉注射CAR-T细胞(1×107个/只)，注射体积为200μl/只，CAR-T细胞注射日记为Day0。小鼠分组与CAR-T细胞注射情况见表5。持续观察并测量小鼠肿瘤体积变化，每周测量记录2次并计算肿瘤抑制率TGI，计算公式同实施例6。
表5.体内抗肿瘤实验分组情况
小鼠肿瘤体积检测结果如图10A和10B所示，图中D3和D4分别表示注射CAR-T细胞后的第3天和第4天。结果表明：通过尾静脉注射后，CLDN18.2-1细胞能够完全抑制G3组小鼠体内肿瘤生长，肿瘤最终完全消失，表现出良好的抑瘤效果。
实施例10：表达嵌合抗原受体细胞(CAR-T细胞)在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验
为了评估CAR-T细胞CLDN18.2-1、CLDN18.2-2、CLDN18.2-3和CLDN18.2-4的抗肿瘤药效，使用小鼠胃癌皮下移植瘤模型进行抗肿瘤药效试验。具体如下：
收集处于对数生长期且生长状态良好的NUGC4-luc细胞，于NPG小鼠(联合免疫缺陷小鼠)皮下接种5×106个细胞。接种肿瘤后第7天测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(公式同实施例6)，根据随机数原则选择肿瘤体积在50mm3左右的小鼠进行随机分组。接种肿瘤后第8天，尾静脉注射CAR-T细胞(1×107个/只)，注射体积为200μl/只，CAR-T细胞注射日记为Day0。小鼠分组与CAR-T细胞注射情况见表6。持续观察并测量小鼠肿瘤体积及体重变化，检测血清内IFN-γ的含量，每周测量记录2次。
表6.体内抗肿瘤实验分组情况

小鼠肿瘤体积检测结果如图11A所示，结果表明：在注射CAR-T细胞后的第45天，CLDN18.2-1和CLDN18.2-3能够完全抑制小鼠肿瘤生长，肿瘤完全消失；CLDN18.2-4能够抑制组内4只小鼠的肿瘤生长，直至肿瘤完全消失。CLDN18.2-2未体现出抑制肿瘤生长的药效，组内小鼠在第34天由于肿瘤体积到安乐死标准被处理。PBS治疗组在第34天由于肿瘤体积到安乐死标准被处理。同时，检测到在分别注射CLDN18.2-3及CLDN18.2-4细胞后，小鼠的体重在第10天下降至最低点，之后逐渐回升，而CLDN18.2-1治疗组小鼠体重变化不明显，呈缓慢增长趋势(如图11B所示)。第34天测量小鼠肿瘤体积，计算肿瘤抑制率TGI％，计算公式同实施例6。如图11C所示，CLDN18.2-1和CLDN18.2-3治疗组抑瘤率均为100％，CLDN18.2-2治疗组抑瘤率达到16％，CLDN18.2-4治疗组抑瘤率达到98％。如图11D所示，各组小鼠血清中IFN-γ的含量在治疗开始后第5天达到最高，然后逐步下降。除PBS治疗组外，其他各组小鼠在第42天仍检测IFN-γ表达。
实施例11：表达嵌合抗原受体细胞(CAR-T细胞)在病人来源肿瘤异种移植模型(Patient-derived Tumor Xenograft Model，PDX)中的抗肿瘤药效试验
为了评估CAR-T细胞CLDN18.2-1、CLDN18.2-3和CLDN18.2-4的抗肿瘤药效，使用病人来源肿瘤皮下异种移植模型进行抗肿瘤药效试验。具体如下：
取GA0006异种移植模型(胃癌模型)荷瘤小鼠肿瘤组织并切成直径为2-3mm的瘤块，接种于NCG小鼠(联合免疫缺陷小鼠)右前肩胛处皮下。在接种肿瘤后第21天，测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(公式同实施例6)，并根据随机数原则选择肿瘤平均体积达到约200mm3左右的小鼠进行随机分组。分组当天记为第0天(即Day0)，并经由尾静脉注射CAR-T细胞(1×107/只)进行治疗，注射体积为200μl/只。
小鼠分组与CAR-T细胞注射情况见表7。持续观察并测量小鼠肿瘤体积及体重变化，每周测量记录2次。
表7.体内抗肿瘤实验分组情况
小鼠肿瘤体积检测结果如图12A所示，结果表明：在注射CAR-T细胞后的第35天，CLDN18.2-1能够抑制小鼠肿瘤生长，其中有2只的肿瘤完全消失；CLDN18.2-3和CLDN18.2-4两组分别有4和5只小鼠肿瘤被抑制，两组均有1只小鼠肿瘤完全消失。同时如图12B所示，各治疗组小鼠均出现体重下降，PBS、CLDN18.2-1和CLDN18.2-3三组治疗组在注射CAR-T细胞后的第7天体重下降至最低点，随后逐渐回升；而CLDN18.2-4治疗组小鼠在第14天体 重显著下降，在第16天有3只动物体重骤降超过20％，被安乐死，余下小鼠体重逐渐回升。第21天和35天分别测量小鼠肿瘤体积，计算肿瘤抑制率TGI％(公式同实施例6)。如图12C所示，第21天CLDN18.2-1治疗组和CLDN18.2-4治疗组抑瘤效果显著，抑瘤率分别达到89％和83％，CLDN18.2-3治疗组抑瘤率为27％；第35天CLDN18.2-1治疗组抑瘤率达到90％，CLDN18.2-3治疗组抑瘤率达到73％，CLDN18.2-4治疗组抑瘤率达到83％。
实施例12：不同剂量表达嵌合抗原受体细胞(CAR-T细胞)在NUGC4-luc小鼠肿瘤模型中的抗肿瘤药效试验
为了评估CAR-T细胞CLDN18.2-1、CLDN18.2-3和CLDN18.2-4的抗肿瘤药效，使用小鼠胃癌皮下移植瘤模型进行抗肿瘤药效试验。具体如下：
收集处于对数生长期且生长良好的NUGC4细胞，于NPG小鼠(联合免疫缺陷小鼠)右前肩胛处皮下接种6×106个细胞。接种肿瘤后第7天，测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(公式同实施例6)，根据随机数原则选择肿瘤体积在60mm3左右的的小鼠进行随机分组。接种肿瘤后第8天，经由尾静脉注射CAR-T细胞(6×106个细胞/只、3×106个细胞/只或1.5×106个细胞/只)进行治疗，注射体积为200ul/只，CAR-T细胞注射日记为第0天(即Day0)。小鼠分组与CAR-T细胞注射情况见表8。持续观察并测量小鼠肿瘤体积及体重变化，每周测量记录2次。
表8.体内抗肿瘤实验分组情况
小鼠肿瘤体积检测结果如图13A所示，结果表明：在注射CAR-T细胞后的第21天，6×106个CLDN18.2-4细胞能够显著抑制小鼠肿瘤生长，其中1只动物肿瘤完全消失；除CLDN18.2-4(1.5×106/只)治疗组外，其余5组在治疗后也表现出抑制小鼠体内肿瘤增长的趋势。同时，CLDN18.2-4(6×106/只)治疗组在第11天体重下降至最低点，之后迅速回升，而其余各组小鼠体重呈缓慢增长趋势(如图13B所示)。在第21天测量小鼠肿瘤体积并计算肿瘤抑制率，计算公式为抑瘤率TGI％(公式同实施例6)。如图13C所示，CLDN18.2-4(6×106/只)治疗组抑瘤率为84％，表现良好的抑制肿瘤生长的效果。CLDN18.2-1(3×106/只)、CLDN18.2-1(6×106/只)和CLDN18.2-4(3×106/只)治疗组抑瘤率也超过20％。
实施例13：不同剂量表达嵌合抗原受体细胞(CAR-T细胞)在病人来源肿瘤异种移植模型中的抗肿瘤药效试验
为了进一步评估CLDN18.2-1和CLDN18.2-4CAR-T细胞的量效关系，使用不同剂量的CLDN18.2-1和CLDN18.2-4CAR-T细胞在病人来源肿瘤皮下异种移植模型进行抗肿瘤药效试验。具体如下：
取GA0006异种移植模型(胃癌模型)荷瘤小鼠肿瘤组织并切成直径为2-3mm的瘤块，接种于NCG小鼠(联合免疫缺陷小鼠)右前肩胛处皮下。接种肿瘤后第18天，测量小鼠肿瘤的长短径a和b，计算小鼠肿瘤体积V(公式同实施例6)，根据随机数原则选择肿瘤平均体积达到100mm3左右的小鼠进行随机分组，分组当天记为第0天(即Day0)，并经由尾静脉注射CAR-T细胞(5×106/只或1×107/只)进行治疗，注射体积为200μl/只。
将小鼠分为5组，每组6只。小鼠分组与CAR-T细胞注射情况见表9。持续观察并测量小鼠肿瘤体积及体重变化，每周测量记录2次。
表9.体内抗肿瘤实验分组情况
小鼠肿瘤体积检测结果如图14A所示，结果表明：在注射CAR-T细胞后的第53天，1×107个CLDN18.2-1细胞能够抑制组内5只小鼠肿瘤生长，并且能够抑制肿瘤复发速度；1×107个CLDN18.2-4细胞能够抑制组内4只小鼠肿瘤生长，其中2只动物的肿瘤完全消失；CLDN18.2-1(5×106/只)和CLDN18.2-4(5×106/只)分别能够抑制组内1只或者2只小鼠肿瘤生长。同时各治疗组小鼠均出现体重下降情况：PBS、CLDN18.2-1(5×106/只)及CLDN18.2-4(5×106/只)治疗组的小鼠体重呈现下降后上升再持续下降的波动趋势，由于治疗后期平均肿瘤体积超过2000mm3，分别在第25天或第32天进行安乐死；而CLDN18.2-1(1×107/只)和CLDN18.2-4(1×107/只)小鼠体重变化不明显，呈缓慢增长趋势(如图14B所示)。第25天测量小鼠肿瘤体积并计算肿瘤抑制率(因PBS组平均肿瘤体积在第25天超过2000mm3，进行安乐死处理)，计算公式为抑瘤率TGI％(公式同实施例6)。如图14C所示，CLDN18.2-4(1×107/只)治疗组抑瘤率达到89％，CLDN18.2-1(1×107/只)治疗组抑瘤率达到85％，CLDN18.2-4(5×106/只)治疗组抑瘤率达到46％，CLDN18.2-1(5×106/只)组抑瘤率达到26％。各治疗组都能很好的抑制小鼠肿瘤增长，表明CLDN18.2-1、CLDN18.2-4在中、高剂量时皆有较好的抗肿瘤活性。

Claims (18)

  1. 一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含信号肽、特异性识别CLDN18.2蛋白的胞外结合区、铰链区、跨膜区、胞内共刺激结构域和胞内信号传导结构域，所述胞外结合区包含特异性识别CLDN18.2的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含SEQ ID NO：9所示重链可变区(VH)中包含的HCDR1、HCDR2和HCDR3，和SEQ ID NO：13所示轻链可变区(VL)中包含的LCDR1、LCDR2和LCDR3；
    优选地，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示序列，LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示序列。
  2. 根据权利要求1所述的嵌合抗原受体，其中所述抗体或其抗原结合片段包含SEQ ID NO：9所示的重链可变区和SEQ ID NO:13所示的轻链可变区；
    优选地，所述抗体或抗原结合片段为scFv的形式，所述scFv具有VH-连接子-VL或VL-连接子-VH的结构；
    优选地，所述连接子具有SEQ ID NO：17或SEQ ID NO：18所示的序列。
  3. 根据权利要求1-2任一项所述的嵌合抗原受体，其中所述信号肽为CD8信号肽；
    优选地，所述CD8信号肽具有SEQ ID NO：29所示的氨基酸序列。
  4. 根据权利要求1-3所述的嵌合抗原受体，其中所述铰链区选自CD28铰链区或CD8α铰链区；
    优选地，所述铰链区包含SEQ ID NO：19、SEQ ID NO：20或SEQ ID NO：21所示的氨基酸序列。
  5. 根据权利要求1-4任一项所述的嵌合抗原受体，其中所述跨膜区选自CD4、CD8α、CD28、PD1和/或4-1BB跨膜区；
    优选地，所述跨膜区为CD28跨膜区或CD8α跨膜区；更优选地，所述跨膜区具有SEQ ID NO：22或SEQ ID NO：23所示的氨基酸序列。
  6. 根据权利要求1-5任一项所述的嵌合抗原受体，其中所述胞内共刺激信号结构域选自CD28或4-1BB胞内共刺激结构域；
    优选地，所述CD28胞内共刺激信号结构域具有如SEQ ID NO：24所示的氨基酸序列，所述4-1BB胞内共刺激结构域具有如SEQ ID NO：25所示的氨基酸序列。
  7. 根据权利要求1-6任一项所述的嵌合抗原受体，其中所述胞内信号传导结构域为CD3ζ胞内信号传导结构域；
    优选地，所述CD3ζ胞内信号传导结构域具有SEQ ID NO:26、SEQ ID NO：27或SEQ ID NO：28所示的序列。
  8. 根据权利要求1–7任一项所述的嵌合抗原受体，其中所述嵌合抗原受体包括顺序连接的CD8信号肽、特异性识别CLDN18.2蛋白的胞外结合区、CD28或CD8α铰链区、CD28或CD8α跨膜结构域、CD28或4-1BB胞内共刺激结合结构域以及CD3ζ胞内信号传导结构域；
    优选地，所述嵌合抗原受体包含SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37或SEQ ID NO：38所述的氨基酸序列，或者包含与上述任一氨基酸序列相比具有至少85％(例如，至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％)序列一致性的氨基酸序列。
  9. 一种分离的核酸分子，其编码权利要求1-8任一项所述的嵌合抗原受体。
  10. 根据权利要求9所述的核酸分子，其中所述核酸分子为DNA或者RNA，所述RNA优选为mRNA。
  11. 包含权利要求9或10所述分离的核酸分子的表达载体。
  12. 表达权利要求1-8任一项所述的嵌合抗原受体、或包含权利要求9-10任一项所述核酸分子、或包含权利要求11所述表达载体的细胞。
  13. 根据权利要求12所述的细胞，其中所述细胞为免疫细胞；优选地，所述免疫细胞选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
  14. 一种制备嵌合抗原受体修饰的免疫细胞的方法，其中所述方法包括将权利要求9-10任一项所述的核酸分子或权利要求11所述的表达载体递送至待修饰的免疫细胞的步骤；
    优选地，递送的方式包括病毒转染或通过阳离子脂质体(例如LNP)递送至所述免疫细胞；优选地，所述方法还包括在递送前，分离和激活所述免疫细胞；优选地，所述免疫细胞选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
  15. 一种药物组合物，其中所述药物组合物包含权利要求1-8任一项所述的嵌合抗原受体、权利要求9-10任一项所述的核酸分子、权利要求11所述的表达载体、权利要求12-13任一项所述的细胞，或通过权利要求14所述的方法制备的产品，以及药学可接受的载体。
  16. 根据权利要求15所述的药物组合物，其用于预防和/或治疗癌症；
    优选地，所述癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
  17. 权利要求1-8任一项所述的嵌合抗原受体、权利要求9-10任一项所述的核酸分子、权利要求11所述的表达载体、权利要求12-13任一项所述的细胞，或权利要求14所述的方法制备的产品，或权利要求15所述的药物组合物在制备预防和/或治疗癌症的组合物中的用途；
    优选地，所述癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
  18. 一种预防和/或治疗癌症的方法，包含向有此需要的患者施用有效量的权利要求1-8任一项所述的嵌合抗原受体、权利要求9-10任一项所述的核酸分子、权利要求11所述的表 达载体、权利要求12-13任一项所述的细胞，或权利要求14所述的方法制备的产品，或权利要求15所述的药物组合物；
    优选地，所述癌症选自胃癌、食管癌、胰腺癌、卵巢癌、原发性腹膜癌、膀胱癌、骨癌、脑癌、乳腺癌、宫颈癌、结肠癌、胶质瘤、头颈癌、肾癌、白血病、急性髓系白血病(AML)、多发性骨髓瘤、肝癌、肺癌、淋巴瘤、黑色素瘤、间皮瘤、髓母细胞瘤、前列腺癌、直肠癌、皮肤癌、睾丸癌、气管癌和外阴癌。
PCT/CN2023/092137 2022-05-06 2023-05-05 靶向cldn18.2的嵌合抗原t细胞受体及其应用 WO2023213280A1 (zh)

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