Application of TBX1 gene expression in tumor treatment

Abstract

The invention provides application of a TBX1 gene as a target spot in tumor treatment. Specifically, the invention provides application of the TBX1 gene expression and / or function inhibitor, the TBX1 gene expression and / or function inhibitor is used for preparing a preparation or a pharmaceutical composition, and the preparation or the pharmaceutical composition is used for exciting / enhancing CD8 + T cell infiltration in tumor tissues; and / or treating a tumor or cancer. Furthermore, by combining inhibition of TBX1 gene expression and / or function with an immune checkpoint inhibitor, the effect of synergistically treating tumors can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor treatment, in particular, to the application of TBX1 gene as a target in tumor treatment. BACKGROUND

[0002] Adaptive immune response plays an important role in tumor occurrence and development, especially the number, type and function of T cells infiltrating the tumor interior are important markers of tumor prognosis. CD8 + The number of T cells is positively correlated with patient prognosis. However, cancer cells can escape killing by immune cells through various mechanisms, one of which is to increase the expression of CD8 + T cell immune checkpoint pathway genes. CD8 + T cells are called exhausted T cells and are in an immune tolerant state, no longer having tumor killing effect. Blocking the immune checkpoint pathway can re-activate the cytotoxic function of CD8 + T cells, effectively killing tumor cells and reducing tumor load.

[0003] Based on these findings, the emerging tumor immunotherapy is a treatment with clinical application value. Among them, the strategy of targeting and inhibiting immune checkpoint molecules PD-1, PD-L1, CTLA-4 and other genes is particularly effective, which has been used in the treatment of various advanced tumors in clinic, including melanoma, certain types of lung cancer, breast cancer, renal cancer, bladder cancer, colorectal cancer, recurrent Hodgkin's lymphoma, etc., which can significantly increase the survival time of patients. However, there are still limitations in current immunotherapy, and the individual differences in drug response are large, which can trigger unexplained inflammatory storm, autoimmune and other fatal reactions in some patients. The cellular and molecular mechanisms leading to these serious side effects are not clear. Therefore, it is of important practical significance and clinical value to further study the molecular mechanisms and targets for effective regulation of tumor infiltrating lymphocytes.

[0004] The lymphatic system runs in parallel with the vascular system, and under physiological conditions, it plays an important role in maintaining body fluid homeostasis, transporting large molecules such as lipids, and immune cell migration. In tumor occurrence and development, lymphatic vessels have dual roles of positive and negative. On the one hand, lymphatic vessels have immune regulatory function to inhibit tumor growth. In the early stage of tumor occurrence, at the stage of cancer cell germination, lymphatic vessels can transport dendritic cells activated by tumor antigens to the draining lymph nodes, and activate the adaptive immune response to kill tumors. On the other hand, in the later stage of tumor occurrence, when the in situ tumor load increases, the rich lymphatic vessel network is the primary channel for cancer cell metastasis. In addition, after tumor formation, the immune regulatory function of lymphatic vessels also changes from activating killing to promoting CD8 +Immune tolerance and inactivation of killer cells such as T cells lead to immune escape from cancer cells. However, the mechanisms by which lymphatic vessels precisely regulate their functional transformation remain unclear. In recent years, the positive role of lymphatic cells in regulating adaptive immunity has gained attention, making them one of the new targets for cancer therapy research (Lund et al., 2016; Fankhauser et al., 2017; Song et al., 2020). Summary of the Invention

[0005] The purpose of this invention is to provide a novel target for tumor therapy, the TBX1 gene, and the application of inhibiting TBX1 gene expression and / or function in tumor therapy.

[0006] In a first aspect, the present invention provides the use of an inhibitor of TBX1 gene expression and / or function for the preparation of a formulation or pharmaceutical composition, said formulation or pharmaceutical composition being used to: (i) stimulate / enhance CD8 expression in tumor tissue. + T-cell infiltration; and / or (ii) treatment of tumors or cancer.

[0007] In another preferred embodiment, the TBX1 gene expression and / or function inhibitor includes nucleic acid reagents, peptide (or protein) reagents, small molecule compounds, or combinations thereof.

[0008] In another preferred embodiment, the nucleic acid reagent includes DNA reagents, RNA reagents, or combinations thereof.

[0009] In another preferred embodiment, the RNA reagent includes (but is not limited to) siRNA, shRNA, sgRNA, and their precursors or expression vectors.

[0010] In another preferred embodiment, the TBX1 gene expression inhibitor stimulates the infiltration of CD8+ cells into the tumor tissue by inhibiting the expression of the TBX1 gene in lymphatic endothelial cells within the tumor microenvironment. + T cell amplification; CD8 + T cells are CD8 cells with cytotoxic capabilities. + T cells.

[0011] In another preferred embodiment, the TBX1 gene function inhibitor stimulates the infiltration of CD8 in tumor tissue by inhibiting the functional activity of TBX1 in lymphatic endothelial cells within the tumor microenvironment. + T cell amplification; CD8 + T cells are CD8 cells with cytotoxic capabilities. + T cells.

[0012] In another preferred embodiment, the tumor or cancer includes solid tumors and hematologic malignancies.

[0013] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, liver cancer, pancreatic cancer, and brain tumors.

[0014] In another preferred embodiment, the tumor or cancer is melanoma.

[0015] A second aspect of the invention provides a pharmaceutical composition comprising: (a) a first active ingredient, an inhibitor of TBX1 gene expression and / or function; (b) a second active ingredient, an inhibitor of an immune checkpoint molecule; and (c) a pharmaceutically acceptable carrier.

[0016] In another preferred embodiment, the TBX1 gene expression inhibitor is used to inhibit the expression of the TBX1 gene, and the TBX1 gene function inhibitor is used to inhibit the functional activity of TBX1.

[0017] In another preferred embodiment, the TBX1 gene expression and / or function inhibitor includes nucleic acid reagents, peptide (or protein) reagents, small molecule compounds, or combinations thereof.

[0018] In another preferred embodiment, the nucleic acid reagent includes DNA reagents, RNA reagents, or combinations thereof.

[0019] In another preferred embodiment, the RNA reagent includes (but is not limited to) siRNA, shRNA, sgRNA, and their precursors or expression vectors.

[0020] In another preferred embodiment, the immune checkpoint molecule inhibitor includes nucleic acid reagents, polypeptide (or protein) reagents, small molecule compounds, or combinations thereof.

[0021] In another preferred embodiment, the immune checkpoint molecule inhibitor is an antibody.

[0022] In another preferred embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, CTLA-4, or combinations thereof.

[0023] A third aspect of the present invention provides the use of the pharmaceutical composition as described in the second aspect of the present invention for the preparation of a medicament for treating tumors or cancer.

[0024] In another preferred embodiment, the tumor or cancer includes solid tumors and hematologic malignancies.

[0025] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, liver cancer, pancreatic cancer, and brain tumors.

[0026] In another preferred embodiment, the tumor or cancer is melanoma.

[0027] A fourth aspect of the present invention provides a medicine box, the medicine box comprising:

[0028] (C1) A first formulation, wherein the first formulation contains an inhibitor of TBX1 gene expression and / or function and a pharmaceutically acceptable carrier;

[0029] (C2) A second formulation, wherein the second formulation contains an immune checkpoint molecule inhibitor and a pharmaceutically acceptable carrier;

[0030] (C3) Specification, which describes a method of using a combination of TBX1 gene expression and / or function inhibitors and immune checkpoint molecule inhibitors to treat tumors or cancer.

[0031] In another preferred embodiment, the first and second formulations are independent of each other.

[0032] In another preferred embodiment, the first and second formulations are lyophilized or liquid formulations.

[0033] In another preferred embodiment, the first and second formulations are injectable formulations.

[0034] In another preferred embodiment, the first formulation is applied before, during, and / or after the application of the second formulation.

[0035] In another preferred embodiment, the tumor or cancer includes solid tumors and hematologic malignancies.

[0036] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, liver cancer, pancreatic cancer, and brain tumors.

[0037] In another preferred embodiment, the tumor or cancer is melanoma.

[0038] A fifth aspect of the present invention provides a method for enhancing CD8 in tumor tissue. + A method for T-cell infiltration, the method comprising the step of administering a TBX1 gene expression inhibitor to a subject.

[0039] In another preferred embodiment, the TBX1 gene expression and / or function inhibitor includes nucleic acid reagents, peptide (or protein) reagents, small molecule compounds, or combinations thereof.

[0040] In another preferred embodiment, the nucleic acid reagent includes DNA reagents, RNA reagents, or combinations thereof.

[0041] In another preferred embodiment, the RNA reagent includes (but is not limited to) siRNA, shRNA, sgRNA, and their precursors or expression vectors.

[0042] In another preferred embodiment, the CD8 + T cells are CD8 cells with cytotoxic capabilities. + T cells.

[0043] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0044] A sixth aspect of the present invention provides a method for treating tumors or cancer, the method comprising administering an inhibitor of TBX1 gene expression and / or function to a subject.

[0045] A seventh aspect of the present invention provides a method for synergistically treating tumors or cancer, the method comprising administering to a subject the pharmaceutical composition of the second aspect of the present invention, or the cassette of the fourth aspect of the present invention.

[0046] In another preferred embodiment, the method includes administering a medicament as described in the fourth aspect of the invention to a subject, wherein the administration of the first formulation is performed before, during, and / or after the subject is administered the second formulation.

[0047] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0048] In another preferred embodiment, the tumor or cancer includes solid tumors and hematologic malignancies.

[0049] In another preferred embodiment, the tumor or cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, liver cancer, pancreatic cancer, and brain tumors.

[0050] In another preferred embodiment, the tumor or cancer is melanoma.

[0051] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0052] Figure 1 Lymphatic vessel-specific knockout of Tbx1 inhibits melanoma growth. (A) Construction of a Tbx1-specific knockout mouse strain (Tbx1cko). (B) Demonstration of a mouse melanoma model. (C) Growth curves of mouse melanoma in control and Tbx1cko mice. (n=10, **p<0.01 Mann-Whitney nonparametric test)

[0053] Figure 2The number of CD8+ T cells infiltrating melanoma cells was significantly increased in Tbx1 knockout mice. (A) Collection of tumor-infiltrating CD8+ T cells + Schematic diagram of T cell experimental methods. (B) Flow cytometry measurement of the proportion of CD8+ T cells in the tumor to the total number of immune cells. (n=3,**p<0.01, Mann-Whitney nonparametric test) (C) Immunofluorescence staining to quantify the proportion of CD8+ T cells (red) in the tumor to the total number of cells. (n=3,**p<0.01, Mann-Whitney nonparametric test)

[0054] Figure 3 Results of Tbx1-specific expression in lymph node endothelial cells. (A) Induction method for Tbx1 lineage tracing using mouse genetics. (B, C) Distribution pattern of Tbx1 lineage cells in draining lymph nodes 11 days after tumor implantation in control and tumor-implanted lymph nodes; Tbx1 lineage cells and lymphatic endothelial cells (green, VEGFR3+) show high overlap. (D) Proportion of Tbx1 lineage-positive cells in various types of lymphatic endothelial cells. (E) Transcriptome analysis of draining lymph node endothelial cells showing differentially expressed genes between the control and Tbx1cko groups. Detailed Implementation

[0055] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that specifically knocking out TBX1 expression in lymphatic endothelial cells can significantly inhibit tumor growth; simultaneously, CD8+ expression within the tumor... + The number of T cells increased significantly. This led to the proposal of stimulating CD8 cell infiltration within tumor tissue by inhibiting TBX1 gene expression and / or function. + T-cell expansion is a novel treatment strategy aimed at killing cancer cells and limiting tumor growth. Furthermore, by combining inhibition of TBX1 gene expression and / or function with immune checkpoint inhibitors, on the one hand, it stimulates the infiltration of CD8 cells into tumor tissue. + T cell expansion, increasing CD8+ in tumor tissue + On the one hand, it affects the number of T cells, and on the other hand, it blocks key molecular pathways such as PD-1, PD-L1, and CTLA-4, and inhibits CD8 cells that do not have tumor-killing functions. + T cells are reactivated, thereby achieving a synergistic therapeutic effect on tumors.

[0056] Based on this, the present invention was completed.

[0057] the term

[0058] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0059] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0060] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0061] As used herein, the terms “subject” and “required subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, and goats.

[0062] As used herein, the term "first active ingredient" refers to an inhibitor of TBX1 gene expression and / or function. The TBX1 gene expression inhibitor refers to a reagent capable of inhibiting TBX1 gene expression, including expression at both the mRNA and protein levels; such reagents include nucleic acid reagents, peptide (or protein) reagents, small molecule compounds, etc. A typical nucleic acid reagent is an RNA reagent, such as siRNA, shRNA, sgRNA, etc., and their precursors or expression vectors. The TBX1 gene function inhibitor refers to a reagent capable of inhibiting the functional activity of TBX1. Such functional activity includes, for example, promoting the expression of genes related to autoimmune tolerance. Such reagents include nucleic acid reagents, peptide (or protein) reagents, small molecule compounds, etc.

[0063] As used in this article, the term "siRNA" (small interfering RNA) refers to a small RNA molecule (approximately 21-25 nucleotides) that can be processed from its precursors (such as dsRNA, shRNA, etc.) by Dicer (an enzyme in the RNase III family that is specific to double-stranded RNA), or it can be synthesized chemically or produced by processing other proteins. siRNA is a major member of siRISC, stimulating the rapid cleavage and degradation of its complementary target mRNA, leading to the silencing of the target gene, thus becoming a key functional molecule in RNAi (RNA interference).

[0064] As used herein, the term "expression cassette" refers to an expression cassette containing the coding sequence of the siRNA precursor of the present invention, as well as a promoter and termination signal operatively linked to said coding sequence, which, after transcription, produces the RNAi precursor of the present invention. As used herein, the term "siRNA precursor" refers to an RNA molecule that can be processed in mammalian cells to produce siRNA; specifically, it is produced by selective processing by Dicer, Ago2, or other similar proteins to generate mature siRNA, which is then used for RNAi.

[0065] The term "shRNA" used in this article is an abbreviation for short hairpin RNA. shRNA consists of two short, inversely complementary sequences separated by a terminal loop sequence, forming a hairpin structure. Transcription is controlled by the intracellular RNA polymerase III promoter, and the shRNA sequence ends with 5-6 T-termini as a transcription terminator for RNA polymerase III. One method for generating small interfering RNA (siRNA) in vivo is to clone the siRNA sequence as part of the short hairpin into a plasmid vector. When introduced into an animal, this hairpin sequence is expressed, forming a double-stranded RNA (shRNA) with a terminal loop structure. This shRNA is recognized and processed by intracellular proteins such as Dicer and Ago2, producing functional siRNA.

[0066] As used in this article, the term "sgRNA" (signal guide RNA) is an important component of the CRISPR gene knockout system. It binds to the Cas9 protein and guides Cas9 to cut genomic DNA, thereby silencing the target gene.

[0067] As used herein, the term "expression vector" refers to a vector capable of transferring a polynucleotide sequence of interest into a target cell. Such vectors can self-replicate or bind to the host cell chromosome (host cells include, for example, prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animal cells, and individual plant cells), and may contain promoters at sites suitable for polynucleotide transcription according to this invention. Expression vectors may contain structural genes and promoters regulating their expression, as well as various regulatory elements that function within the host cell. It is well known in the art that the type of expression vector and the types of regulatory elements used in living organisms (such as animals) can vary depending on the type of host cell used. A typical expression vector is a viral vector. There are no particular limitations on the viral vectors used in this invention; any viral vector capable of utilizing the ability of viruses to deliver their genome to introduce genetic material into other cells for infection can be used. This can occur in whole living organisms or in cell culture. Examples include lentiviral vectors, adenovirus vectors, herpesvirus vectors, and poxvirus vectors.

[0068] As used herein, the term "second active ingredient" refers to an immune checkpoint molecule inhibitor, particularly a small molecule targeting inhibitor and an antibody-based targeting inhibitor. Preferably, the immune checkpoint molecules include PD-1, PD-L1, CTLA-4, etc.

[0069] As used herein, the terms “combination of active ingredients of the present invention”, “combination of substances of the present invention”, and “combination of pharmaceutical ingredients of the present invention” are used interchangeably to refer to the combination of the first and second active ingredients described above.

[0070] TBX1

[0071] The TBX1 gene encodes a T-box transcription factor located in the human 22q11.2 region of the genome and is the main pathogenic gene for 22q11.2 microdeletion syndrome. The incidence of this disease in newborns is as high as 1 in 3000, with 75% of affected infants exhibiting congenital heart disease, accompanied by facial abnormalities, thymus agenesis, and other congenital defects. However, many individuals carrying the TBX1 genetic defect are asymptomatic. Previous studies have found that TBX1 is widely expressed in the lymphatic vessels of adult mammals. In Tbx1-deficient mice, cytotoxic CD8+ expression was observed in the heart after myocardial infarction. + A significant increase in T cell count led to severe inflammatory responses and tissue adhesions, resulting in increased scar area and further impaired cardiac ejection function, exacerbating heart failure symptoms. Endothelial cell-specific transcriptomics and immunoprecipitation combined with high-throughput sequencing (ChIP-seq) were used to detect the transcriptional regulatory function of Tbx1, revealing that Tbx1 protein promotes the expression of genes related to autoimmune tolerance. These results indicate that inhibiting Tbx1 gene expression in lymphatic endothelial cells can prevent immune tolerance and stimulate CD8+ expression. + The killing function of T cells.

[0072] The TBX1 gene has a gene ID of 6899 in the NCBI database, and its expressed mRNA sequence has four isoforms: NM_005992.1, NM_080646.2, NM_080647.1, and NM_001379200.1. The mouse Tbx1 gene has a gene ID of 21380 in the NCBI database, and its expressed mRNA sequence has four isoforms: NM_011532.2, NM_001285472.1, NM_001285476.1, and NM_001373938.1. As used herein, TBX1 gene expression inhibitors can target any of the above sequences or their variants, provided that the sequence can effectively inhibit TBX1 gene expression at the mRNA and / or protein levels.

[0073] Pharmaceutical Composition

[0074] The present invention also provides a composition that can be used for the synergistic treatment of tumors or cancer, and can be used to inhibit tumor growth and / or metastasis.

[0075] The pharmaceutical composition of the present invention comprises: an effective amount of an inhibitor of TBX1 gene expression and / or function, an effective amount of an inhibitor of immune checkpoint molecules, and a pharmaceutically acceptable carrier.

[0076] Furthermore, the pharmaceutical compositions of the present invention may also include optional chemotherapeutic agents for solid tumors. These chemotherapeutic agents include (but are not limited to) cisplatin, paclitaxel, doxorubicin, etc.

[0077] Typically, the TBX1 gene expression and / or function inhibitors or immune checkpoint molecule inhibitors of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium, wherein the pH is typically about 5-8, preferably about 6-8.

[0078] As used herein, "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. It typically refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0079] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and / or cells and is acceptable to humans and / or animals.

[0080] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injectables, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of this invention can also be formulated as sustained-release formulations.

[0081] Furthermore, the combination of active ingredients of the present invention can also be used in conjunction with other therapeutic agents (such as antitumor agents or immunomodulators).

[0082] When using a pharmaceutical composition, it is to administer a safe and effective amount of the combination of active ingredients (including a first active ingredient (or a formulation thereof) and / or a second active ingredient (or a formulation thereof)) to a mammal.

[0083] It should be understood that the effective amounts of the first active ingredient (or its formulation) and / or the second active ingredient (or its formulation) in the combination of active ingredients described in this invention may vary depending on the administration method and the severity of the tumor, etc. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; tumor severity, patient weight, patient immune status, route of administration, etc.

[0084] Typically, for the first active ingredient, the safe and effective amount is usually at least about 10 nanograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, this dose is about 50 nanograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0085] For the second active ingredient, the safe and effective amount is generally at least about 10 nanograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, this dose is about 50 nanograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0086] There are no particular limitations on the administration method of the pharmaceutical composition described in this invention. Representative examples include (but are not limited to): intravenous injection, subcutaneous injection, intramuscular injection, etc.

[0087] medicine box

[0088] This invention provides a medicine box, the medicine box comprising:

[0089] (C1) A first formulation, wherein the first formulation contains an inhibitor of TBX1 gene expression and / or function and a pharmaceutically acceptable carrier;

[0090] (C2) A second formulation, wherein the second formulation contains an immune checkpoint molecule inhibitor and a pharmaceutically acceptable carrier;

[0091] (C3) Specification, which describes a method of using a combination of TBX1 gene expression and / or function inhibitors and immune checkpoint molecule inhibitors to treat tumors or cancer.

[0092] The first formulation containing an inhibitor of TBX1 gene expression and / or function includes (but is not limited to): a lyophilized formulation, a liquid formulation, or an intravenous injection solution.

[0093] The second formulation containing an immune checkpoint molecule inhibitor includes (but is not limited to): lyophilized form, liquid formulation, tablet, capsule, suppository, or intravenous solution.

[0094] In this invention, polypeptide or protein formulations are typically lyophilized or injectable.

[0095] Typically, the kit contains one or more (e.g., at least two) unit dosage forms containing inhibitors of TBX1 gene expression and / or function, and one or more (e.g., at least two) unit dosage forms containing inhibitors of immune checkpoint molecules; preferably 4-10 of each.

[0096] As used herein, the term "unit dosage form" refers to a dosage form in which a composition is prepared for a single administration for ease of use, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0097] The specification provided by this invention may include the following description: The method of using the kit is to simultaneously use a unit dosage form containing a TBX1 gene expression inhibitor and a unit dosage form containing an immune checkpoint molecule inhibitor.

[0098] The medicine box provided by the present invention is prepared by the following steps: placing a preparation containing an inhibitor of TBX1 gene expression and / or function, a preparation containing an inhibitor of immune checkpoint molecules, and the instruction manual together to form a medicine box.

[0099] The formulation containing an inhibitor of TBX1 gene expression and / or function is preferably a unit dosage form containing both an inhibitor of TBX1 gene expression and TBX1 gene function, and the formulation containing an immune checkpoint molecule inhibitor is preferably a unit dosage form containing an immune checkpoint molecule inhibitor.

[0100] The preferred step is to place at least one unit dosage form containing an inhibitor of TBX1 gene expression and / or function, and at least one unit dosage form containing an inhibitor of immune checkpoint molecules, together with the instructions, to form a medicine box.

[0101] Therapeutic applications

[0102] This invention provides a CD8 inhibitor that enhances the killing ability within tumor tissue. + Methods for T-cell infiltration include the step of administering a TBX1 gene expression and / or function inhibitor to the subject.

[0103] The present invention also provides a method for treating tumors or cancer, comprising the step of administering an inhibitor of TBX1 gene expression and / or function to a subject. Further, the present invention also provides a method for synergistic treatment of tumors or cancer, the method comprising administering to a subject the pharmaceutical composition described in the second aspect of the present invention, or the kit described in the fourth aspect of the present invention.

[0104] The subjects include humans or non-human mammals. Furthermore, the subjects have tumors or cancers. These tumors or cancers include solid tumors and hematologic malignancies, such as melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, etc.

[0105] The beneficial effects of this invention include:

[0106] (1) The role of the TBX1 gene in tumor-related research has not been reported. This invention is the first to discover a new use for the TBX1 gene, which can stimulate the infiltration of CD8 in tumor tissues by inhibiting the expression and / or function of the TBX1 gene. + T cell proliferation can kill cancer cells and limit tumor growth.

[0107] (2) The mechanism of existing immune checkpoint inhibitor therapy is to target and block key molecular pathways such as PD-1, PD-L1, and CTLA-4, thereby inhibiting CD8, which does not have tumor-killing function. + T cell reactivation. The effectiveness of these drugs relies on the presence of a certain number of CD8+ cells in the tumor microenvironment. + T cells. This invention can significantly increase the infiltration of CD8+ cells into tumor tissue. + Increasing the number of T cells provides a new combination therapy approach to enhance the effectiveness of tumor immunotherapy.

[0108] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Materials, reagents, instruments, etc., used in the embodiments are commercially available unless otherwise specified.

[0109] Experimental methods

[0110] 1. Methods for knocking out TBX1 expression in lymphatic endothelial cells

[0111] Prox1 is obtained through mating. CreER2T / +Tbx1 flox / flox Tbx1 knockout in lymphatic endothelium was induced in mice (Tbx1cko) by intraperitoneal injection of Tamoxifen. The control group consisted of Tbx1flox / flox mice injected with an equal amount of Tamoxifen, or Prox1 mice injected with corn oil. CreER2T / + Tbx1 flox / flox Strain of mice.

[0112] 2. Constructing a mouse melanoma implantation model

[0113] After hair removal on one side of the mouse's back, 1x10 mouse melanoma cell line B16F10 cells were injected subdermally. 5 Each melanoma was implanted with a 50 μL injection volume. The control group (without implantation) received an equal volume of DMEM. Tumor size was measured every two days after implantation. Successful melanoma implantation resulted in distinct black patches; tumor volume was calculated by measuring the length, width, and height of these patches.

[0114] 3. Quantitative assay of CD8+ T cells with cytotoxicity inside melanoma

[0115] 3.1 Flow cytometry

[0116] Tumor tissue was cut from the skin, placed in ice-cold PBS solution, and minced to less than 1 mm. 3 The cells were digested with a mixture of collagenase IV and dispersant enzyme, and then incubated at 37°C with shaking for approximately 1 hour. During digestion, the digestate was removed every 10 minutes and repeatedly pipetted until the tissue was digested into a single-cell suspension. This suspension was then filtered through a 70μm filter before being used in subsequent experiments. To enrich immune cells, anti-mouse CD45 microbeads were used to label the cells, followed by magnetic bead sorting. The isolated single-cell suspension was incubated at 4°C with antibodies against CD8, CD4, CD3e, CD45, CD19, and Mrc1 for 1 hour before being analyzed using a BD FACSCanto II flow cytometer. Data processing was performed using Flowjo V10. CD8+ T cells were defined as a community of CD45+CD3e+CD8+CD4-CD19-Mrc1-.

[0117] 3.2 Immunofluorescence staining

[0118] After sectioning the tumor tissue, nine sections were taken from each sample at the corresponding locations and immunofluorescence staining was performed using the CD8+ T cell-specific antibody CD8A. The effect of inhibiting Tbx1 expression on invasive cytotoxic T cells within the tumor was confirmed by comparing the differences in the number and spatial distribution of CD8+ T cells within the control group and the Tbx1cko group.

[0119] Example 1: Lymphatic vessel-specific knockout of Tbx1 can inhibit melanoma growth.

[0120] Three days before constructing the mouse melanoma implantation model, Prox1 was used. CreER2T / + Tbx1 flox / flox Tbx1 knockout in lymphatic vessels of strain mice (Tbx1cko) Figure 1 (A) After injection of melanoma cells, tumor size was observed every two days, and a tumor growth curve was plotted. Figure 1 (B) We found that in the control group, the tumors entered the exponential growth phase 11 days after implantation. We continued observation until day 19 post-implantation, when the experiment was terminated due to excessive tumor burden in the control group mice. The results showed that the tumor volume observed on days 11, 13, 15, 17, and 19 post-implantation was significantly smaller in Tbx1cko mice than in the control group (B). Figure 1 (C)

[0121] Example 2: The number of CD8+ T cells infiltrating melanoma cells was significantly increased in Tbx1 knockout mice.

[0122] To investigate the mechanism by which Tbx1cko mice inhibit tumor growth, the cytotoxic CD8+ cells within the tumor were analyzed on day 11 post-implantation. + Quantification of T cells ( Figure 2 (A). Immunofluorescence and flow cytometry analysis showed that ( Figure 2 In B and C), Tbx1cko tumor infiltration CD8 + The number of T cells increased significantly, indicating that knocking out Tbx1 expression in lymphatic endothelium promotes the production of more cytotoxic CD8 cells. + T cells enter the tumor, thereby inhibiting tumor growth.

[0123] Example 3: Gene expression and lymphocyte type in lymph node lymphoendothelial cells in a Tbx1-regulated tumor burden model.

[0124] To investigate the expression pattern of Tbx1, immunofluorescence staining was performed on the draining lymph nodes at the tumor site on day 11 post-tumor implantation using Tbx1 lineage-traced mice. Figure 3 (A). The results showed that the draining lymph nodes significantly increased in size after tumor resection, and Tbx1 was specifically expressed in the lymph node endothelial cells. Figure 3 Tbx1cko mouse endothelial cell transcriptome analysis revealed a series of abnormal expression of lymphatic endothelial-specific genes in the draining lymph nodes. Figure 3 The presence of E in the gene suggests that it is a potential target gene regulated by Tbx1.

[0125] discuss

[0126] The tumor microenvironment and draining lymph nodes contain abundant lymphatic vessels expressing TBX1. Based on findings in a cardiac injury model, the inventors hypothesize that inhibiting TBX1 expression in lymphatic vessels may stimulate CD8+ expression in the tumor microenvironment. + T cell count and cytotoxic function, thereby killing cancer cells. In this invention, a mouse melanoma implantation model was used to study the role of TBX1 in inhibiting tumor growth. Experimental results showed that specific knockout of TBX1 expression in lymphatic endothelial cells significantly inhibited tumor growth. Simultaneously, CD8+ expression within the tumor was reduced. + The number of T cells increased significantly. This invention proposes that the TBX1 gene plays a role in inhibiting tumor growth and stimulating CD8 cell cytotoxicity within tumor cells. + New functions in T cells.

[0127] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of an inhibitor of TBX1 gene expression and / or function, characterized in that, for the preparation of a medicament or pharmaceutical composition for: (i) priming / enhancing CD8 + T cell infiltration in tumor tissue; and / or (ii) treating a tumor or cancer.

2. Use according to claim 1, characterized in that, The TBX1 gene expression and / or function inhibitor includes a nucleic acid agent, a polypeptide (or protein) agent, a small molecule compound, or a combination thereof.

3. Use according to claim 1, characterized in that, The tumor or cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, kidney cancer, bladder cancer, colorectal cancer, Hodgkin's lymphoma, liver cancer, pancreatic cancer, and brain tumor.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition includes: (a) a first active ingredient, a TBX1 gene expression and / or function inhibitor; (b) a second active ingredient, an immune checkpoint molecule inhibitor; and (c) a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, wherein The immune checkpoint molecule inhibitor includes a nucleic acid agent, a polypeptide (or protein) agent, a small molecule compound, or a combination thereof.

6. The pharmaceutical composition of claim 4, wherein The immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, CTLA-4, or a combination thereof.

7. Use of a pharmaceutical composition according to claim 4, characterized in that, A medicament for treating a tumor or cancer.

8. A kit characterized in that, The kit includes: (C1) a first agent containing a TBX1 gene expression and / or function inhibitor and a pharmaceutically acceptable carrier; (C2) a second agent containing an immune checkpoint molecule inhibitor and a pharmaceutically acceptable carrier; (C3) an instruction describing a method of using the TBX1 gene expression and / or function inhibitor and the immune checkpoint molecule inhibitor in combination for treating a tumor or cancer.

9. A method of enhancing CD8 + T cell infiltration in a tumor tissue, the method comprising the step of administering to a subject an inhibitor of TBX1 gene expression and / or function.

10. The method of claim 9, wherein, The CD8 + T cells are CD8 + T cells.

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