Chimeric antigen receptors containing CLDN18.2 single-domain antibodies and their applications

Abstract

Chimeric antigen receptors comprising CLDN18.2 single domain antibodies and their applications. The constructed chimeric antigen receptors have good specificity, have higher safety, lower toxicity in in vivo use, and can provide a treatment regimen for patients with gastric cancer highly expressing the CLDN18.2 protein, who are currently treated with radiotherapy, chemotherapy and surgery.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to patent application CN2022105948645, filed on 27 May 2022, the entire contents of which are incorporated herein by reference.

[0002] [Technical field] The present invention relates to the biopharmaceutical technology, and more specifically to a chimeric antigen receptor containing a CLDN18.2 single-domain antibody and its applications. [Background technology]

[0003] Currently, the treatment principle for advanced gastric cancer remains a comprehensive approach primarily involving systemic chemotherapy, including systemic chemotherapy, surgical resection, radiation therapy, targeted therapy, immunotherapy, and herbal medicine. While the survival time of patients with advanced gastric cancer is clearly extended as chemotherapy drugs and regimens are continuously optimized, it remains difficult for patients to achieve a median survival time of more than one year.

[0004] Recent advances in immunotherapy have brought about significant changes in the field of oncology, particularly in immune checkpoint therapies represented by CTLA-4 and PD1 / PDL1 pathway inhibitors, and adoptive cell therapies represented by CAR-T. Adoptive cell therapies include TIL, NK, TCR-T, and CAR-T / NK / NKT / TIL / Mφ. Among these, CD19-targeted CAR-T therapy has achieved excellent clinical efficacy in B-cell tumors, and in 2017, two CAR-T products were approved by the FDA for use in the treatment of B-cell leukemia or lymphoma.

[0005] While targeted therapies and immune checkpoint inhibitors have benefited certain populations, finding other targets for gastric cancer at different stages of progression is urgently needed.

[0006] Claudins are a family of proteins that function to maintain tight junctions that regulate intercellular molecular exchange. They are widely distributed in gastric, pancreatic, and lung tissues and can be used for diagnosis and treatment. The CLDN18.2 isotype is gastric-specific, and since Sahin discovered that CLDN18.2 is a highly selective molecule and is widely expressed only in cancer cells, it has become an ideal target. CLDN18.2 is normally embedded in the gastric mucosa, and monoclonal antibodies in normal tissues do not make contact with it. The development of malignant tumors causes disruption of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells, making it a specific target. Thus, CLDN18.2 confers specificity to targeted therapy. It is expressed in 80% of gastrointestinal adenocarcinomas, 60% of pancreatic cancers, and multiple cancers including biliary tract cancer, ovarian cancer, and lung cancer.

[0007] Currently, global product types for CLDN18.2 include monoclonal antibodies, bispecific antibodies, CAR-T receptors, and ADCs. Of these, monoclonal antibodies are the most numerous in development. The fastest-growing drug currently being developed for CLDN18.2 is IMAB362 (Zolbetuximab, claudiximab), developed by Ganymed GmbH in Germany. IMAB362 is a human-mouse chimeric antibody that specifically recognizes and binds to the extracellular domain ECL1 of the claudin18.2 protein, but does not bind to any other claudin family members (including claudin18.1). It has already progressed to Phase III clinical trials.

[0008] While CAR-T therapy has achieved significant progress in hematological malignancies, its efficacy in treating solid tumors remains low. One important reason for this is that solid tumors lack favorable targets like those found in hematological malignancies. Furthermore, conventional CAR-T receptor sequences are derived from mice, resulting in relatively strong affinity. This leads to solid tumor CAR-T therapy consistently exhibiting stronger on-target and off-tumor effects, resulting in relatively greater side effects. Additionally, the relatively large molar mass of mouse-derived scfv and its high immunogenicity make it easier for patients to form anti-antibodies in vivo. As a result, CAR-T is rapidly eliminated by antibodies produced by the host in vivo, preventing it from providing sustained tumor relief in patients. This is another reason for the low efficacy of CAR-T therapy in solid tumors. [Overview of the Initiative]

[0009] One of the objectives of the present invention is to provide a chimeric antigen receptor in which the extracellular domain comprises a CLDN18.2 single-domain antibody, and the amino acid sequence of the heavy chain complementarity-determining region of the single-domain antibody is shown in SEQ ID NOs: 1 to 3.

[0010] The present invention also relates to nucleic acids and vectors related to the above-mentioned chimeric antigen receptor.

[0011] The present invention further provides immune cells that express the above-mentioned chimeric antigen receptor.

[0012] The present invention further relates to a pharmaceutical composition containing the above-mentioned immune cells.

[0013] The present invention further relates to the application of the immunoglobulin variable domain or immune cells in the manufacture of drugs for killing CLDN18.2-positive tumor cells.

[0014] The inventors of this invention unexpectedly discovered through testing that superior technical effects can be obtained when the selected single-domain antibody is constructed on a chimeric antigen receptor. The constructed chimeric antigen receptor exhibits good specificity, higher safety and lower toxicity in in vivo use, and may offer a better treatment regimen for patients with advanced gastric cancer that highly expresses the CLDN18.2 protein, currently treated with radiotherapy and surgery. [Brief explanation of the drawing]

[0015] To more clearly illustrate specific embodiments of the present invention or technical concepts in the prior art, the following briefly introduces the drawings used in describing specific embodiments or the prior art. Of course, the drawings in the following description represent only a few embodiments of the present invention, and those skilled in the art can obtain further drawings based on these without any creative effort.

[0016] [Figure 1] This is a diagram showing the configuration of the IMC002 CAR-T. [Figure 2] This is IHC staining of CLDN18.2 in normal human tissue. [Figure 3] This is the expression of CLDN18.2 in human gastric cancer tissue. [Figure 4] This is the expression of CLDN18.2 in different target cells. [Figure 5] This involves detecting CAR expression. [Figure 6] This involves detecting the ability of CAR-T cells to kill target cells. [Figure 7] This involves detecting the ability of target cells to secrete IFN-γ after CAR-T cell catalysis. [Figure 8] These are the test results for CAR-T cell toxicity, such as that of 21047. [Figure 9] 21050 These are the test results for CAR-T cell toxicity. [Figure 10A-C]Inhibition of the growth of NUGC4-luc subcutaneous transplanted tumors in mice in vivo by CAR-T cells such as 21047. [Figure 10D] Inhibition of the growth of NUGC4-luc subcutaneous transplanted tumors in mice in vivo by CAR-T cells such as 21047. [Figure 11] Binding of 21047 and primary cells (strains). [Figure 12] Killing ability of 21047 CAR-T against human and mouse lung tissues. [Figure 13] Killing ability of CAR-T such as 21047 against human primary cells / tissues. [Figure 14] Functional verification of several mutants after replacing different domains with 21047 CAR-T.

Modes for Carrying Out the Invention

[0017] Here, references to embodiments of the present invention are provided in detail, and one or more examples thereof are described below. Each example provided is for illustrative purposes and does not limit the present invention. In fact, it is obvious to those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or depicted as part of one embodiment can be used in another embodiment to generate further embodiments.

[0018] Unless otherwise noted, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. For further guidance, the following definitions are used to better understand the teachings of the present invention. In the specification of the present invention, the terms used herein are only used for the purpose of explaining specific embodiments and are not intended to limit the present invention.

[0019] As used herein, the selection of terms “and / or,” “or / and,” and “and / or” includes any one of two or more relatedly listed items, and also includes any and all combinations of relatedly listed items, the such any and all combinations include any two relatedly listed items, any more relatedly listed items, or any combination of all relatedly listed items. It should be explained that when at least three items are connected by a combination of at least two conjunctions selected from “and / or,” “or / and,” and “and / or,” it should be understood in this application that such technical proposals definitely include technical proposals that are connected by a “logical AND,” and definitely include technical proposals that are connected by a “logical OR.” For example, “A and / or B” includes three parallel technical proposals: A, B, and A+B. Furthermore, for example, the technical proposal "A, and / or B, and / or C, and / or D" includes any one of A, B, C, and D (i.e., all technical proposals connected by "theoretical sum"), as well as any and all combinations of A, B, C, and D, i.e., any two or any three combinations of A, B, C, and D, as well as any four combinations of A, B, C, and D (i.e., all technical proposals connected by "theoretical product").

[0020] As used in this invention, the terms “contains,” “includes,” and “includes” are inclusive or open synonyms that do not exclude any additional members, elements, or method steps not cited.

[0021] In this invention, the numerical range indicated by an endpoint includes all numerical values ​​and fractions contained within that range, as well as the referenced endpoint.

[0022] In this invention, unless otherwise specified, the terms "multiple" and "multiple types" mean that the number is two or more.

[0023] In the present invention, technical features described in an open format include both closed-format technical proposals composed of enumerated features and open-format technical proposals that encompass the enumerated features.

[0024] The present invention relates to a chimeric antigen receptor whose extracellular domain contains a CLDN18.2 single-domain antibody, and whose amino acid sequence of the heavy chain complementarity-determining region of the single-domain antibody is shown in SEQ ID NOs: 1-3.

[0025] As used herein, “chimeric antigen receptor (CAR)” refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide derived from the extracellular domain, and at least one intracellular domain. “Chimeric antigen receptor (CAR)” may also be referred to as “chimeric receptor” or “chimeric immune receptor (CIR).” “Extracellular domain capable of binding to an antigen” refers to any oligopeptide or polypeptide capable of binding to a particular antigen. “Intracellular domain” refers to any oligopeptide or polypeptide known to function intracellularly as a domain that transmits signals causing activation or inhibition of biological processes.

[0026] A single-domain antibody (sdAb) is an antibody that lacks the antibody light chain and possesses only the heavy chain variable region. Due to its small molecular weight, it is also called a nanobody. [Note: nanobody] TM (Nanobody TM Nanobodies TM ) and nanoclones TM (Nanoclone TM ) is a trademark of Ablynx NV. Single-domain antibodies may or may not have a constant region. In some contexts, V H Section H may be shared with Section H.

[0027] Single-domain antibodies can be obtained by activating the immune system of a dromedary, Bactrian camel, alpaca, llama, or shark with a specific antigen, and then isolating mRNA that produces a heavy-chain antibody. It has a constant region which may be derived from a dromedary, Bactrian camel, alpaca, llama, or shark. In some embodiments, it has a constant region CH1-CH5 (e.g., IgNAR). In some embodiments, it has a constant region CH2 and CH3 (hcIgG).

[0028] As further described herein, the amino acid sequence and structure (but not limited to) of a single-domain antibody include four framework regions or "FRs" isolated by three complementarity-determining regions or "CDRs".

[0029] In some embodiments, the single-domain antibody described above is humanized.

[0030] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a CDR sequence from the original animal (mouse, rabbit, etc.) into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. This overcomes the heterogeneous reactions induced by the presence of a large amount of the original animal protein component in chimeric antibodies. Such framework sequences can be obtained from a common DNA database containing germline antibody gene sequences or from disclosed references. For example, germline DNA sequences of human heavy chain and light chain variable region genes can be obtained from the "VBase" human germline sequence database (www.mrccpe.com.ac.uk / vbase) and can be found in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity due to reduced immunogenicity, activity can be maintained by performing the fewest possible reverse or reverse mutations on the above human antibody variable region framework sequence. The humanized antibody of the present invention further includes a humanized antibody that has undergone affinity maturation to the CDR by phage display. The human antibody variable region framework is selected by design, and for example, the heavy chain FR region sequence of the antibody heavy chain variable region is derived from a combination sequence of human germline heavy chain IGHV1-18*01 and hjh6.1, or a combination sequence of human germline heavy chain IGHV1-3*01 and hjh6.1, and the light chain FR region sequence of the antibody light chain variable region is derived from a combination sequence of human germline light chain IGKV1-39*01 and hjk4.1. In order to avoid a decrease in activity due to decreased immunogenicity, the fewest possible reverse mutations can be made to the human antibody variable region so as to maintain activity.

[0031] In some embodiments, the amino acid sequence of the single-domain antibody is shown in SEQ ID NO: 4.

[0032] Modified forms of single-domain antibodies are also within the scope of the present invention, for example, by covalent bonding of polyethylene glycol or other suitable polymers. Variants of single-domain antibodies are also within the scope of the present invention, among which the CDR1-CDR3 variants may each contain up to three amino acid mutations (e.g., substitution, deletion or addition of one, two or three amino acids, or any combination thereof) compared to any one of the complementarity-determining region combinations shown in SEQ ID NOs. 1-3, preferably the mutations are conservative mutations. "Conservative substitution" means substituting an amino acid in the protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation and rigidity, etc.) so that the protein can be frequently modified without changing its biological activity.

[0033] Conservative substitutions and those generally considered to be substitutions include the mutual substitution of aliphatic amino acids Ala, Val, Leu, and Ile; the exchange of hydroxyl residues Ser and Thr; the exchange of acidic residues Asp and Glu; the substitution between amide residues Asn and Gln; the exchange between basic residues Lys and Arg; and the substitution between aromatic residues Phe and Tyr. Those skilled in the art generally know that single amino acid substitutions in the non-essential region of a polypeptide do not fundamentally alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p224, (4th edition)). Furthermore, substitutions of amino acids with similar structure or function are less likely to disrupt biological activity.

[0034] In some embodiments, the chimeric antigen receptor includes a hinge region, a transmembrane region, and an intracellular signaling region.

[0035] As used herein, the “region” or “domain” contained in the above-mentioned chimeric antigen receptor refers to a region in a polypeptide that can fold into a specific structure independently of other regions. These “regions” or “domains” may be sequences of mouse or other animal origin, preferably human sequences. Furthermore, unless otherwise distinguished or emphasized, “regions” or “domains” should be understood to be known sequences and may be full-length or partially active sections.

[0036] In some embodiments, the hinge region is selected from the hinge regions of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, and CD7, or selected from the constant regions of CH3 and CH2-CH3. Preferably, the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 6 or SEQ ID NO: 7, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 8.

[0037] The transmembrane region includes the α, β, or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), and 4-1B. B(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ , IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103 It may also be one selected from ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C. In some embodiments, the transmembrane region is preferably a transmembrane region of CD8a, CD28, CD4, ICOS, CD7, CD2, CD80, CD40, OX40, CD27, LFA-1, 4-1BB, ICOS, CD3ζ, or CD3ε, more preferably the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 9, and preferably the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO: 10.

[0038] In some embodiments, the intracellular signaling region includes a CD3ζ signaling domain, and a preferred amino acid sequence is shown in SEQ ID NO: 11.

[0039] In some embodiments, the intracellular signaling region further comprises a protein or its intracellular signaling region (or referred to as a co-stimulatory region) selected from those shown in the table below, and in some embodiments, the intracellular signaling region further comprises one or more proteins or their intracellular signaling regions selected from CD28, 4-1BB, OX40, ICOS, CD27, MYD88, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80, and CD3ε.

[0040] Exemplary intracellular signaling regions and corresponding exemplary sequences used in the present invention are shown in the table below.

[0041] [Table 1] TIFF0007863857000002.tif220125 TIFF0007863857000003.tif212125

[0042] In some specific embodiments, the intracellular signaling region further comprises CD28, the amino acid sequence of which is shown in SEQ ID NO: 12, or OX40, the amino acid sequence of which is shown in SEQ ID NO: 13, or 4-1BB, the amino acid sequence of which is shown in SEQ ID NO: 14, or ICOS, the amino acid sequence of which is shown in SEQ ID NO: 15.

[0043] In some embodiments, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 5.

[0044] Variants of sequence numbers 4 / 5 are also within the scope of the present invention, and the variants may have, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in sequence numbers 4 / 5. The variants may also have the conservative substitutions described above.

[0045] A further aspect of the present invention relates to an isolated nucleic acid capable of expressing the above-mentioned chimeric antigen receptor.

[0046] In this specification, nucleic acids include variants of their conserved substitutions (e.g., degenerate codon substitutions) and complementary sequences, including variants that are more efficiently expressed in desired host cells through codon optimization. Nucleic acids are typically RNA or DNA, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. When nucleic acids are functionally related with another nucleic acid sequence, the nucleic acids are "effectively ligated". For example, when a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively ligated to the coding sequence. When ligated into a vector, it is preferable to use DNA nucleic acids.

[0047] The present invention further relates to a vector containing the above-mentioned nucleic acid.

[0048] The term "vector" refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it enables the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements it contains can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, CRISPR / CAS plasmids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or artificial chromosomes derived from P1 (PACs), phages such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomabaculoviruses (e.g., SV40). In some embodiments, the vectors described in the present invention include regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRESs), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences).

[0049] The present invention further relates to immune cells expressing the above-mentioned chimeric antigen receptor. The immune cells are, for example, one or more of the following: T cells, B cells, NK cells, macrophages, dendritic cells, etc.

[0050] In some embodiments, the immune cells are T cells.

[0051] The T cells may be one or more of the subclasses well known in the field, such as helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδT cells.

[0052] A further aspect of the present invention relates to a pharmaceutical composition containing the above-mentioned immune cells.

[0053] The above pharmaceutical compositions may further contain pharmaceutically acceptable carriers. As used herein, “pharmaceutically acceptable carriers” include any material that, when combined with the active ingredient, allows the ingredient to maintain its biological activity and not react with the target immune system. Examples include, but are not limited to, standard drug carriers (e.g., phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions)) and any one of various types of wetting agents. Exemplary diluents used for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing, 2005).

[0054] A further aspect of the present invention relates to the application of the above-mentioned immune cells in the manufacture of drugs for killing CLDN18.2-positive tumor cells.

[0055] In some embodiments, the drug is used for the prevention or treatment of gastric cancer.

[0056] A further aspect of the present invention relates to a method for treating a tumor in a patient in need thereof, comprising administering a therapeutically effective amount of the above-mentioned immune cells or pharmaceutical composition to the patient.

[0057] The tumor is preferably a solid tumor, and in the present invention, "solid tumor" includes tumors arising from any lesion of bone, bone junctions, muscles, lungs, trachea, heart, spleen, arteries, veins, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, cecum, liver, gallbladder, pancreas, parotid gland, sublingual gland, uronephritis, ureter, bladder, urethra, ovaries, fallopian tubes, uterus, vagina, vulva, scrotum, testes, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland, parathyroid gland, adrenal gland, pituitary gland, pineal gland, pancreatic islets, thymus, or gonads.

[0058] In some embodiments, tumor cells in a tumor are CLDN18.2 positive.

[0059] In some embodiments, the tumor is gastric cancer, pancreatic cancer, or esophageal cancer.

[0060] It should be understood that the proposed treatment methods also include the administration of other immunotherapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenovirus vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), and antibodies (e.g., those that bind to tumor-associated antigens or patient-specific neo-tumor antigens), stem cell grafts (e.g., xenovirus or autologous), and tumor-targeting cytokines (e.g., NHS-IL12, where IL-12 is conjugated to tumor-targeting antibodies or fragments thereof).

[0061] "Patient" refers to mammals, including but not limited to humans, monkeys, pigs and other farm animals, sporting animals, pets, primates, horses, dogs, cats, pandas, and rodents (including mice, rats, and guinea pigs).

[0062] Embodiments of the present invention will be described in detail below with reference to examples. These examples are for illustrative purposes only and do not limit the scope of the present invention. In the following examples, for experimental methods where specific conditions are not specified, it is preferable to refer to the guidelines provided in the present invention, to follow experimental manuals or common conditions in the art, to refer to other experimental methods known in the art, or to follow conditions proposed by the manufacturer.

[0063] In the following specific examples, unless otherwise specified, minor deviations may exist within the range of weighing accuracy regarding the measurement parameters of the raw material components. Regarding temperature and time parameters, acceptable deviations due to the test accuracy or operating accuracy of the equipment are permitted. [Examples]

[0064] 1. Research on the expression status of CLDN18.2 target tissues The human CLDN18 gene has two selectable exons, giving rise to two distinct protein isomers, CLDN18.1 and CLDN18.2 (only eight amino acids differ between the extracellular domains of CLDN18.1 and CLDN18.2). The two isotypes have different lineage functions: CLDN18.1 is primarily expressed in lung tissue, while CLDN18.2 exhibits primarily gastric specificity. The CLDN18.2 protein is expressed in short-cycle differentiated cells rather than in the stem cell region of the gastric mucosa, making it a highly selective gastric lineage marker.

[0065] To study the distribution of CLDN18.2 in normal human tissues and organs, we detected CLDN18.2 expression in major normal human organs using a CLDN18.2-specific IHC antibody. As shown in Figure 2, CLDN18.2 was mainly expressed in gastric tissue, expressed in small amounts in pancreatic tissue, and hardly expressed in other tissues.

[0066] To further investigate the expression status of CLDN18.2 in gastric tumor tissue, we detected CLDN18.2 expression in human gastric tumor tissue using a CLDN18.2-specific antibody. The detection results showed that CLDN18.2 was expressed relatively highly in more than 30% of gastric cancer cell membranes and was highly expressed in glandular epithelial cells of normal gastric tissue and paragastric cancerous tissue. The results are shown in Figure 3.

[0067] 2. CLDN18.2 Research on the in vitro function of CAR-T cells The inventors first studied the in vitro target cell and cytokine secretion killing ability of CLDN18.2 CAR-T cells induced by different extracellular regions such as 21008, 21009, 21011, 21047, and 21050 (sequences shown in SEQ ID NOs. 16, 17, 18, 4, and 19, respectively). The fragments adopted from other parts of these CARs are the same as those in SEQ ID NO. 5, i.e., the fragments obtained by splicing SEQ ID NOs. 6-8. Since CLDN18.2 differs from the extracellular region of another isotype, CLDN18.1, by only seven amino acids, the inventors first constructed cell lines expressing CLDN18.2 and CLDN18.1 and identified the expression levels of CLDN18.2 and CLDN18.1 in different target cells to study whether CAR-T cells can recognize the difference between CLDN18.2 and CLDN18.1. Flow cytometry (FCM) staining revealed, as shown in Figure 4, that gastric cancer cell line NUGC4 cells highly expressed CLDN18.2, N87-luc-18.1 cells moderately expressed CLDN18.1 and did not express CLDN18.2, and 293T cells did not express either CLDN18.1 or CLDN18.2.

[0068] To investigate whether CAR-T cells such as 21008, 21009, 21011, 21047, and 21050 are specifically activated by CLDN18.2 target cells, the inventors constructed several CAR-T cells and identified CAR expression, and the results are shown in Figure 5.

[0069] The CAR-T cells constructed as described above were co-incubated with target cells, and the ability of effector cells to lyse target cells was detected using the LDH method. As shown in Figure 6, all five CAR-T cells tested were able to clearly kill CLDN18.2-positive target cells NUGC4, while simultaneously not killing negative target cells N87-luc-CLDN18.1 and 293T. This indicates that the five CAR-T cells can specifically recognize CLDN18.2 but not CLDN18.1.

[0070] After co-incubating the CAR-T cells constructed as described above with target cells, the release of the cytokine IFN-γ was detected. As shown in Figure 7, when five types of CAR-T cells, including 21047, were co-incubated with CLDN18.2-positive target cells NUGC4, their IFN-γ secretion capacity was activated, and the secretion level reached over 5000 pg / mL. However, when co-incubated with negative target cells N87-luc-18.1 and 293T, cytokine secretion was lower than 100 pg / mL, indicating that the CAR-T cells under test can be specifically activated by NUGC4, while they cannot be activated when co-incubated with negative target cells N87-luc-18.1 and 293T.

[0071] 3. CLDN18.2 Study on the in vivo safety of single-dose administration of CAR-T cells The inventors further studied the in vivo safety of five CAR-Ts, including 21008, 21009, 21011, 21047, and 21050.

[0072] In in vivo experiments, the inventors compared four CAR-T cells, 21008, 21009, 21011, and 21047, and in an NSG mouse model, they found that 5 × 10⁻⁶ cells were present. 6CAR-T was administered once at a dose of cells / mouse. After infusion, the mice were observed twice a week for their condition and their body weights were measured. Throughout the experimental period, no obvious abnormalities were observed in the mice in the 21047 CAR-T 5M group, and the body weights of the mice did not significantly decrease throughout the study period (Figure 8). In 21011, obvious weight loss occurred and then gradually recovered. At the end of the study, the inventors collected the vital organs of each mouse and performed H&E staining. The pathological results are shown in the following table.

[0073]

Table 2

[0074] In addition to the 21047 and unT groups, the organs of the mice in the 21008, 21009, and 21011 treatment groups all showed certain tissue damage or obvious histological changes (including the brain, heart, liver, lungs, kidneys, and stomach). In short, 21047 CAR-T cells have good safety in the mouse model.

[0075] In another experiment, the inventors compared the safety of single-dose administration of 21050 and unT in the NSG mouse model at a dose of 5×10 6 cells / mouse. After infusion, the mice were observed twice a week for their condition and their body weights were measured. Throughout the experimental period, obvious abnormalities were observed in the mice in the 21050 CAR-T 5M group, the body weights of the mice significantly decreased throughout the study period, and deaths occurred (Figure 9A / B), indicating that 21050 also has obvious toxicity in the mouse model.

[0076] 4. Study on the in vivo efficacy of CLDN18.2 CAR-T cells To study the ability of five CAR-Ts such as 21047 to remove tumor cells in vivo, the inventors constructed a subcutaneous transplanted tumor model of NUGC4-luc cells based on NCG immunodeficient mice and studied the efficacy of CAR-T. Specifically, using a 1 mL syringe, 100 μL (cell number: 5×10 6The human gastric cancer cell line NUGC4-luc was inoculated subcutaneously into the back of NCG mice to establish a subcutaneous transplant tumor model, and CAR-T cell injection was started when the tumor reached approximately 50-150 mm3. As shown in Figure 10, 1 × 10⁶ cells were injected into different groups of mice. 6 CAR-T cells or untransduced T cells (unT) were reinjected. Subsequently, tumor growth, mouse body weight, and health status were continuously monitored, and finally, subcutaneous tumor growth curves and mouse body weight gain curves were plotted using Graph Pad Prism 7.0 graphics software (mean ± sem). As shown in Figure 10A, at the end of the study, both molecules 21050 and 21047 achieved tumor clearance. On the other hand, neither molecules 21008 nor 21009 achieved complete tumor clearance. The inventors further compared tumor sizes at D35 and found that the order of tumor size was 21050 < 21047 < 21009 = 21008 (Figure 10B). Furthermore, the tumor size in the 21008 and 21009 treatment groups was not significantly different from that of the unT group, indicating a relatively weaker drug effect (Figure 10B). During the study period, the body weight of the 21047 treatment group showed no significant change, while the 21008, 21009, 21011, and 21050 groups all experienced significant weight loss, deterioration of condition, or death in one or more mice (Figures 10C and 10D). These results demonstrate that 21047 possesses both a clear safety advantage and clear therapeutic efficacy compared to other molecules.

[0077] 5. CLDN18.2 In vitro safety study of CAR-T To further investigate the nonspecific binding of 21047 to primary cells, the inventors incubated 21047 diluted to various concentrations with CLDN18.2-positive target cells NUGC4 or primary cell lines, then detected MFI by flow cytometry. Using IMAB362 as a positive control antibody and isotype as a negative control antibody, the results, as shown in Figure 11, demonstrated that 21047 and IMAB362 bound normally to CLDN18.2-positive target cells NUGC4 but did not bind to primary cells such as colon, ARPE-19, Beas-2B, and Intestine.

[0078] Since lung tissue cells express CLDN18.1, the inventors studied the cytokine release capacity upon killing human lung tissue, lung cancer, paracancerous tissue, and mouse lung tissue to investigate whether CLDN18.2 CAR-T cells are toxic to lung tissue. As shown in Figure 12, when 21047 CAR-T cells were co-incubated with human and mouse lung tissue, the IFN-γ secretion level did not increase significantly. Simultaneously, when co-incubated with 293-CLDN18.2 cells, the IFN-γ secretion level reached a high of over 10,000 pg / mL. As a negative control, when co-incubated with N87-luc-18.1, the IFN-γ secretion level did not increase significantly. These results indicate that 21047 CAR-T cells did not exhibit significant toxicity to lung tissue.

[0079] To further verify the safety of 21047 CAR-T, the inventors analyzed the killing ability of CAR-T cells, including 21047, prepared by PBMCs from three different source donors, against human primary tissues / cells (lines), including Beas-2B (human primary lung cell line), ARPE-19 (human retinal epithelial cells), primary colon cells (human primary colon cells), HIBEpiC (human intrahepatic bile duct epithelial cells), primary intestine cells (human primary small intestine cells), human lung tissue, and mouse lung tissue. As shown in Figure 13A / C, after co-incubating 21011 and 21050 with primary tissue / cell cells, CAR-T cells showed a clear increase in IFN-γ in some cells, such as colon cells or primary lung cells, suggesting that 21011 and 21050 may have potential tissue toxicity. On the other hand, after co-incubating 21047 CAR-T and unT (Untransduced T cells) cells with human primary cell cells, IFN-γ secretion levels did not increase significantly. Simultaneously, when co-incubated with CLDN18.2-positive cells NUGC4, IFN-γ secretion levels reached high levels of over 5000 pg / mL. The results are shown in Figure 13B / D. These results indicate that 21047 CAR-T cells do not nonspecifically bind to or activate human lung, eye, colon, small intestine, or hepatic bile duct epithelial cells, suggesting relatively good safety.

[0080] 6. In vitro function studies of CLDN18.2 CAR-T in different signaling domains To study the effects of different hinge region, transmembrane domain, and intracellular signaling domain combinations on CAR-T function, the inventors further constructed four different hinge region, transmembrane domain, and intracellular domain combinations based on 21047, the structures of which are shown in the table below.

[0081] [Table 3]

[0082] In vitro, the ability to kill target cells and cytokine secretion after target cell elimination were compared between different structures, and the results are shown in Figure 14. The results showed that CAR-T molecules with different hinge region, transmembrane region, and costimulatory domain structures could all kill positive target cells and secrete cytokines such as IL2 and IFN-γ normally. Molecules such as 21047lib-35B (CD8-OX40-CD3ζ structure), 21047lib-41B (CD28-41BB-CD3ζ structure), and 21047lib-42B (CD8-ICOS-CD3ζ structure) did not show significant differences in target cell elimination ability and cytokine secretion ability compared to the original 21047 (CD28-CD3ζ structure).

[0083] The above examples merely illustrate some embodiments of the present invention, and although the description is relatively specific and detailed, it should not be interpreted as limiting the scope of the patent of the present invention. Furthermore, for those skilled in the art, several modifications and improvements are possible without departing from the spirit of the present invention, and all of these are included within the scope of the claims. Accordingly, the scope of the patent claims of the present invention is based on the appended claims, and the specification and drawings can be used to illustrate the content of the claims.

Claims

1. A chimeric antigen receptor, wherein its extracellular domain contains a CLDN18.2 single-domain antibody, and the amino acid sequences of the heavy chain complementarity-determining regions 1 to 3 of the single-domain antibody are shown in SEQ ID NOs: 1 to 3, respectively. Chimeric antigen receptor.

2. The aforementioned single-domain antibody is humanized. The chimeric antigen receptor according to feature 1.

3. The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:

4. The chimeric antigen receptor according to feature 2.

4. Including the hinge region, transmembrane region and intracellular signaling region, A chimeric antigen receptor according to any one of claims 1 to 3.

5. The hinge region is selected from the hinge regions of CD8, CD28, IgG1, IgG4, 4-1BB, ICOS, OX40, CD40, CD80, CD7, or the steady-state regions of CH3, CH2-CH3. The chimeric antigen receptor according to feature 4.

6. The aforementioned transmembrane region is selected from the transmembrane regions of CD8a, CD28, CD4, ICOS, CD7, CD2, CD80, CD40, OX40, CD27, LFA-1, 4-1BB, ICOS, CD3ζ, or CD3ε. The chimeric antigen receptor according to feature 4.

7. The intracellular signaling region includes a CD3ζ signaling domain. The chimeric antigen receptor according to feature 4.

8. The intracellular signaling region further comprises one or more selected from the proteins CD28, 4-1BB, OX40, ICOS, CD27, MYD88, KIR2DS2, DAP10, DAP12, CD3ζ, TLRs, CD2, LFA-1, CD8α, CD40, CD80, and CD3ε or their intracellular signaling regions. The chimeric antigen receptor according to feature 4.

9. The amino acid sequence is shown in Sequence ID No.

5. The chimeric antigen receptor according to feature 4.

10. Isolated nucleic acids, This can be obtained by expressing the chimeric antigen receptor described in any one of claims 1 to 3. Isolated nucleic acid characterized by the following features.

11. A nucleic acid comprising the nucleic acid described in claim 10, vector.

12. Expressing a chimeric antigen receptor according to any one of claims 1 to 3, immune cells.

13. Selected from T cells, B cells, NK cells, macrophages, and dendritic cells, The immune cell according to feature 12.

14. The T cells are selected from helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells and γδT cells, The immune cell according to feature 13.

15. A pharmaceutical composition, The immune cells described in claim 12, Pharmaceutical composition.

16. A drug for killing CLDN18.2-positive tumor cells, comprising the immune cells described in claim 12.

17. The aforementioned drug is used for the prevention or treatment of gastric cancer, pancreatic cancer, or esophageal cancer. The drug according to claim 16.

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