TRuC-T cells for improving safety of targeted CLDN18.2 immune cells and application of TRuC-T cells

Abstract

The invention discloses TRuC-T cells capable of improving safety of targeted CLDN18.2 immune cells and application of the TRuC-T cells. The TRuC-T cells comprise AB011TRuC-T and F2H TRuC-T. According to the TRuC-T cells, the effective killing capacity on tumor cells is reserved, meanwhile, toxicity to normal stomach tissue is remarkably reduced, the technical problem of stomach side effects in an existing CLDN18.2 targeted therapy is effectively solved, and the TRuC-T cells can be used for improving the safety of the targeted CLDN18.2 immune cells. The invention provides a better choice for precise treatment of CLDN18.2 positive solid tumors, and has important scientific significance and clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a type of TruC-T cell that enhances the safety of targeting CLDN18.2 immune cells and its application. Background Technology

[0002] CLDN18.2 (tight junction protein 18.2) is an important member of the Claudin protein family. Under normal physiological conditions, it is expressed at low levels in gastric mucosal differentiated epithelial cells, responsible for maintaining tight junctions and barrier function. However, CLDN18.2 expression is significantly upregulated in various solid tumors, such as in approximately 70%-80% of gastric cancer, 60% of pancreatic cancer, and some esophageal, ovarian, and lung cancer patients. This tumor-specific expression pattern makes it an ideal target for solid tumor therapy. Based on this, CLDN18.2 CAR-T therapy has emerged. This therapy involves genetically engineering the patient's T cells to express a chimeric antigen receptor (CAR) targeting CLDN18.2, thereby precisely recognizing and killing tumor cells.

[0003] Although CLDN18.2 CAR-T therapy has made breakthroughs in the treatment of solid tumors, its potential toxicity to normal gastric tissue cannot be ignored. Since CLDN18.2 is still expressed at low levels in normal gastric mucosa, CAR-T cells may inadvertently damage healthy gastric mucosa, leading to the following side effects: (1) Gastric mucosal damage: In the phase I clinical trial of CT041, 8 patients (8.2%) experienced gastric mucosal damage, including 7 cases of grade 1-2 gastric erosion and 1 case of grade 3 gastric erosion (which healed after treatment). This damage was caused by CAR-T cells recognizing and attacking residual CLDN18.2 in normal gastric mucosa, manifesting as symptoms such as upper abdominal pain and gastrointestinal bleeding. (2) Hypoalbuminemia: CLDN18.2 targeted therapy (including CAR-T, monoclonal antibodies and ADCs) is often accompanied by hypoalbuminemia, which may be related to increased gastrointestinal permeability or mucosal edema leading to protein loss. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the art with a type of TRuC-T cell that improves the safety of targeting CLDN18.2 immune cells and its application.

[0005] This invention addresses the technical challenge of gastric side effects by constructing CLDN18.2 CAR-T modified TruC-T cells, preserving the original antibody's effectiveness in specifically recognizing tumors. Furthermore, increasing the dosage enhances the tumor-killing effect of TruC-T cells without altering their ability to combat gastric side effects. This provides new insights and approaches for the clinical treatment of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, and other types of CLDN18.2-positive cancers.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0007] A first aspect of the present invention provides a TCR fusion construct (TRuC) targeting CLDN18.2.

[0008] Furthermore, the TCR fusion construct comprises an antibody targeting CLDN18.2, a linker, and a TCR complex subunit fusion portion.

[0009] Furthermore, the amino acid sequence of the antibody targeting CLDN18.2 is shown in SEQ ID NO:2 or SEQ ID NO:3;

[0010] Optionally, the nucleotide sequence of the antibody targeting CLDN18.2 is shown in SEQ ID NO:12 or SEQ ID NO:13.

[0011] Furthermore, the linker is G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6 or EAAAK;

[0012] Optionally, the linker is a G4S;

[0013] Optionally, the amino acid sequence of the G4S is shown in SEQ ID NO:9;

[0014] Optionally, the nucleotide sequence of the G4S is shown in SEQ ID NO:19;

[0015] Optionally, the TCR complex subunit fusion portion is CD3ε, CD3γ, or CD3δ;

[0016] Optionally, the TCR complex subunit fusion portion is CD3ε;

[0017] Optionally, the amino acid sequence of CD3ε is shown in SEQ ID NO:10;

[0018] Optionally, the nucleotide sequence of CD3ε is shown in SEQ ID NO:20;

[0019] Optionally, the TCR fusion construct further comprises a signal peptide;

[0020] Optionally, the signal peptide is the effervescent signal peptide CD8a SP or the effervescent signal peptide GMCSF SP;

[0021] Optionally, the signal peptide is the exomembrane signal peptide CD8a SP;

[0022] Optionally, the amino acid sequence of the exomembrane signal peptide CD8a SP is shown in SEQ ID NO:1;

[0023] Optionally, the nucleotide sequence of the exomembrane signal peptide CD8a SP is shown in SEQ ID NO:11;

[0024] Optionally, the TCR fusion construct further comprises cytokines, p40, and / or TGF-β-RII;

[0025] Optionally, the cytokines are IL-2, IL-7, IL-10, IL-12, IL-15 and / or IL-21;

[0026] Optionally, the TCR fusion construct is obtained by sequentially connecting a signal peptide, an antibody targeting CLDN18.2, a linker, and a TCR complex subunit fusion moiety.

[0027] Optionally, the TCR fusion construct is obtained by sequentially connecting the exomembrane signal peptide CD8a SP, the antibody targeting CLDN18.2, G4S, and CD3ε.

[0028] Optionally, the TCR fusion construct is obtained by sequentially connecting a signal peptide, an antibody targeting CLDN18.2, a linker, a TCR complex subunit fusion moiety, the cytokine, p40, and / or TGF-β-RII.

[0029] Optionally, the TCR fusion construct is obtained by sequentially connecting the exomembrane signal peptide CD8a SP, the antibody targeting CLDN18.2, G4S, CD3ε, the cytokine, p40 and / or TGF-β-RII.

[0030] In some embodiments, those skilled in the art may modify the combination type and sequence of the signal peptide, the antibody targeting CLDN18.2, the linker, and the TCR complex subunit fusion portion according to actual circumstances or needs. Regardless of the form of modification, it is included within the scope of protection of this invention.

[0031] In some embodiments, the TCR fusion constructs provided by the present invention include not only the TCR fusion constructs with amino acid sequences as described in the first aspect of the present invention, but also mutants of the TCR fusion constructs described in the first aspect of the present invention.

[0032] In some embodiments, mutants of the TCR fusion construct include amino acid sequences having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity or homology with the amino acid sequence of the TCR fusion construct described in the first aspect of the present invention, and retaining the biological activity of the TCR fusion construct. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. That is, mutants obtained by mutating the amino acid / nucleotide sequence corresponding to the TCR fusion construct described in the first aspect of the present invention are also included within the scope of protection of the present invention.

[0033] In this invention, the TCR fusion construct, similar to TRuC, is a structure that links an antibody to a CD3 complex (CD3γ, CD3δ, CD3ε). It has the characteristic of recognizing specific targets through antibodies and using the TCR-CD3 complex to stimulate the activation signal of natural T cells to kill target cells.

[0034] In some embodiments, the p40 is a common subunit of the cytokines IL-12 (interleukin-12) and IL-23, encoded by the p40 gene, and has a molecular weight of approximately 40 kDa.

[0035] In some embodiments, the TGF-β-RII (transforming growth factor-β receptor II) is a transmembrane serine / threonine kinase receptor belonging to the TGF-β receptor superfamily. It is mainly responsible for binding ligands TGF-β (including TGF-β1, TGF-β2, and TGF-β3) and initiating downstream signal transduction.

[0036] A second aspect of the present invention provides a nucleic acid molecule.

[0037] Furthermore, the nucleic acid molecule encodes the TCR fusion construct described in the first aspect of this invention;

[0038] Optionally, the nucleotide sequence encoding the antibody targeting CLDN18.2 in the TCR fusion construct according to the first aspect of the invention is shown in SEQ ID NO:12 or SEQ ID NO:13;

[0039] Optionally, the nucleotide sequence encoding G4S in the TCR fusion construct described in the first aspect of the invention is shown in SEQ ID NO:19;

[0040] Optionally, the nucleotide sequence encoding CD3ε in the TCR fusion construct described in the first aspect of the invention is shown in SEQ ID NO:20;

[0041] Optionally, the nucleotide sequence of the exophase signal peptide CD8a SP in the TCR fusion construct described in the first aspect of the invention is shown in SEQ ID NO:11.

[0042] A third aspect of the present invention provides an expression carrier.

[0043] Furthermore, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention;

[0044] Optionally, the vector is a plasmid, phage particle, granule, artificial chromosome, or virus-derived vector;

[0045] Optionally, the vector from which the virus originates may be a lentiviral vector, a retroviral vector, adeno-associated virus vector, adenovirus vector, poxvirus vector, herpesvirus vector, or baculovirus vector.

[0046] In some embodiments, the nucleic acid molecules provided by this invention can be cloned into many types of vectors. For example, the nucleic acid molecule can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0047] A fourth aspect of the present invention provides a host cell modified with a TCR fusion construct.

[0048] Furthermore, the host cell modified by the TCR fusion construct comprises the TCR fusion construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention;

[0049] Optionally, the host cell is a mammalian cell;

[0050] Optionally, the host cell is an immune cell;

[0051] Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, γδT cells, CTL cells, monocytes, myeloid cells, neutrophils, dendritic cells, macrophages, mast cells, and / or any combination thereof.

[0052] In some embodiments, the immune cells include, but are not limited to, leukocytes, lymphocytes (T cells, B cells, natural killer (NK) cells) derived from hematopoietic stem cells (HSCs) generated in the bone marrow, and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In a specific embodiment of the invention, the immune cells are T cells.

[0053] A fifth aspect of the present invention provides a host cell population modified with a TCR fusion construct.

[0054] Furthermore, the host cell population modified by the TCR fusion construct includes the host cells modified by the TCR fusion construct as described in the fourth aspect of the present invention;

[0055] Optionally, the host cell population modified by the TCR fusion construct may further include immune cells that do not contain the TCR fusion construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention.

[0056] Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, γδT cells, CTL cells, monocytes, myeloid cells, neutrophils, dendritic cells, macrophages, mast cells, and / or any combination thereof.

[0057] A sixth aspect of the present invention provides a derivative or pharmaceutical composition or biological agent.

[0058] Furthermore, the derivative is a TCR fusion construct of the first aspect of the present invention labeled with a detectable marker or a nucleic acid molecule of the second aspect of the present invention, a TCR fusion construct of the first aspect of the present invention or a nucleic acid molecule of the second aspect of the present invention conferring antibiotic resistance, or a TCR fusion construct of the first aspect of the present invention or a nucleic acid molecule of the second aspect of the present invention bound to or conjugated with a therapeutic agent;

[0059] Optionally, the detectable marker is a fluorescent dye, colloidal gold, chemiluminescent marker, or chemiluminescent catalyst;

[0060] Optionally, the chemiluminescent label is luminol and its derivatives, isoluminol and its derivatives, acridine ester, acridine ester derivative, adamantane, rare earth elements, or bipyridine ruthenium complex;

[0061] Optionally, the chemiluminescent catalyst is horseradish peroxidase or alkaline phosphatase;

[0062] Optionally, the antibiotic resistance gene is a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene;

[0063] Optionally, the therapeutic agent is a radionuclide, cytokine, gold nanoparticles, viral particles, liposomes, magnetic nanoparticles, prodrug-activated enzymes, or chemotherapeutic agents.

[0064] Alternatively, the cytokine is IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF or M-CSF;

[0065] Optionally, the chemotherapeutic agent is cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxalate platinum, or oxaliplatin;

[0066] Optionally, the pharmaceutical composition comprises the TCR fusion construct according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the expression vector according to the third aspect of the present invention, the host cell modified by the TCR fusion construct according to the fourth aspect of the present invention, and / or the host cell population modified by the TCR fusion construct according to the fifth aspect of the present invention.

[0067] Optionally, the biological agent comprises the pharmaceutical composition;

[0068] Optionally, the dosage form of the biological agent is an injection, oral preparation, cream, gel, drop, or lyophilized powder.

[0069] In some implementations, the detectable markers include, but are not limited to: fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes.

[0070] In some embodiments, the fluorescent pigment is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, or polydinoflavin-chlorophyll protein.

[0071] In some embodiments, the avidin is biotin, egg albumin, streptavidin, yolk avidin, or avidin-like substances.

[0072] In some implementations, the radioactive isotope is radioactive iodine, radioactive cesium, radioactive iridium, or radioactive cobalt.

[0073] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically or physiologically acceptable combinations of carriers, diluents, or excipients. Such compositions may comprise: buffer solutions, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions of the present invention can be formulated for oral, intravenous, topical, enteral, and / or parenteral administration.

[0074] In some embodiments, the biological agent comprises the pharmaceutical composition as described above. In some embodiments, the present invention does not particularly limit the specific dosage form, route of administration, and dosage of the biological agent, and those skilled in the art can make conventional selections based on the actual circumstances of the subjects. In some embodiments, the route of administration includes, but is not limited to: intravenous, intramuscular, subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, rectal, and intraperitoneal administration.

[0075] In some embodiments, the biological agent further comprises buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants; and preservatives.

[0076] The seventh aspect of the present invention provides any of the following methods:

[0077] (1) A method for preparing host cells modified with the TCR fusion construct according to the fourth aspect of the present invention, the method comprising the following steps: providing host cells to be modified, introducing the nucleic acid molecule according to the second aspect of the present invention or the expression vector according to the third aspect of the present invention into the host cells, thereby obtaining the host cells modified with the TCR fusion construct;

[0078] (2) A method for stimulating a mammalian target cell population or tissue to produce an immune response, the method comprising the steps of administering to a mammal an effective amount of host cells modified with the TCR fusion construct of the fourth aspect of the present invention and / or a host cell population modified with the TCR fusion construct of the fifth aspect of the present invention.

[0079] In some embodiments, the host cell to be modified is a mammalian cell, preferably an immune cell, which includes, but is not limited to: T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages or any combination thereof, more preferably T cells.

[0080] In some implementations, the introduced method is calcium phosphate transfection, DEAE-glucan-mediated transfection, microinjection, electroporation, TALEN, ZFN method, liposome-mediated transfection, lentiviral infection, retroviral infection, adenovirus infection, transposon technology, or CRISPR-Cas9 technology.

[0081] The present invention also provides a method for treating and / or preventing CLDN18.2-positive related diseases, the method comprising administering to a subject in need a therapeutic and / or preventative effective amount of a host cell population modified with the TCR fusion construct of the fifth aspect of the present invention and / or a derivative or pharmaceutical composition or biological agent of the sixth aspect of the present invention.

[0082] In some embodiments, the subject includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. In some embodiments, the subject is preferably a human.

[0083] In some implementations, the effective amount includes a therapeutically effective amount and a preventatively effective amount. The therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount can vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments. The preventatively effective amount refers to an amount sufficient to prevent, stop, or delay the onset of the disease.

[0084] In some implementations, the treatment and / or prevention refers to the complete or partial improvement or reduction of the disease or condition, or symptom, adverse effects or consequences, or associated phenotype. This includes, but is not limited to: preventing the onset or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease condition, and alleviating or improving prognosis.

[0085] The eighth aspect of the present invention provides for any of the following applications:

[0086] (1) The use of the TCR fusion construct of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the host cell modified by the TCR fusion construct of the fourth aspect of the present invention, the host cell population modified by the TCR fusion construct of the fifth aspect of the present invention, and / or the derivative of the sixth aspect of the present invention in the preparation of a medicament for the treatment and / or prevention of CLDN18.2 positive related diseases;

[0087] (2) The use of the TCR fusion construct of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the host cell modified by the TCR fusion construct of the fourth aspect of the present invention, the host cell population modified by the TCR fusion construct of the fifth aspect of the present invention, and / or the derivative or pharmaceutical composition of the sixth aspect of the present invention in the preparation of a biological agent for the treatment and / or prevention of CLDN18.2 positive related diseases;

[0088] (3) The use of the TCR fusion construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in host cells modified with the TCR fusion construct for the treatment and / or prevention of CLDN18.2-positive related diseases;

[0089] (4) The use of the TCR fusion construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in preparing host cell populations modified with TCR fusion constructs for the treatment and / or prevention of CLDN18.2-positive related diseases;

[0090] (5) The use of the TCR fusion construct described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in the preparation of a kit for preparing host cells modified with the TCR fusion construct for the treatment and / or prevention of CLDN18.2-positive related diseases;

[0091] (6) The use of the TCR fusion construct of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the host cell modified by the TCR fusion construct of the fourth aspect of the present invention, the host cell population modified by the TCR fusion construct of the fifth aspect of the present invention, and / or the derivatives or pharmaceutical compositions or biological agents of the sixth aspect of the present invention in the preparation of a medicament for treating and / or preventing CLDN18.2 positive related diseases and reducing or avoiding damage to normal gastric mucosal tissue by immunocellular therapy;

[0092] (7) The use of the TCR fusion construct of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the host cell modified by the TCR fusion construct of the fourth aspect of the present invention, the host cell population modified by the TCR fusion construct of the fifth aspect of the present invention, and / or the derivatives or pharmaceutical compositions or biological agents of the sixth aspect of the present invention in the preparation of biological agents for the treatment and / or prevention of CLDN18.2 positive related diseases and for reducing or avoiding damage to normal gastric mucosal tissue by immunocellular therapy;

[0093] Optionally, the CLDN18.2 positive associated diseases are gastric cancer, pancreatic cancer, colorectal cancer, bile duct cancer, esophageal cancer, ovarian cancer, lung cancer, breast cancer, liver cancer, head and neck cancer, bronchial cancer, bone cancer, testicular cancer, kidney cancer, bladder cancer, endometrial cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, or melanoma.

[0094] In this invention, the CLDN18.2-positive related diseases include solid tumors, hematologic malignancies, or combinations thereof; more preferably, the CLDN18.2-positive related diseases include gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, lung cancer, bone cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colorectal cancer, anal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, etc. In some embodiments, the CLDN18.2-positive related diseases of this invention are not limited to the specific diseases listed above, and any disease and / or symptom related to CLDN18.2 expression is within the scope of protection of this invention.

[0095] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0096] This invention constructs TruC-T cells (AB011 TruC-T and F2HT TruC-T) targeting CLDN18.2, which significantly reduces toxicity to normal gastric tissue while retaining effective tumor cell killing ability. In vitro experiments show that TruC-T cells, like their corresponding CAR-T structures, can specifically kill CLDN18.2-overexpressing tumor cells. In vivo experiments show that the gastric mucosa of mice in the TruC-T group remained intact, similar to the PBS control group, while the CAR-T group showed significant gastric mucosal erosion. The TruC-T cells provided by this invention effectively solve the technical problem of gastric side effects in existing CLDN18.2-targeted therapies. Furthermore, increasing the infusion dose of TruC-T enhances its tumor-killing effect without aggravating gastric damage, further improving the efficacy and safety of the treatment. This invention provides a better option for the precision treatment of CLDN18.2-positive solid tumors and has significant scientific and clinical application value. Attached Figure Description

[0097] Figure 1 : A schematic diagram of the structure corresponding to the method of constructing CLDN18.2 CAR-T modified TruC-T cells;

[0098] Figure 2Schematic diagrams of the structures corresponding to CD28 CAR-T, 41BB CAR-T, and TruC-T;

[0099] Figure 3 Schematic diagram of plasmid structure;

[0100] Figure 4 : Results of in vitro killing experiments;

[0101] Figure 5 : Results of in vivo experiments;

[0102] Figure 6 : Results of CAR / TRuC-T quantity detection and cell typing in the spleen and tumors of mice on day 18 after CAR / T injection;

[0103] Figure 7 : Results of HE staining of the stomach;

[0104] Figure 8 Experimental results corresponding to different dosage groups;

[0105] Figure 9 Comparison of gastric damage control results among different dosage groups. Detailed Implementation

[0106] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0107] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0108] Example AB011: Construction and functional verification of TRuC-T and F2H TRuC-T cells.

[0109] 1. In vitro experiments

[0110] (1) Experimental methods

[0111] according to Figure 2 The schematic diagram shown illustrates the design and synthesis of AB011 and F2H antibodies corresponding to different types of CAR-T and TruC-T structures into the pCDH-EF1a lentiviral expression plasmid (see...). Figure 3 (The diagram shows the plasmid structure.) The structures and corresponding sequences of the designed and synthesized AB011 CAR, AB011 TRuC, F2H CAR, and F2H TRuC are as follows:

[0112] 1) AB011 CAR: It is formed by the sequential tandem of SP (signal peptide), scFv (AB011), CD8 hinge region, CD8 transmembrane domain, 41BB (same as 4-1BB) co-stimulatory signal transduction domain, and CD3ζ (same as CD3z) intracellular signal transduction domain;

[0113] The amino acid sequences corresponding to SP, AB011, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, respectively.

[0114] The nucleotide sequences corresponding to SP, AB011, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively.

[0115] 2) AB011 TRuC: It is obtained by sequentially connecting SP (signal peptide), scFv (AB011), G4S linker, and CD3ε (same as CD3e) TCR signal transduction related domains;

[0116] The amino acid sequences corresponding to SP, AB011, G4S linker, and CD3ε are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0117] The nucleotide sequences corresponding to SP, AB011, G4S linker, and CD3ε are shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0118] 3) F2H CAR: It is formed by the sequential tandem of SP (signal peptide), scFv (F2H), CD8 hinge region, CD8 transmembrane domain, 41BB (same as 4-1BB) co-stimulatory signal transduction domain, and CD3ζ (same as CD3z) intracellular signal transduction domain;

[0119] The amino acid sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, respectively.

[0120] The nucleotide sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively.

[0121] 4) F2H TRuC: It is obtained by sequentially connecting SP (signal peptide), scFv (F2H), G4S linker, and CD3ε (same as CD3e) TCR signal transduction related domains;

[0122] The amino acid sequences corresponding to SP, F2H, G4S linker, and CD3ε are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0123] The nucleotide sequences corresponding to SP, F2H, G4S linker, and CD3ε are shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0124] The obtained lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. Cell culture supernatant was harvested 48-72 hours after transfection, concentrated, filtered, and the resulting CAR lentivirus was stored at -80°C for later use. Peripheral blood was collected from patients or healthy volunteers via leukoablation, and PBMCs were obtained by Ficoll density gradient centrifugation. EasySep was used to analyze the PBMCs.TM The HumanT Cell Isolation Kit (STEMCELL, #17951) is used to isolate T cells. It is formulated with 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult. TM After normal activation of T cells using X-vivo (lonza) medium containing human CD3 / CD28 / CD2 TCell Activator (STEMCELL, #10970) antibody for 2-3 days, lentivirus transduction was performed. The medium was then replaced with one lacking the activating antibody, and amplification was continued for 9-11 days. The positivity rate of CAR-T or TruC-T cells was measured, and the positivity rate of each group was adjusted to a consistent level by mixing in T cells from the same batch.

[0125] A Panc01 pancreatic cancer cell line overexpressing CLDN18.2 (i.e., the Panc01-CLDN18.2 cell line) was constructed using the PLV3-CMV overexpression system as target cells. Target cells were seeded into RTCA-specific E-plates, and baseline scanning was performed after adding culture medium to record the initial cell index. Once the target cells adhered and proliferated to a suitable density, the four groups of CAR / T RuC-T cells were added to the wells at a preset effector-to-target ratio (E:T = 1:1) and co-incubated with the target cells. Cell impedance changes were monitored in real-time using the RTCA system. The killing efficiency of CAR-T cells (16 hours) was evaluated by analyzing the cell index change curves.

[0126] (2) Experimental Results

[0127] The results are as follows Figure 4 As shown, the results indicate that for the same scFv, the targeted killing effect still exists even when the CAR-T structure is changed to the TruC-T structure.

[0128] 2. In vivo experiments

[0129] (1) Experimental methods

[0130] NCG mice were inoculated with 5×10 mice via subcutaneous tumor implantation. 6 Panc01-CLDN18.2 cells / 100μL / animal were seeded into tumors. Eight days later, the above four groups of CAR / TRuC-T cells were injected via the tail vein. 1×10 6Tumor size and mouse weight were assessed weekly, and CAR / TRuC-T cell counts and genotypes were determined using venous blood samples from mice. Flow cytometry was performed using the following antibodies to differentiate T cell types: APC / Cyanine7 anti-human CD3 (BioLegend, Catalog#300426), PE / Cyanine7 anti-human CD8 (BioLegend, Catalog#344712), APC anti-human EGFR Antibody (BioLegend, Catalog#352906) (CAR / TRuC marker), FITC anti-human CD45RA Antibody (BioLegend, Catalog#304106), and PE anti-human CD197 (CCR7) Antibody (BioLegend, Catalog#353204).

[0131] After the experiment, the mice were euthanized and their tissues were collected. The stomach tissue of the mice was fixed and stained with hematoxylin and eosin (HE). The tumor and spleen tissues of the mice were collected, and after cell grinding and separation, flow cytometry was performed to detect the proportion of CD3 positive and CAR / TRuC positive cells.

[0132] (2) Experimental Results

[0133] The results are as follows Figure 5 As shown, the results indicated that the body weight of mice in the AB011 and F2H CAR-T groups decreased significantly in the later stages, while the body weight of mice in the TRuC group was not significantly different from that of the PBS control group. Monitoring of the number of CAR / TRuC-T cells in the peripheral blood of mice after CAR / TRuC-T injection revealed that T cells in all groups significantly increased over time, with the AB011 and F2H CAR-T groups showing greater expansion than the TRuC-T group. Monitoring of tumor size in mice after CAR / TRuC-T injection, and the tumor size at the time of tissue sampling on the last day (four mice in the PBS group died prematurely), showed that tumor size decreased in all CAR / TRuC-T groups.

[0134] The results are as follows Figure 6 As shown, the results indicated that on day 18 after CAR / T injection, the number of CAR / T cells in the spleen and tumor of mice and the cell typing results showed that all CAR / T groups could be significantly expanded in mice, and a higher proportion of CD8+TRuC-T cells infiltrated the tumor.

[0135] HE staining results of the stomach Figure 7As shown, the results indicated that the gastric mucosa of mice in the CAR-T injection group showed significant erosive damage, while the gastric mucosa of mice in the TruC-T group was intact and similar to that of the control group PBS, indicating that TruC-T did not significantly kill or destroy CLDN18.2 cells at the tight junction of the stomach while killing tumors.

[0136] 3. Experiments with different dosage groups

[0137] (1) Experimental methods

[0138] F2H TRuC-T, F2H-41BB CAR-T, and F2H-CD28 CAR-T were reconstructed, where 41BB and CD28 are two different co-stimulatory signal transduction domains in the CAR-T structure. The above CAR-Ts were then used to perform RTCA killing experiments on Panc01-CLDN18.2 cells. The composition and corresponding sequences of F2H TRuC, F2H-41BB CAR, and F2H-CD28 CAR in the constructed F2H TRuC-T, F2H-41BB CAR-T, and F2H-CD28 CAR-T are as follows:

[0139] 1) F2H TRuC: It is obtained by sequentially connecting SP (signal peptide), scFv (F2H), G4S linker, and CD3ε (same as CD3e) TCR signal transduction related domains;

[0140] The amino acid sequences corresponding to SP, F2H, G4S linker, and CD3ε are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0141] The nucleotide sequences corresponding to SP, F2H, G4S linker, and CD3ε are shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0142] 2) F2H-41BB CAR: It is formed by the sequential tandem of SP (signal peptide), scFv (F2H), CD8 hinge region, CD8 transmembrane domain, 41BB (same as 4-1BB) co-stimulatory signal transduction domain, and CD3ζ (same as CD3z) intracellular signal transduction domain;

[0143] The amino acid sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8, respectively.

[0144] The nucleotide sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, 41BB co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:18, respectively.

[0145] 3) F2H-CD28 CAR: It is formed by sequentially connecting SP (signal peptide), scFv (F2H), CD8 hinge region, CD8 transmembrane domain, CD28 co-stimulatory signal transduction domain, and CD3ζ (same as CD3z) intracellular signal transduction domain.

[0146] The amino acid sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, CD28 co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0147] The nucleotide sequences corresponding to SP, F2H, CD8 hinge region, CD8 transmembrane domain, CD28 co-stimulatory signal transduction domain, and CD3ζ intracellular signal transduction domain are shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:18, respectively.

[0148] 5×10 NCG mice were administered via subcutaneous tumor-bearing method. 6 Five days after seeding tumors with 100 μL / particle of Panc01-CLDN18.2 cells, 1 × 10⁻⁶ F₂H TRuC-T, 1 × 10⁻⁶ F₂H-41BB CAR-T, and 1 × 10⁻⁶ F₂H-CD28 CAR-T were injected via tail vein. 6 cells / cell, set up the increased dose group F2H-TRuC-T*5 infusion of corresponding cells 5×10 6Cells / mouse. After the experiment, mice were euthanized and tissue samples were collected. The stomach tissue of the mice was fixed and stained with hematoxylin and eosin (HE).

[0149] (2) Experimental Results

[0150] The results of the RTCA kill experiment are as follows: Figure 8 As shown, the results indicated that all three cell types—F2H TRuC-T, F2H-41BB CAR-T, and F2H-CD28 CAR-T—had a killing effect on the tumor cells. Compared to the WT group, all experimental groups showed some therapeutic effect, with the F2H-TRuC-T*5 group showing a slightly better therapeutic effect than the F2H-CD28 CAR-T group. Furthermore, HE staining results revealed that the high-dose TruC-T group still showed significantly better control of gastric damage than the CAR-T group. Figure 9 ).

Claims

1. A TCR fusion construct (TRuC) targeting CLDN18.2, characterized in that, The TCR fusion construct comprises an antibody targeting CLDN18.2, a linker, and a TCR complex subunit fusion portion.

2. The TCR fusion construct according to claim 1, characterized in that, The amino acid sequence of the antibody targeting CLDN18.2 is shown in SEQ ID NO:2 or SEQ ID NO:3; Optionally, the nucleotide sequence of the antibody targeting CLDN18.2 is shown in SEQ ID NO:12 or SEQ ID NO:

13.

3. The TCR fusion construct according to claim 1, characterized in that, The linker is G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6 or EAAAK; Optionally, the linker is a G4S; Optionally, the amino acid sequence of the G4S is shown in SEQ ID NO:9; Optionally, the nucleotide sequence of the G4S is shown in SEQ ID NO:19; Optionally, the TCR complex subunit fusion portion is CD3ε, CD3γ, or CD3δ; Optionally, the TCR complex subunit fusion portion is CD3ε; Optionally, the amino acid sequence of CD3ε is shown in SEQ ID NO:10; Optionally, the nucleotide sequence of CD3ε is shown in SEQ ID NO:20; Optionally, the TCR fusion construct further comprises a signal peptide; Optionally, the signal peptide is the effervescent signal peptide CD8a SP or the effervescent signal peptide GMCSF SP; Optionally, the signal peptide is the exomembrane signal peptide CD8a SP; Optionally, the amino acid sequence of the exomembrane signal peptide CD8a SP is shown in SEQ ID NO:1; Optionally, the nucleotide sequence of the exomembrane signal peptide CD8a SP is shown in SEQ ID NO:11; Optionally, the TCR fusion construct further comprises cytokines, p40, and / or TGF-β-RII; Optionally, the cytokines are IL-2, IL-7, IL-10, IL-12, IL-15 and / or IL-21; Optionally, the TCR fusion construct is obtained by sequentially connecting a signal peptide, an antibody targeting CLDN18.2, a linker, and a TCR complex subunit fusion moiety. Optionally, the TCR fusion construct is obtained by sequentially connecting the exomembrane signal peptide CD8a SP, the antibody targeting CLDN18.2, G4S, and CD3ε. Optionally, the TCR fusion construct is obtained by sequentially connecting a signal peptide, an antibody targeting CLDN18.2, a linker, a TCR complex subunit fusion moiety, the cytokine, p40, and / or TGF-β-RII. Optionally, the TCR fusion construct is obtained by sequentially connecting the exomembrane signal peptide CD8a SP, the antibody targeting CLDN18.2, G4S, CD3ε, the cytokine, p40 and / or TGF-β-RII.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the TCR fusion construct according to any one of claims 1-3; Optionally, the nucleotide sequence encoding the antibody targeting CLDN18.2 in the TCR fusion construct according to any one of claims 1-3 is shown in SEQ ID NO:12 or SEQ ID NO:13; Optionally, the nucleotide sequence encoding G4S in the TCR fusion construct according to any one of claims 1-3 is shown in SEQ ID NO:19; Optionally, the nucleotide sequence encoding CD3ε in the TCR fusion construct according to any one of claims 1-3 is shown in SEQ ID NO:20; Optionally, the nucleotide sequence encoding the membrane exit signal peptide CD8a SP in any one of claims 1-3 is shown in SEQ ID NO:

11.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4; Optionally, the vector is a plasmid, phage particle, granule, artificial chromosome, or virus-derived vector; Optionally, the vector from which the virus originates may be a lentiviral vector, a retroviral vector, adeno-associated virus vector, adenovirus vector, poxvirus vector, herpesvirus vector, or baculovirus vector.

6. A host cell modified with a TCR fusion construct, characterized in that, The host cell modified by the TCR fusion construct comprises the TCR fusion construct according to any one of claims 1-3, the nucleic acid molecule according to claim 4, and / or the expression vector according to claim 5; Optionally, the host cell is a mammalian cell; Optionally, the host cell is an immune cell; Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, γδT cells, CTL cells, monocytes, myeloid cells, neutrophils, dendritic cells, macrophages, mast cells, and / or any combination thereof.

7. A host cell population modified with a TCR fusion construct, characterized in that, The host cell population modified by the TCR fusion construct comprises the host cells modified by the TCR fusion construct of claim 6; Optionally, the host cell population modified by the TCR fusion construct further includes immune cells that do not contain the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, and / or the expression vector of claim 5; Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, γδT cells, CTL cells, monocytes, myeloid cells, neutrophils, dendritic cells, macrophages, mast cells, and / or any combination thereof.

8. A derivative, pharmaceutical composition, or biological agent, characterized in that, The derivative is a TCR fusion construct of any one of claims 1-3 or a nucleic acid molecule of claim 4 labeled with a detectable marker, a TCR fusion construct of any one of claims 1-3 or a nucleic acid molecule of claim 4 conferring antibiotic resistance, or a TCR fusion construct of any one of claims 1-3 or a nucleic acid molecule of claim 4 that is bound to or conjugated with a therapeutic agent; Optionally, the detectable marker is a fluorescent dye, colloidal gold, chemiluminescent marker, or chemiluminescent catalyst; Optionally, the chemiluminescent label is luminol and its derivatives, isoluminol and its derivatives, acridine ester, acridine ester derivative, adamantane, rare earth elements, or bipyridine ruthenium complex; Optionally, the chemiluminescent catalyst is horseradish peroxidase or alkaline phosphatase; Optionally, the antibiotic resistance gene is a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene; Optionally, the therapeutic agent is a radionuclide, cytokine, gold nanoparticles, viral particles, liposomes, magnetic nanoparticles, prodrug-activated enzymes, or chemotherapeutic agents. Alternatively, the cytokine is IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF or M-CSF; Optionally, the chemotherapeutic agent is cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxalate platinum, or oxaliplatin; Optionally, the pharmaceutical composition comprises the TCR fusion construct according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell modified with the TCR fusion construct according to claim 6, and / or the host cell population modified with the TCR fusion construct according to claim 7; Optionally, the biological agent comprises the pharmaceutical composition; Optionally, the dosage form of the biological agent is an injection, oral preparation, cream, gel, drop, or lyophilized powder.

9. The following method, characterized in that, The method includes: (1) A method for preparing a host cell modified with the TCR fusion construct according to claim 6, the method comprising the following steps: providing a host cell to be modified, introducing the nucleic acid molecule according to claim 4 or the expression vector according to claim 5 into the host cell to obtain the host cell modified with the TCR fusion construct; (2) A method for stimulating a mammalian target cell population or tissue to generate an immune response, the method comprising the steps of administering to a mammal an effective amount of host cells modified with the TCR fusion construct of claim 6 and / or a population of host cells modified with the TCR fusion construct of claim 7.

10. The following application, characterized in that, The applications include: (1) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell modified by the TCR fusion construct of claim 6, the host cell population modified by the TCR fusion construct of claim 7, and / or the derivative of claim 8 in the preparation of a medicament for the treatment and / or prevention of CLDN18.2 positive related diseases; (2) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell modified by the TCR fusion construct of claim 6, the host cell population modified by the TCR fusion construct of claim 7, and / or the derivative or pharmaceutical composition of claim 8 in the preparation of a biological agent for the treatment and / or prevention of CLDN18.2 positive related diseases; (3) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, and / or the expression vector of claim 5 in host cells modified with a TCR fusion construct for the treatment and / or prevention of CLDN18.2-positive related diseases; (4) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, and / or the expression vector of claim 5 in preparing host cell populations modified with TCR fusion constructs for the treatment and / or prevention of CLDN18.2-positive related diseases; (5) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, and / or the expression vector of claim 5 in the preparation of a kit for preparing host cells modified with a TCR fusion construct for the treatment and / or prevention of CLDN18.2-positive related diseases; (6) The use of the TCR fusion construct of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell modified by the TCR fusion construct of claim 6, the host cell population modified by the TCR fusion construct of claim 7, and / or the derivative or pharmaceutical composition or biological agent of claim 8 in the preparation of a medicament for treating and / or preventing CLDN18.2 positive related diseases and reducing or avoiding damage to normal gastric mucosal tissue by immunocellular therapy; (7) The use of any one of the TCR fusion constructs of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell modified by the TCR fusion construct of claim 6, the host cell population modified by the TCR fusion construct of claim 7, and / or the derivatives or pharmaceutical compositions or biological agents of claim 8 in the preparation of biological agents for the treatment and / or prevention of CLDN18.2 positive related diseases and for reducing or avoiding damage to normal gastric mucosal tissue by immunocellular therapy; Optionally, the CLDN18.2 positive associated diseases are gastric cancer, pancreatic cancer, colorectal cancer, bile duct cancer, esophageal cancer, ovarian cancer, lung cancer, breast cancer, liver cancer, head and neck cancer, bronchial cancer, bone cancer, testicular cancer, kidney cancer, bladder cancer, endometrial cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, or melanoma.

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