Chimeric antigen receptor comprising a CLDN18.2 single domain antibody and its application

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 to Related Applications] This application claims the priority of the patent application CN2022105948645 filed on May 27, 2022, the entire content of which is incorporated herein by reference.

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

Background Art

[0003] Currently, the treatment principle for advanced gastric cancer is still comprehensive treatment mainly based on systemic chemotherapy, including systemic chemotherapy, surgical resection, radiotherapy, targeted therapy, immunotherapy, and traditional Chinese medicine therapy. As the continuous optimization of chemotherapeutic drugs and chemotherapy regimens progresses, the survival period of advanced gastric cancer patients is clearly extended, but it is still difficult for the median survival period of patients to exceed 1 year.

[0004] The development of immunotherapy in recent years has brought profound changes in the field of tumor treatment, especially in immune checkpoint therapy represented by CTLA - 4, PD1 / PDL1 pathway inhibitors and adoptive cell therapy represented by CAR - T. Adoptive cell therapy includes TIL, NK, TCR - T, CAR - T / NK / NKT / TIL / Mφ, etc. Among them, CAR - T therapy targeting CD19 has achieved excellent clinical effects in B - cell tumors, and in 2017, two CAR - T products were approved by the FDA for the treatment of B - cell leukemia or lymphoma.

[0005] Targeted therapy and immune checkpoint inhibitors have brought benefits to specific populations, but there is no waiting time to find other targets at the advanced stage of gastric cancer.

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

[0007] Currently, global product types against CLDN18.2 include monoclonal antibodies, bispecific antibodies, CAR-T, and ADC. Among them, the number of monoclonal antibodies under development is the largest. Currently, among the drugs developed against CLDN18.2, the one progressing most rapidly is IMAB362 (Zolbetuximab, claudiximab) developed by German company Ganymed. IMAB362 is a human-mouse chimeric antibody that can specifically recognize and bind to the extracellular domain ECL1 of the claudin18.2 protein but does not bind to any other claudin family members (including claudin18.1). Currently, it has already moved into Phase III clinical trials.

[0008] CAR-T has achieved significant progress in hematological malignancies, but its efficacy in the treatment of solid tumors is low. One of the important reasons is that solid tumors do not have good targets like those in hematological malignancies, and the receptor sequences of conventional CAR-T are derived from mice and have relatively strong affinity. Therefore, solid tumor CAR-T always has a stronger on-target off-tumor effect and relatively large side effects. In addition, the molar mass of mouse-derived scfv is relatively large and the immunogenicity is relatively high, so it is easy to form anti-antibodies in patients in vivo, and as a result, CAR-T is rapidly removed by antibodies generated by the host in vivo, and it cannot continuously relieve tumors in patients, which is also one of the reasons for the low efficacy of CAR-T in solid tumors.

Summary of the Invention

[0009] One object of the present invention is to provide a chimeric antigen receptor whose extracellular domain contains 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 chimeric antigen receptor.

[0011] The present invention further provides immune cells expressing the above chimeric antigen receptor.

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

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

[0014] The inventors of the present invention have unexpectedly found through tests that excellent technical effects can be obtained when the selected single-domain antibody of the present invention is constructed into a chimeric antigen receptor. The constructed chimeric antigen receptor has good specificity, has higher safety and lower toxicity in in vivo use, and can currently provide a better treatment regimen for patients with advanced gastric cancer highly expressing the CLDN18.2 protein who are being treated with radiotherapy, chemotherapy and surgery.

Brief Description of Drawings

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Of course, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10A-C

Figure 10D

Figure 11

Figure 12

Figure 13

Figure 14

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 apparent 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 defined, 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 for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0019] As used herein, the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items, where any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted 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", in this application, it is understood that the technical solution must include technical solutions connected by "logical product" and must also include technical solutions connected by "logical sum". For example, "A and / or B" includes three parallel technical solutions: A, B, and A + B. Also, for example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., technical solutions connected by "logical sum"), and also includes any and all combinations of A, B, C, and D, that is, combinations of any two or any three of A, B, C, and D, and also includes the combination of A, B, C, and D (i.e., technical solutions connected by "logical product").

[0020] As used in the present invention, the terms "comprising", "including", and "containing" are synonymous and are inclusive or open-ended, and do not exclude additional members, elements, or method steps not recited.

[0021] In the present invention, a numerical range indicated by endpoints includes all numerical values and fractions within the range, as well as the recited endpoints.

[0022] In the present invention, descriptions such as "a plurality" and "a plurality of types" mean that the number is two or more unless otherwise specifically limited.

[0023] In the present invention, technical features described in an open format include closed technical solutions consisting of the recited features, and also include open technical solutions including the recited features.

[0024] The present invention relates to a chimeric antigen receptor, the extracellular domain of which comprises a CLDN18.2 single domain antibody, and the amino acid sequences of the heavy chain complementarity determining regions of the single domain antibody are shown in SEQ ID NOs: 1 to 3.

[0025] As used herein, a "chimeric antigen receptor (CAR)" refers to a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. A "chimeric antigen receptor (CAR)" may also be referred to as a "chimeric receptor" or "chimeric immune receptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function within a cell as a domain that transmits a signal that causes activation or inhibition of a biological process.

[0026] Single domain antibodies (sdAbs) are antibodies that lack the antibody light chain and have only the heavy chain variable region. Due to their small molecular weight, they are also called nanobodies. TM (Nanobody TM , Nanobodies TM ) and Nanoclone TM (Nanoclone TM ) are trademarks of Ablynx NV. Single domain antibodies may or may not have constant regions. In some contexts, V H May be common with H section.

[0027] Single-domain antibodies can be obtained by activating the immune system of camels, dromedaries, alpacas, llamas or sharks with a specific antigen and then isolating the mRNA that produces the heavy-chain antibodies. It has a constant region that can be derived from camels, dromedaries, alpacas, llamas or sharks. In some embodiments, it has constant regions CH1-CH5 (e.g., IgNAR). In some embodiments, it has constant regions CH2 and CH3 (hcIgG).

[0028] As further described herein, the amino acid sequences and structures of single-domain antibodies (but not limited thereto) include four framework regions or "FRs" isolated by three complementarity-determining regions or "CDRs".

[0029] In some embodiments, the single-domain antibodies are humanized.

[0030] The term "humanized antibody" is also called a CDR-grafted antibody, and refers to an antibody produced by transplanting the CDR sequences of an animal origin (such as mouse, rabbit, etc.) into a human antibody variable region framework, that is, different types of human germline antibody framework sequences. Chimeric antibodies can overcome the heterologous reactions induced by having a large amount of the first animal origin protein components. Such framework sequences can be obtained from a common DNA database containing germline antibody gene sequences or disclosed references. For example, the germline DNA sequences of human heavy 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, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing the least number of reverse mutations or revertant mutations on the above human antibody variable region framework sequences. The humanized antibodies of the present invention further include humanized antibodies after affinity maturation of the CDRs by phage display. The human antibody variable region framework is selected by design. For example, among them, the heavy chain FR region sequence of the above antibody heavy chain variable region is derived from the combined sequence of human germline heavy chain IGHV1-18*01 and hjh6.1, or the combined sequence of human germline heavy chain IGHV1-3*01 and hjh6.1, and among them, the light chain FR region sequence of the above antibody light chain variable region is derived from the combined sequence of human germline light chain IGKV1-39*01 and hjk4.1. In order to avoid a decrease in activity associated with a decrease in immunogenicity, the least number of reverse mutations can be performed on the above human antibody variable region so that the activity is maintained.

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

[0032] Modified forms of single-domain antibodies are also within the scope of the claims of the present invention and are modified, for example, by covalent attachment of polyethylene glycol or other suitable polymers. Variants of single-domain antibodies are also within the scope of the present invention, among which the variants of the above CDR1 to CDR3 can each contain up to three amino acid mutations (for example, substitution, deletion or addition of one, two or three amino acids, or any combination thereof) compared to any one of the combinations of complementarity-determining regions shown in SEQ ID NOs: 1 to 3, and preferably, the above mutations are conservative mutations. "Conservative substitution" means substituting an amino acid in a protein with another amino acid having similar characteristics (such as charge, side-chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) to make it frequently changeable without changing the biological activity of the protein.

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

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

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

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

[0037] The transmembrane region may be any one selected from the α, β, or ζ chain of the T cell receptor, 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), 4-1BB (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, 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 the 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. 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 the CD3ζ signaling domain, and the preferred amino acid sequence is shown in SEQ ID NO: 11.

[0039] In some embodiments, the intracellular signaling region further comprises a protein selected from those shown in the following table or its intracellular signaling region (or co-stimulatory region), and in some embodiments, the intracellular signaling region 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.

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

[0041] [Table 1] TIFF2025518100000003.tif220125 TIFF2025518100000004.tif212125

[0042] In some specific embodiments, the intracellular signaling region further comprises CD28 whose amino acid sequence is shown in SEQ ID NO: 12, or comprises OX40 whose amino acid sequence is shown in SEQ ID NO: 13, or comprises 4-1BB whose amino acid sequence is shown in SEQ ID NO: 14, or comprises ICOS whose amino acid sequence 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 SEQ ID NO: 4 / 5 are also within the scope of the claims of the present invention, and the variants can 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 to the sequence shown in SEQ ID NO: 4 / 5. The variants may also have the conservative substitutions described above.

[0045] According to a further aspect of the invention, it further relates to an isolated nucleic acid capable of expressing the chimeric antigen receptor.

[0046] As used herein, nucleic acids include variants of conservative substitutions thereof (e.g., substitution of degenerate codons) and complementary sequences, and also include variants that are more efficiently expressed in a desired host cell by codon optimization. Nucleic acids are usually RNA or DNA, including fragments thereof such as genes, cDNA molecules, mRNA molecules, and oligonucleotides. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked". For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence. When ligating to a vector, it is preferred to use DNA nucleic acid.

[0047] The invention further relates to a vector containing the nucleic acid.

[0048] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, and the genetic elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, artificial chromosomes such as plasmids, phagemids, CRISPR / CAS plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as λ phage or M13 phage, 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, papillomavirus baculoviruses (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 (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and polyU sequences).

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

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

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

[0052] According to a further aspect of the present invention, it further relates to a pharmaceutical composition comprising the above immune cells.

[0053] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier agent. As used herein, "pharmaceutically acceptable carrier agent" includes any material that, when combined with the active ingredient, enables the above components to maintain biological activity and not react with the immune system of the subject. Examples include, but are not limited to, any one of standard drug carrier agents (e.g., phosphate buffered saline aqueous solution, water, emulsion (e.g., oil / water emulsion)), and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carrier agents 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, 21th edition, Mack Publishing, 2005).

[0054] According to a further aspect of the present invention, it further relates to the application of the above immune cells in the manufacture of a drug for killing CLDN18.2-positive tumor cells.

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

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

[0057] The tumor is preferably a solid tumor. In the present invention, "solid tumor" includes tumors arising from lesions in any of bone, bone joint, muscle, lung, trachea, heart, spleen, artery, vein, capillary, lymph node, lymphatic vessel, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, cecum, liver, gallbladder, pancreas, parotid gland, sublingual gland, urogenital kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerve, thyroid, parathyroid, adrenal gland, pituitary gland, pineal gland, pancreatic islet, thymus, gonad.

[0058] In some embodiments, the tumor cells in the 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 contemplated treatment methods include 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 called ALT-803, from Altor Biosciences), and antibodies (e.g., those binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell grafts (e.g., allogeneic or autologous) and tumor-targeted cytokines (e.g., NHS-IL12, where IL-12 is conjugated to a tumor-targeting antibody or fragment thereof), and other immunotherapeutic entities, particularly preferably also including the administration of immunotherapeutic entities.

[0061] "Patient" is a mammal including, but not limited to, human, monkey, pig and other farm animals, sports animals, pets, primates, horse, dog, cat, panda, rodents (including mouse, rat, guinea pig, etc.).

[0062] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. These examples are only for explaining the present invention 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, and it is also possible to follow the experimental manuals or normal conditions in the field, refer to other experimental methods known in the field, or follow the conditions proposed by the manufacturer.

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

Example

[0064] 1. Research on the expression status of CLDN18.2 target tissue The human CLDN18 gene has two alternative first exons and generates two different protein isoforms, CLDN18.1 and CLDN18.2 (there is only an 8 - amino - acid difference in the extracellular region 1 between CLDN18.1 and CLDN18.2). The two isoforms have different tissue functions. CLDN18.1 is mainly expressed in lung tissue, while CLDN18.2 mainly shows gastric specificity. The CLDN18.2 protein is expressed in short - cycle differentiated cells without being expressed in the stem cell region of the gastric mucosa as a highly selective gastric lineage marker.

[0065] To study the distribution of CLDN18.2 in human normal tissues and organs, the expression of CLDN18.2 in major human normal organs was detected using a CLDN18.2 - specific IHC antibody. The results showed that, as shown in Figure 2, CLDN18.2 was mainly expressed in gastric tissue, slightly expressed in pancreatic tissue, and hardly expressed in other tissues.

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

[0067] 2. Research on the in vitro function of CLDN18.2 CAR-T cells First, the inventors studied the killing ability of CLDN18.2 CAR-T cells induced by different extracellular regions (sequences are shown in SEQ ID NO: 16, 17, 18, 4, and 19 in order), such as 21008, 21009, 21011, 21047, and 21050, on target cells and cytokine secretion in vitro. The fragments used in other parts of these CARs are the same as SEQ ID NO: 5, that is, the fragments obtained by splicing SEQ ID NO: 6-8. Since CLDN18.2 differs from the extracellular region of another isoform, CLDN18.1, by only seven amino acids, in order to study whether CAR-T can recognize the difference between CLDN18.2 and CLDN18.1, 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. By flow cytometry (FCM) staining, the results are as shown in Figure 4. The gastric cancer cell line NUGC4 cells highly expressed CLDN18.2, N87-luc-18.1 moderately expressed CLDN18.1 and did not express CLDN18.2, and 293T cells did not express CLDN18.1 and CLDN18.2.

[0068] To study 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 the expression of CAR. 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 Fig. 6, all five types of CAR-T tested were able to clearly kill CLDN18.2-positive target cells NUGC4, and at the same time did not kill negative target cells N87-luc-CLDN18.1 and 293T, indicating that the five types of CAR-T cells could specifically recognize CLDN18.2 and did not recognize 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 Fig. 7, after incubating five types of CAR-T such as 21047 with CLDN18.2-positive target cells NUGC4, the secretion ability of IFN-γ was activated, and the secretion level of IFN-γ reached 5000 pg / mL or more. After incubating with negative target cells N87-luc-18.1 and 293T, the cytokine secretion was lower than 100 pg / mL, indicating that the CAR-T tested could be specifically activated by NUGC4, while it could not be activated after incubating with negative target cells N87-luc-18.1 and 293T.

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

[0072] In the in vivo experiment, the inventors compared four types of CAR-T, 21008, 21009, 21011, and 21047. In the NSG mouse model, the inventors used 5×10 6A single dose of CAR-T was administered at a dose of cells / mouse. After infusion, the condition of the mice was observed twice a week 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 important 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 a single dose of 21050 and unT of CAR-T in the NSG mouse model at a dose of 5×10 6 cells / mouse. After infusion, the condition of the mice was observed twice a week 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 tumor cell elimination ability in vivo of five CAR-Ts such as 21047, 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 of ) was inoculated subcutaneously into the back of NCG mice to establish a subcutaneous transplanted tumor model, and the injection of CAR-T cells was started when the tumor reached about 50-150 mm3. As shown in Figure 10, 1×10 6 CAR-T or non-transduced T cells (unT) were reinjected into mice in different groups. Subsequently, the tumor growth status, body weight, and health status of the mice were continuously monitored, and finally, the mouse subcutaneous tumor growth curve and mouse body weight gain curve were drawn using Graph Pad Prism 7.0 graphic software (mean±s.e.m.). As shown in Figure 10A, at the end of the study, both the 21050 and 21047 molecules achieved tumor clearance. On the other hand, neither the 21008 nor the 21009 molecule achieved complete tumor clearance. When the inventors further compared the tumor sizes at D35, the order of tumor sizes was 21050 < 21047 < 21009 = 21008 (Figure 10B). Moreover, the tumor sizes in the 21008 and 21009 treatment groups were not significantly different from those in the unT group, indicating relatively weak drug efficacy (Figure 10B). During the study period, the body weight of the 21047 treatment group did not change significantly, while in the 21008, 21009, 21011, and 21050 groups, obvious weight loss, deterioration of condition, or death occurred in one or more mice (Figures 10C and 10D). From the above results, compared with other molecules, 21047 was shown to have obvious safety advantages and obvious drug efficacy.

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

[0078] Since lung tissue cells express CLDN18.1, in order to study whether CLDN18.2 CAR-T is toxic to lung tissue, the inventors studied the cytokine release ability during the killing of human lung tissue, lung cancer, peritumoral tissue and mouse lung tissue. As shown in Figure 12, after 21047 CAR-T cells were incubated with human and mouse lung tissues, the IFN-γ secretion level did not increase significantly. At the same time, when incubated with 293-CLDN18.2 cells, the IFN-γ secretion level reached as high as 10000 pg / mL or more. As a negative control, when incubated with N87-luc-18.1, the IFN-γ secretion level did not increase significantly. The above results indicate that 21047 CAR-T did not show obvious toxicity to lung tissue.

[0079] To further verify the safety of 21047 CAR-T, the inventors analyzed the killing ability of CAR-T cells such as 21047 prepared from PBMC of three different donors against human primary tissues / cells (strains) including Beas-2B (human lung primary 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 Figures 13A / C, after co-incubating 21011 and 21050 with primary tissues / cells (strains), in some cells such as colon cells or lung primary cells, CAR-T showed an obvious increase in IFN-γ, indicating that there may be potential tissue toxicity in 21011 and 21050. On the other hand, after co-incubating 21047 CAR-T and unT (Untransduced T cells) cells with human primary cells (strains), the IFN-γ secretion level did not increase significantly. At the same time, when co-incubated with CLDN18.2-positive cell NUGC4, the IFN-γ secretion level reached as high as 5000 pg / mL or more. The results are shown in Figures 13B / D. The above results indicate that 21047 CAR-T does not bind or activate non-specifically to human lung, eye, colon, small intestine, and intrahepatic bile duct epithelial cells, and its safety is relatively good.

[0080] 6. In vitro function study of CLDN18.2 CAR-T in different signal domains To study the effect of different combinations of hinge regions, transmembrane regions, and intracellular signal domains on CAR-T function, the inventors further constructed four different combinations of hinge regions, transmembrane regions, and intracellular domains based on 21047, and the structures are shown in the following table.

[0081]

Table 3

[0082] In vitro, the killing ability of different structures against target cells and the cytokine secretion after target cell killing were compared, and the results are shown in Figure 14. The above results indicated that CAR-Ts with different hinge region, transmembrane region and co-stimulatory domain structures could all kill positive target cells and normally secrete cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). Molecules such as 21047lib-35B (CD8-OX40-CD3ζ structure), 21047lib-41B (CD28-41BB-CD3ζ structure), and 21047lib-42B (CD8-ICOS-CD3ζ structure) showed no significant difference in target killing ability and cytokine secretion ability compared with the original 21047 (CD28-CD3ζ structure).

[0083] The above examples merely illustrate some embodiments of the present invention. Although the description is relatively specific and detailed, it should not be construed as limiting the patent scope of the present invention. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the idea of the present invention, and all of these are included in the claims of the present invention. Therefore, the patent claims of the present invention are based on the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A chimeric antigen receptor, wherein its 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, Chimeric antigen receptor.

2. The single-domain antibody is humanized, The chimeric antigen receptor according to claim 1, characterized in that.

3. The amino acid sequence of the single-domain antibody is shown in SEQ ID NO: 4, The chimeric antigen receptor according to claim 2, characterized in that.

4. Comprising a hinge region, a transmembrane region, and an intracellular signaling region, The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that.

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

6. The 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 claim 4, characterized in that.

7. The intracellular signaling region comprises a CD3ζ signaling domain, The chimeric antigen receptor according to claim 4, characterized in that.

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 claim 4, characterized in that.

9. Its amino acid sequence is shown in SEQ ID NO: 5, The chimeric antigen receptor according to claim 4, characterized in that.

10. An isolated nucleic acid, Which can be obtained by expressing the chimeric antigen receptor according to any one of claims 1 to 9, An isolated nucleic acid, characterized in that.

11. A vector comprising the nucleic acid according to claim 10, Vector.

12. An immune cell that expresses the chimeric antigen receptor according to any one of claims 1 to 9, Immune cell.

13. Selected from T cells, B cells, NK cells, macrophages, and dendritic cells, The immune cell according to claim 12, characterized in that...

14. The T cell is selected from helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, MAIT cells, NKT cells, and γδ T cells, The immune cell according to claim 13, characterized in that...

15. A pharmaceutical composition, comprising the immune cell according to any one of claims 12 to 14, The pharmaceutical composition.

16. Use of the immune cell according to any one of claims 12 to 14 in the manufacture of a drug for killing CLDN18.2-positive tumor cells.

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

Images