Application of a new immune checkpoint, the PIEZO1 pathway, in tumor T cell therapy

Abstract

This invention relates to the fields of biology, medicine, and clinical medicine, and specifically to the application of a novel immune checkpoint pathway, PIEZO1, in tumor T-cell therapy. Specifically, the invention provides the use of PIEZO1 inhibitors in treating cancer, enhancing T-cell killing capacity, and improving the therapeutic efficacy of immune checkpoint inhibitors. The novel immune checkpoint PIEZO1 and its downstream pathways described in this application have pioneered innovative strategies for tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the fields of biology, medicine, and clinical medicine, and specifically relates to the application of a new immune checkpoint PIEZO1 pathway in tumor T cell therapy. Background Art

[0002] Cancer immunotherapy is defined as an approach to fighting cancer by generating or enhancing an immune response against cancer cells. Over the past decade, two types of immunotherapy have shown promising results in cancer treatment: using immune checkpoint inhibitors to enhance natural anti-tumor activity and adoptive cell therapy to deliver specific anti-tumor immune cells.

[0003] Currently, the most widely used type of immunotherapy is monoclonal antibodies targeting regulatory immune checkpoint molecules that inhibit T cell activation, specifically cytotoxic T lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed death ligand 1 (PD-L1). Immune checkpoint blockade (ICB) has achieved great success in treating a variety of cancer types, such as metastatic melanoma, non-small cell lung cancer, and renal cancer. Although this treatment modality has been successfully applied to many solid tumors, because its main mechanism relies on enhancing the patient's existing population of potential tumor-reactive T cells, significant responses to current ICB therapies are limited to a small number of cancer patients, and most responding patients subsequently relapse. Therefore, in cancer types with poor immunogenicity, immune checkpoint therapy alone may fail.

[0004] This highlights the urgent need to explore more effective strategies by identifying new immune checkpoints. So far, a lot of effort has been spent on finding T cell immune checkpoints based on biochemical signals. We need to explore new T cell immune checkpoints in addition to biochemical signals and use them as targets in future clinical tumor immunotherapy. Summary of the Invention

[0005] The present invention discovered that PIEZO1 is a potential mechanical immune checkpoint in cytotoxic T cells, which can be manipulated to enhance the traction force exerted by T cells and subsequently enhance their cytotoxicity against cancer cells, thereby killing tumor cells, thus providing a new treatment method for cancer.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] Applications of PIEZO1

[0008] In a first aspect, the present invention provides the use of a PIEZO1 inhibitor in the preparation of the following products:

[0009] 1) Products for treating cancer,

[0010] 2) Products that enhance T cell killing ability,

[0011] 3) Products that enhance the therapeutic efficacy of immune checkpoint inhibitors (ICIs).

[0012] The products described in the present invention include drugs, drug compositions, drug combinations, etc.

[0013] More preferably, the immune checkpoint is PD1, and the immune checkpoint inhibitor is an antibody targeting PD1.

[0014] Specifically, the killing ability is reflected in traction force, enhanced cytotoxicity, and the degree of tumor infiltration.

[0015] Drug combination compositions

[0016] On the other hand, the present invention provides a drug combination composition for treating cancer, wherein the drug combination composition comprises a PIEZO1 inhibitor and an immune checkpoint inhibitor (ICIs).

[0017] In a specific embodiment, the dosage forms of the PIEZO1 inhibitor and the immune checkpoint inhibitor in the drug combination composition are the same or different.

[0018] In a specific embodiment, the PIEZO1 inhibitor and the immune checkpoint inhibitor in the drug combination composition are administered simultaneously or sequentially, specifically, at intervals of 0, 1, 2, 3, 4, 5, 6, 7 or more days.

[0019] The dosage form and mode of administration of the pharmaceutical combination composition of the present invention are not particularly limited. Representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration. Representative dosage forms include tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations, aerosols, films, injections, intravenous drips, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, pellets, nasal preparations, pulmonary preparations, eye drops, and the like.

[0020] In a specific embodiment, the dosage ratio of the PIEZO1 inhibitor and the immune checkpoint inhibitor in the drug combination composition is 1:0.1-2; specifically including 3:1, 2:1, 1:1, 1:2, and 1:3.

[0021] More preferably, the immune checkpoint is PD1, and the immune checkpoint inhibitor is an antibody targeting PD1.

[0022] Preferably, the pharmaceutical combination composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises any one or more combinations of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0023] T cell preparation method and product application

[0024] On the other hand, the present invention provides a method for preparing T cells with high killing ability, the method comprising the step of pretreating T cells with a PIEZO1 inhibitor, or the method comprising the step of obtaining T cells with reduced PIEZO1 expression after knocking down PIEZO1.

[0025] Preferably, the pretreatment refers to a step of pretreatment (culturing) in a culture medium containing a PIEZO1 inhibitor.

[0026] More specifically, the content of the PIEZO1 inhibitor in the culture medium may be 0.01-10 μM or more, for example, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM.

[0027] Preferably, the method for knocking down PIEZO1 includes RNA interference technology (RNAi), antisense oligonucleotide (ASO) technology, CRISPR technology, TALEN technology, ZFN technology, and Cre-loxP gene recombination technology.

[0028] Preferably, the reduced PIEZO1 expression may comprise a reduction in PIEZO1 expression of at least 1%-100%, such as 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to a control, including individuals without the disease or condition, or internal controls (e.g., housekeeping genes), or the median expression level of a biomarker in samples from a patient group / population.

[0029] Preferably, the method is performed in vitro.

[0030] Preferably, the method is for non-therapeutic purposes.

[0031] Specifically, the highly cytotoxic T cells have enhanced traction force, enhanced cytotoxicity, and enhanced tumor infiltration compared to control T cells, and have a better therapeutic effect in treating cancer.

[0032] At the same time, the present invention provides T cells prepared by the above method.

[0033] Preferably, the T cells are solubilized in a suitable solution to maintain cell activity.

[0034] At the same time, the present invention also provides the use of T cells prepared by the above method in treating cancer and preparing cancer drugs.

[0035] Treatment

[0036] In another aspect, the present invention provides a method for treating cancer, comprising administering a PIEZO1 inhibitor or T cells prepared by the aforementioned method to a patient.

[0037] Preferably, the treatment method can also be used in combination with other cancer treatment methods.

[0038] Preferably, the treatment methods for other cancers include surgical therapy, chemotherapy, radiotherapy, gene therapy, immunotherapy, etc.

[0039] Specifically, the chemotherapy is administered to the patient with chemical drugs (chemo drugs), such as: thiotepa, cyclosphosphamide, difluoromethylornithine (DMFO), retinoic acid, piposulfan, dolastatin, busulfan, improsulfan, clodronate, camptothecin, bryostatin, capecitabine, carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, and pharmaceutically acceptable salts, acids or derivatives of any one of the above.

[0040] Specifically, immunotherapy refers to a treatment method that artificially enhances or suppresses the body's immune function to treat a disease, targeting a low or high immune state. Tumor immunotherapy aims to activate the body's immune system and rely on its own immune function to kill cancer cells and tumor tissue. It generally relies on the use of immune effector cells and molecules to target and destroy cancer cells.

[0041] Specifically, the gene therapy refers to administering therapeutic polynucleotides to a patient. Viral vectors for expressing polynucleotides are well known in the art and include eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpes virus, lentivirus, poxvirus (including vaccinia virus) and papillomavirus (including SV40). Alternatively, the administration of polynucleotides can be with lipid-based carriers such as liposomes.

[0042] Specifically, the radiotherapy involves ionizing radiation to the tumor, causing extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The ionizing radiation includes x-ray radiation, ultraviolet radiation, infrared radiation, gamma-ray radiation, or microwave radiation.

[0043] Specifically, the surgical treatment includes resection, which refers to the removal, excision and / or destruction of all or part of the cancerous tissue. Surgical treatment also includes laser surgery, cryosurgery, electrosurgery and microscope-controlled surgery.

[0044] General Concepts

[0045] The immune checkpoints described in this invention refer to programmed death receptors and their ligands. Immune checkpoint blockade therapy based on programmed death receptors and their ligands inhibits the binding of programmed death receptors and their ligands, thereby increasing the host immune system's aggressiveness against tumor cells.

[0046] Preferably, the immune checkpoints include PD1, CTLA4, VISTA, IDO, CD137, TIGIT, TIM3, BTLA, CD27L, CD40, LAG-3, CD270, GITR, SIRPα, etc.

[0047] The immune checkpoint inhibitors (ICIs) described in the present invention are inhibitors targeting the above-mentioned immune checkpoints.

[0048] As used herein, the term "inhibitor" refers to a substance that targets, reduces, or inhibits at least one activity of a specific target gene or protein. In the present invention, it particularly refers to active substances that target, reduce, or inhibit PIEZO1 and immune checkpoints.

[0049] Preferably, the inhibitor is artificially synthesized or naturally occurring.

[0050] Preferably, the inhibitor includes an agent that reduces the expression of the target gene or protein through the following technologies: RNA interference technology (RNAi), antisense oligonucleotide (ASO) technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology.

[0051] Preferably, the inhibitor further includes compounds or antibodies that specifically target target genes or proteins.

[0052] Preferably, the PIEZO1 inhibitor includes an agent that reduces PIEZO1 expression through the following technologies: RNA interference technology (RNAi), antisense oligonucleotide (ASO) technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology.

[0053] Preferably, the PIEZO1 inhibitor also includes compounds and antibodies that specifically target PIEZO1.

[0054] Preferably, the PIEZO1 inhibitor includes GsMTx4, D-GsMTx4, and GsMTx4 TFA.

[0055] Most preferably, the PIEZO1 inhibitor is GsMTx4.

[0056] Preferably, the PIEZO1 inhibitor is an agent specifically targeting PIEZO1 used in RNA interference technology; more specifically, it is the shRNA used in the specific embodiments of the present invention.

[0057] Preferably, the PD1 inhibitor is an antibody targeting PD1.

[0058] The antibodies of the present invention include polyclonal antibodies or monoclonal antibodies that are complete antibodies or shortened antibodies (e.g., F(ab')2 (Fab', Fab or Fv fragments) antibodies, chimeric antibodies, humanized antibodies or fully humanized antibodies. Such antibodies can be produced according to well-known antibody or antiserum production methods.

[0059] The cancers described herein include, but are not limited to, cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), rectal cancer, breast cancer, squamous cell carcinoma of the skin, colon cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, transitional cell carcinoma of the bladder, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, mesothelioma, neuroendocrine tumor (e.g., pancreatic neuroendocrine tumor or carcinoma, gastric neuroendocrine tumor or carcinoma, intestinal neuroendocrine tumor or carcinoma).

[0060] More specifically, the cancers described herein include cancers of the head, neck, eyes, mouth, larynx, esophagus, trachea, larynx, pharynx, chest, bone, lung, colon, rectum, stomach, prostate, bladder, uterus, cervix, breast, ovary, testicles, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain or central nervous system, pleura, peritoneum, and neuroendocrine system.

[0061] Preferably, the cancer is melanoma, colon cancer, gastric cancer, liver cancer.

[0062] Most preferably, the specific embodiments of the present invention have been verified for melanoma and colon cancer, demonstrating that T cells treated with PIEZO1 inhibitors have a higher therapeutic effect on melanoma and colon cancer, and can further enhance the therapeutic effect when used in combination with PD1 inhibitors.

[0063] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

[0064] In certain embodiments, the subject (preferably a human) suffers from cancer or an autoimmune disease, or is suspected of being at risk of suffering from such a disease.

[0065] As used herein, "treating" means alleviating or alleviating at least one symptom associated with such a condition, or slowing or reversing the progression of such a condition. Within the meaning of the present invention, the term "treating" also means inhibiting or delaying the onset of a condition (i.e., the period before the clinical manifestation of the disease) and / or reducing the risk of the disease developing or worsening. For example, the term "treating" in relation to cancer may refer to eliminating or reducing a patient's tumor burden, or preventing, delaying or inhibiting metastasis, etc.

[0066] The T cells described herein include autologous T cells or allogeneic T cells. Preferably, the autologous T cells are derived from human umbilical cord blood or peripheral blood. Preferably, the T cells may also be mature commercial cell line products. Specifically, the T cells may also be genetically engineered to express exogenous proteins, such as TCR-T cells or CAR-T cells.

[0067] Preferably, the T cells include CD8 + T, CD4 + T, TCR-T or CAR-T cells; more preferably, the T cells are CD8 + T cells.

[0068] Preferably, the T cells are prepared as an injection for administration, and the injection includes injection at any site, such as intradermal injection, subcutaneous injection, intramuscular injection, and intravenous injection.

[0069] The cytotoxic T lymphocytes (CTL cells) of the present invention are generally referred to as CD8 + T cells are key components of the adaptive immune system and play an important role in the immune system's defense against pathogens such as viruses, bacteria, and tumors.

[0070] In summary, the solution of this application has the following effects:

[0071] 1. This application describes a novel immune checkpoint mediated by PIEZO1, which clarifies a new mechanism by which T cells participate in tumor immune killing and rationally explains a new type of T cell immune checkpoint in addition to biochemical signals, and has good prospects for clinical application.

[0072] 2. Adding the PIEZO1 inhibitor GsMT×4 to T cells or knocking down PIEZO1 can enhance the cytotoxicity of T cells and strengthen their tumor immune killing and infiltration function, thereby inhibiting tumor growth.

[0073] 3. The new immune checkpoint PIEZO1 and its downstream pathway in this application have pioneered an innovative strategy for tumor immunotherapy, targeting the PIEZO1 mechanical channel pathway. By inhibiting it, the traction force of T cells is increased, and the immune killing ability is enhanced. At the same time, the combination of GsMT×4 and αPD-1 greatly improves the function of T cells to recognize and kill tumors, opening up ideas for the development of clinical targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1A -B is the detection result of T cell cytotoxicity against B16F10 cells and MC38 cells after treating T cells with the PIEZO1 inhibitor GsMT×4 and knocking down PIEZO1. A is B16F10 cells and B is MC38 cells.

[0075] Figure 2 This is the result of detecting cell traction force after T cells were treated with PIEZO1 inhibitor GsMT×4.

[0076] Figure 3 This figure shows the results of the detection of the degree of T cell tumor infiltration after treating mouse T cells with the PIEZO1 inhibitor GsMT×4 and adoptively treating B16F10 tumor mice.

[0077] Figure 4A -B is the result of detecting the tumor volume and survival time of mice after adoptive treatment with T cells after PIEZO1 inhibition using GsMT×4.

[0078] Figure 5 This figure shows the results of detecting the tumor volume of mice after adoptive treatment with T cells after knocking down PIEZO1.

[0079] Figure 6A -B is a graph showing the results of detecting tumor volume and survival of mice after treatment with T cells treated with GsMT×4 and / or αPD-1. DETAILED DESCRIPTION

[0080] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0081] Various cell lines, drugs and experimental animals used in the following examples:

[0082] B16F10 murine melanoma cell line and MC38 murine colon cancer cell line were purchased from ATCC;

[0083] GxMT×4 was purchased from Selleck;

[0084] C57BL / 6J mice were purchased from the Medical Laboratory Animal Center of the Chinese Academy of Medical Sciences;

[0085] CD45.1 + OT-1TCR mice are composed of OT-1 transgenic mice and CD45.1 + Mice are bred;

[0086] OVA-B16 tumor mice were created in our own laboratory;

[0087] αPD-1 was purchased from Bio X Cell.

[0088] Example 1: T cell cytotoxicity is enhanced after inhibition and knockdown of PIEZO1

[0089] 1. Experimental steps

[0090] We used pmel1 TCR transgenic CD8 cells pretreated for 24 hours with GsMT×4 and Yoda1 (GsMT×4-1μM and Yoda1-2μM, inhibiting or activating PIEZO1, respectively). + T cells (CD8 + T cells were cultured in IMDM medium containing 10% fetal bovine serum, 50 μM β-mercaptoethanol, and 50 U / ml IL-2 and co-cultured with B16F10 cells or MC38 cells at a ratio of 20:1 for 4 hours.

[0091]

[0092] At the same time, sense and antisense sequences were synthesized for the targets of shRNA shown in SEQ ID NO.1 and SEQ ID NO.4, and Piezo1-shRNAs lentiviral vectors were constructed. T cells were transduced to obtain Piezo1-shRNAs Pmel-1 T cells, and Piezo1-shRNAs Pmel-1 T cells were co-cultured with B16F10 cells. Flow cytometry was used to detect apoptosis of CD45- tumor cells.

[0093] 2. Experimental results

[0094] Inhibition or knockdown of PIEZO1 levels in the B16F10 melanoma cell line enhanced T cell cytotoxicity and promoted tumor cell apoptosis ( Figure 1A ), and the same results were obtained in MC38 mouse colon cancer cells ( Figure 1B ).

[0095] Example 2: T cell traction force is enhanced after PIEZO1 inhibition

[0096] 1. Experimental steps

[0097] The glass bottom of the confocal dish was pretreated with APTES (Sigma-Aldrich). After polymerization, the gel surface was covered with a fluorescent bead solution for 15 minutes to fix it. The gel was activated with sulfamethoxazole (Sanpah) and coated with polylysine (PLL) overnight at 4°C. Then, 5 μg / mL anti-CD3ε antibody was applied at 37°C for 60 minutes. Cells were incubated on the PAA gel for 30 minutes to ensure activation. Phase contrast and fluorescent bead fluorescence images of the cells were taken to measure the displacement field generated during T cell activation.

[0098] 2. Experimental results

[0099] Mouse CD8 cells pretreated with the PIEZO1 inhibitor GsMT×4 prepared in Example 1 + T cells significantly increased their pulling force in both resting and activated states ( Figure 2 ).

[0100] Example 3: GsMT×4 Increases T Cell Tumor Infiltration

[0101] 1. Experimental steps:

[0102] From CD45.1 + Obtaining activated CD8 T cells in OT-1 TCR mice + T cells (obtained by the literature: Nat Immunol. 2021 Mar; 22(3): 358-369.) were pretreated with GsMT×4 (1 μM) for 24 hours and adoptively transferred into C57BJ / 6L mice bearing OVA-B16 tumors via tail vein injection. CD45.1 + The degree of tumor infiltration was detected by T cell flow cytometry.

[0103] 2. Experimental results:

[0104] In the CD45.1 + Obtaining activated CD8 T cells in OT-1 TCR mice + Flow cytometry analysis of tumor CD45.1 in C57BJ / 6L mice treated with T cells + T cell infiltration increased significantly ( Figure 3 ).

[0105] Example 4: GsMT×4 inhibits T cells after PIEZO1, inhibits tumor growth in mice and prolongs their survival

[0106] 1. Experimental steps:

[0107] OT-1CD8 cells were pretreated with GsMT×4 (1 μM) for 24 hours. +T cells were adoptively transferred into OVA-B16 tumor mice, and the tumor growth and survival of the mice were recorded.

[0108] 2. Experimental results:

[0109] GsMT×4 significantly delayed the growth of B16F10 tumor in mice ( Figure 4A ) and prolonged the survival of mice ( Figure 4B ).

[0110] Example 5: T cells with PIEZO1 knockdown have an inhibitory effect on mouse tumor growth

[0111] 1. Experimental steps:

[0112] In OT-1CD8 + A PIEZO1-shRNA stably transfected cell line was constructed in T cells and adoptively transferred into OVA-B16 tumor-bearing mice, and the tumor growth of the mice was recorded and observed.

[0113] 2. Experimental results:

[0114] OT-1CD8 cells with PIEZO1 knockdown + T cells have a significant inhibitory effect on the growth of B16F10 tumors in mice ( Figure 5 ).

[0115] Example 6: The combination of GsMT×4 and αPD-1 can better enhance the inhibitory function of T cells on tumor growth.

[0116] 1. Experimental steps:

[0117] OT-1CD8 + After T cells were stimulated with or without GsMT×4 (1 μM) for 24 hours, these T cells were injected into mice through the tail vein. Some mice were also given anti-PD-1 antibodies through the abdominal cavity (200 μg per mouse, once every three days, for a total of three times), and the tumor growth of the mice was recorded and observed.

[0118] 2. Experimental results:

[0119] Compared with the control group, OT-1CD8 + T cells have a significant inhibitory effect on OVA-B16 tumors in mice ( Figure 6A ), and the survival time of mice treated with combined drugs was significantly prolonged ( Figure 6B ).

Claims

1. A drug combination for treating cancer, comprising T cells pretreated with a PIEZO1 inhibitor and an immune checkpoint inhibitor; The PIEZO1 inhibitor is GsMTx4; The immune checkpoint inhibitor is an antibody targeting PD1; The cancer is melanoma; The pretreatment refers to the step of culturing in a culture medium containing a PIEZO1 inhibitor; The content of the PIEZO1 inhibitor in the culture medium is 1-10 μM; The T cells are CD8 + T cells; The combined use of T cells pretreated with the PIEZO1 inhibitor and immune checkpoint inhibitors can further enhance the effect of cancer treatment.

2. The pharmaceutical combination composition according to claim 1, further comprising a pharmaceutically acceptable carrier or excipient.

3. Use of the drug combination composition according to claim 1 in the preparation of a drug for treating cancer; The cancer is melanoma.

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