Tumor immunotherapy polypeptide and application thereof

Abstract

The invention relates to a novel anti-tumor active peptide, a pharmaceutical composition and application of the novel anti-tumor active peptide in preparation of anti-tumor drugs. The amino acid sequence of the active peptide is RHSGSYLVTSV. The invention also discloses a fusion peptide which is formed by linking a cell-penetrating peptide to the N tail end of the active peptide. The invention also discloses a pharmaceutical composition comprising the active peptide and other anti-tumor active components. The invention also discloses application of the active peptide or the pharmaceutical composition in preparation of medicines for treating tumors. The active peptide disclosed by the invention can be used for remarkably inhibiting the growth of tumors in animal bodies and improving the treatment effect of an immune checkpoint inhibitor PD-1 antibody, so that the aim of tumor immunotherapy is fulfilled. Therefore, the polypeptide provided by the invention can be used as a candidate drug for tumor immunotherapy, and has good potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a polypeptide for tumor immunotherapy. Background Technology

[0002] According to the 2020 Global Cancer Data Report released by the International Agency for Research on Cancer (IARC) of the World Health Organization, colorectal cancer ranks third in incidence among all malignant tumors globally, and its mortality rate is second only to lung cancer. Currently, surgery remains the primary treatment for colorectal cancer, showing significant effectiveness even in early-stage patients. However, the early diagnosis rate of colorectal cancer in my country is significantly lower than in Europe and the United States, with most patients diagnosed at middle or late stages. Traditional treatments such as surgery, chemotherapy, and radiotherapy are less effective for advanced, especially late-stage, colorectal cancer, contributing significantly to my country's heavy cancer burden. Therefore, finding more effective treatments and targets for colorectal cancer is a crucial research direction for oncologists in my country. However, targeted therapies currently face numerous challenges, including individualized treatment options, rational drug use, and drug resistance, which significantly limit their therapeutic efficacy. Therefore, more effective treatment methods are urgently needed to provide new directions for cancer treatment.

[0003] In recent years, as the fourth anti-tumor strategy following surgery, chemotherapy, and radiotherapy, tumor immunotherapy has achieved many remarkable results in clinical practice and has become a focal point in the field of cancer treatment. Due to its outstanding efficacy and innovation, tumor immunotherapy was ranked first among the top ten scientific breakthroughs of 2013 by the prestigious international academic journal *Science*. In 2015, *Science* again listed tumor immunotherapy research as one of the five potential breakthroughs in the natural sciences, demonstrating its immense potential in the field of anti-tumor therapy. Currently, novel tumor immunotherapy methods mainly include immune checkpoint inhibition therapy, adoptive immune cell therapy, and tumor vaccines. Among these, immune checkpoint inhibition therapy is a new type of anti-cancer immunotherapy that is currently attracting worldwide attention and is being widely researched. The discovery of T-cell inhibitory receptors, represented by PD-1 and CTLA-4, has revolutionized cancer treatment. Their targeted antibodies, nivolumab and ipilimumab, have shown significant efficacy in the clinical treatment of melanoma and some lung cancer patients. Furthermore, treatment combining immune checkpoint inhibitors with radiotherapy and chemotherapy has also shown significant inhibitory effects on tumors such as triple-negative breast cancer. In 2018, American scientist James P. Allison and Japanese scientist Tasuku Honjo were awarded the Nobel Prize in Physiology or Medicine for their contributions to the discovery of therapies for cancer through negative immune regulation. It can be said that immunotherapy has provided new hope for previously incurable tumors.

[0004] In colorectal cancer, while immune checkpoint inhibitors combined with chemotherapy achieve a 40% objective response rate (ORR) in the treatment of DNA mismatch repair deficient (dMMR) / microsatellite highly unstable (MSI-H) metastatic CRC, this group accounts for less than 15% of advanced CRC patients. Furthermore, this therapy does not achieve an ORR in the 85% of microsatellite stable (MSS) CRC patients. In other cancer patients, the response rate to cancer immunotherapy is only around 10%-30%. Therefore, the proportion of patients who truly benefit from immunotherapy is currently very small. Mutations in the APC (adenomatous polyposis coli) gene are closely related to the development of colorectal cancer; approximately 80% of colorectal cancer patients carry APC mutations. Summary of the Invention

[0005] Based on the aforementioned technical background, the present invention aims to provide a highly effective tumor-inhibiting active monomeric peptide. This peptide is derived from a partial peptide segment of endogenous APC protein. Further research has confirmed that the active peptide can promote the infiltration of cytotoxic T cells in tissues, achieving the purpose of tumor immunotherapy, and holds promise for application in the development of anti-tumor related products.

[0006] Based on the above-mentioned technical effects, the present invention provides the following technical solution:

[0007] According to one aspect of the present invention, an active peptide for enhancing tumor immunotherapy is provided, the amino acid sequence of which is as follows:

[0008] (1)RHSGSYLVTSV; or

[0009] (2) is an amino acid sequence that still has the same physiological activity after adding, deleting, or replacing one or more amino acids in (1).

[0010] According to certain embodiments of the present invention, the active peptide is a fusion peptide.

[0011] According to certain embodiments of the present invention, the N-terminus or C-terminus of the fusion peptide includes a membrane-penetrating peptide or a targeted membrane-penetrating peptide.

[0012] According to certain embodiments of the present invention, the membrane-penetrating peptide is any one of natural proteins, chimeric peptides, and artificially synthesized peptides.

[0013] According to certain embodiments of the present invention, the membrane-penetrating peptide is selected from any one of iRGD, RGD-4C, cRDG, NGR, TCP-1, pVEC, ANHP, DIV1, DV3, PEGA, HAP-1, HAP-2, F3, Pep42, TAT, R9, MPG, MPGΔNLS, Stearyl-R8, Transportan, and Pep1.

[0014] According to certain embodiments of the present invention, the membrane-penetrating peptide or targeted membrane-penetrating peptide is connected to the active peptide by one of the following methods: encapsulation, electrostatic interaction, or covalent bonding.

[0015] According to certain embodiments of the present invention, the fusion peptide further includes a linker arm for connecting the membrane-penetrating peptide or the targeted membrane-penetrating peptide to the active peptide.

[0016] According to certain embodiments of the present invention, the linker arm is a short peptide having 1 to 6 amino acids.

[0017] According to certain embodiments of the present invention, the active peptide further includes polyethylene glycol modification.

[0018] According to certain embodiments of the present invention, the polyethylene glycol is modified at the N-terminus or C-terminus of the active peptide.

[0019] According to one aspect of the present invention, a pharmaceutical composition for treating tumors is provided, the pharmaceutical composition comprising the active peptide and a pharmaceutically acceptable carrier.

[0020] According to certain embodiments of the present invention, the pharmaceutical composition further includes other antitumor active ingredients.

[0021] According to certain embodiments of the present invention, the other antitumor active ingredients include immune checkpoint inhibitors.

[0022] According to certain embodiments of the present invention, the immune checkpoint inhibitor is selected from PD-1 inhibitors or PD-L1 inhibitors.

[0023] According to certain embodiments of the present invention, the PD-1 inhibitor is selected from PD-1 antibodies.

[0024] According to one aspect of the present invention, the present invention provides the use of the said active peptide or the said pharmaceutical composition in the preparation of a medicament for treating tumors.

[0025] According to certain embodiments of the present invention, the tumor is selected from colorectal tumors, gastric cancer, breast cancer, melanoma, lung cancer, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

[0026] In a first aspect, the present invention provides an active peptide, the amino acid sequence of which is as follows:

[0027] (1)RHSGSYLVTSV; or

[0028] (2) is an amino acid sequence that still has the same physiological activity after adding, deleting, or replacing one or more amino acids in (1).

[0029] According to certain embodiments of the present invention, the sequence of the active peptide is RHSGSYLVTSV, its structure is shown in Formula 1 below, its isoelectric point is 8.75, and its hydrophobic index is -0.027 kcal·mol⁻¹. -1 .

[0030]

[0031] APC is a tumor suppressor gene, but current understanding is largely limited to its inhibitory effect on the Wnt signaling pathway. Over the past 20 years, many strategies have been proposed for drug research targeting the Wnt signaling pathway, but no breakthrough has been achieved. This invention designs a method to screen for highly active fractions from the APC protein. The aforementioned APC11 has a suitable molecular weight, meeting the molecular weight requirements of various drug formulations for active ingredients. Furthermore, it has been verified that the aforementioned active peptide has good inhibitory effects on colorectal cancer, breast cancer, and melanoma. This process is mainly achieved through immune regulation. In some tumors, the aforementioned active peptide shows better efficacy in anti-tumor immunotherapy than PD-1 antibodies.

[0032] Furthermore, this invention also verified that the above-mentioned peptide, when used in a colorectal cancer model, can promote the infiltration of CD8-positive T cells into tumor tissue. Simultaneously, this active peptide also exhibits anti-tumor effects in breast cancer and melanoma. Moreover, this active peptide has a synergistic effect with the immune checkpoint inhibitor PD-1 antibody.

[0033] In a preferred embodiment, the active peptide further includes a modified form of the active peptide, including but not limited to salt-forming modifications to improve physical properties such as water solubility, active group modifications to improve pharmacological activity, or modifications with luminescent or tracer groups.

[0034] More preferably, the active peptide further includes modifications to the active peptide, including but not limited to salt-forming modifications to improve physical properties such as water solubility, active group modifications to improve pharmacological activity, or modifications with luminescent groups or tracer groups.

[0035] Further preferred are any one of iRGD, RGD-4C, cRDG, NGR, TCP-1, pVEC, ANHP, DIV1, DV3, PEGA, HAP-1, HAP-2, F3, Pep42, TAT, R9, MPG, MPGΔNLS, Stearyl-R8, Transportan, and Pep1.

[0036] More preferably, the connection between the transmembrane peptide and the active peptide is one of encapsulation, electrostatic interaction, or covalent bonding.

[0037] Preferably, the fusion peptide further includes a linker arm for connecting the membrane-penetrating peptide and the active peptide.

[0038] More preferably, the linker arm is a short peptide with 1 to 6 amino acids.

[0039] In a second aspect, the present invention provides a pharmaceutical composition comprising the active peptide described in the first aspect.

[0040] Preferably, the pharmaceutical composition includes other components with antitumor activity.

[0041] Preferably, the pharmaceutical composition further includes a buffer reagent or the like for maintaining the activity of the polypeptide.

[0042] Preferably, the pharmaceutical composition further includes a pharmaceutically necessary carrier.

[0043] In a third aspect, the present invention provides an antitumor product comprising the active peptide described in the first aspect or the pharmaceutical composition described in the second aspect.

[0044] Preferably, the anti-tumor products include anti-tumor drugs, anti-tumor health products, and anti-tumor model drugs.

[0045] The anti-tumor products include, but are not limited to, preparations for anti-colorectal tumors, anti-gastric cancer, anti-breast cancer, anti-melanoma, anti-lung cancer, anti-liver cancer, anti-uterine tumors, anti-leukemia, anti-lymphoma, and anti-multiple myeloma.

[0046] In a fourth aspect, the present invention provides a medicament for enhancing the infiltration of immune cells in tumor tissue, the product comprising the active peptide described in the first aspect or the pharmaceutical composition described in the second aspect.

[0047] Preferably, the drugs that enhance the infiltration of immune cells in tumor tissue include, but are not limited to, drugs used to regulate the infiltration of CD8-positive T cells.

[0048] According to certain embodiments of the present invention, certain peptide segments of the APC protein can be used for pharmaceutical applications in the preparation of drugs that inhibit the binding of PTPN13 and STAT1 proteins.

[0049] Advantages of this invention

[0050] 1) This invention is the first to discover that APC deficiency can inhibit the response of epithelial cells to interferon-γ, reduce the expression of antigen-presenting molecules on the surface of tumor cells, and promote immune escape of tumor cells. Silencing APC expression can inhibit the phosphorylation of STAT1, a downstream molecule of interferon-γ, and the expression of antigen-presenting molecules. Further research suggests that APC can bind to PTPN13 (human gene ID: 5783, mouse gene ID: 19249).

[0051] 2) By constructing and screening active peptides of APC proteins of different lengths, this invention discovered that the active peptide composed of 11 amino acids at the C-terminus of APC (hereinafter named APC11) can bind to the PDZ2 domain of PTPN13. Verification using fluorescence polarization experiments revealed that APC11 exhibits the strongest binding affinity to PDZ2, with a binding constant of 6 μmol.

[0052] 3) This invention further utilizes protein co-crystallization technology to analyze and find that the last three amino acids of the active peptide APC11 can insert between the α-helix and β-sheet of PDZ2. In particular, the terminal valine has a strong hydrophobic interaction with S21 of PDZ2, playing a key role in their binding. After mutating the last valine to alanine, the mutant APC11M cannot bind to PDZ2.

[0053] 4) In cell experiments, this invention found that APC11 significantly inhibits the binding of the PDZ2 domain of PTPN13 to STAT1 protein, while its mutant does not. Introducing the bioactive peptide APC11 into tumor cells using a transmembrane peptide significantly increases STAT1 phosphorylation and promotes the expression of major histocompatibility antigen class I (MHC-I) on the tumor cell surface, while its mutant APC11M has no effect. In animal models, tumor cells introduced with this bioactive peptide significantly inhibited tumor growth in immunocompetent mice, but had no effect on tumor growth in immunodeficient mice lacking T cells. Analysis of subcutaneous tumor tissue from immunocompetent mice revealed that the introduction of the bioactive peptide APC11 significantly increased the infiltration of CD8-positive T cells in the tumor.

[0054] 5) In order to realize the application of this active peptide in vivo, the present invention modifies the active peptide with polyethylene glycol to obtain PEG-APC11, because it can functionally provide infiltration of CD8 positive T cells in tumor tissue. The present invention also attempts to explore its relationship with the immune checkpoint inhibitor PD-1 antibody.

[0055] 6) By intraperitoneally injecting PEG-APC11 and PD-1 antibodies into mice, this invention found that PEG-APC11 significantly increased the survival time of mice in APC-deficient CT26 and MC38 cells. Further trials in other tumors also showed that PEG-APC11 significantly improved the survival time of mice inoculated with melanoma and breast cancer cells, demonstrating its effectiveness even in tumor cells with intact APCs.

[0056] 7) Finally, the present invention also linked the active peptide APC11 and the targeted membrane-penetrating peptide iRGD to nanoparticles for animal experiments, and found that nanoparticles linked with the active peptide APC11 could significantly inhibit the growth of colorectal cancer, melanoma and breast cancer cells in mice.

[0057] This invention relates to a novel antitumor bioactive peptide, a pharmaceutical composition, and its application in the preparation of antitumor drugs. The bioactive peptide is derived from a partial peptide segment of the endogenous APC (human gene ID: 324, mouse gene ID: 11789) protein, specifically the nucleotide sequence cgccattctgggtcttaccttgtgacatctgtt and the corresponding amino acid sequence RHSGSYLVTSV. This bioactive peptide can precisely target the tumor immunosuppressive protein PTPN13 (human gene ID: 5783, mouse gene ID: 19249), with a binding constant of 6 μmol to its PDZ2a domain. At micromolar concentrations, it can significantly inhibit the binding of PTPN13 to STAT1 (human gene ID: 6772, mouse gene ID: 20846) protein, inhibiting the dephosphorylation of STAT1 by PTPN13. In animals, it can significantly inhibit tumor growth and improve the therapeutic effect of the immune checkpoint inhibitor PD-1 antibody. Thus, it achieves the goal of tumor immunotherapy. Therefore, the peptide of this invention can serve as a candidate drug for tumor immunotherapy and has good potential clinical application value.

[0058] definition

[0059] APC

[0060] The APC (adenomatous polyposis coli) gene, initially discovered and named after patients with colorectal adenomatous polyps, is a tumor suppressor gene. Located on chromosome 5q21-22, it has 15 exons, with a high frequency of loss of heterozygosity in the 5q21 region. APC proteins are primarily known as tumor suppressor factors regulating Wnt signaling; they are also important cytoskeletal proteins. Mutations in the APC gene are associated with colorectal cancer, various neurological disorders, and intellectual disabilities.

[0061] The APC gene encodes a 2843-amino acid protein with a molecular weight of 314 kDa, normally located in the cytoplasm. The APC protein has multiple functional regions. Its first 17 amino acids mediate homodimer formation by forming an α-helix, allowing truncated APC proteins to link with wild-type APC proteins through this region. The middle region contains seven repeats, an AM repeat, a phosphorylation site, and a β-catenin binding site. The C-terminus contains a β-catenin-degrading site and a cytoskeletal microtubule-binding site.

[0062] PTPN13

[0063] The ptpn13 (protein tyrosine phosphatase non-receptor type 13) gene belongs to the family of non-transmembrane proteins and is located on chromosome 4q21.3. The protein it encodes is the largest in molecular weight of the protein tyrosine phosphatase (PTP) family, with a size of 270 kDa, and is also known as PTP-BL, FAP-1, PTP-BAS, and PTP1E.

[0064] The PTPN13 protein contains, from its N-terminus to its C-terminus, a KIND domain, a FERM domain, followed by PDZ1, PDZ2, PDZ3, PDZ4, and PDZ5 domains, with a catalytic phosphatase domain at the C-terminus. The function of the N-terminal KIND domain remains unclear. The FERM domain is crucial for guiding enzyme entry into the cell membrane and regulating the interactions of PTPN13 with other proteins. The PDZ1–5 domains are responsible for protein-protein interactions, acting as a scaffold.

[0065] Colorectal cancer

[0066] Colorectal cancer is a cancer originating from the epithelial cells of the large intestine. It is the third most common cancer worldwide and the second leading cause of cancer-related deaths globally. Currently, based on histological classification, it mainly includes adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma, with adenocarcinoma being the most common. Based on the tumor's microsatellite stability, it is divided into microsatellite stable and microsatellite unstable types.

[0067] CT26 cells

[0068] CT26 cells are mouse colorectal cancer cells induced by N-nitroso-N-methylcarbamate (NMU), characterized by their ease of implantation and metastasis. Genomic, transcriptomic, and immunomic studies have revealed homozygous Kras mutations (p.G12D) and (Cdkn2a) in CT26 cells. These cells exhibit high expression of proliferation and stem cell markers, while differentiation markers are absent. CT26 cells share molecular characteristics with invasive and refractory human colorectal cancer cells, classifying them as microsatellite stable colorectal cancer.

[0069] Membrane-penetrating peptides

[0070] Cell-penetrating peptides are polypeptide molecules with a size of no more than 30 amino acids that can penetrate cell membranes. They are rich in positive charges and can also transport functional molecules that cannot penetrate the membrane into cells. Due to their small and short polypeptide properties, they can be normally broken down after entering the cell, exhibiting good biocompatibility and low cytotoxicity.

[0071] Immune checkpoints

[0072] Immune checkpoints are inhibitory signaling pathways in the immune system, regulated by ligand / receptor interactions. They play a crucial role in maintaining autoimmune tolerance and regulating the duration and magnitude of pathological immune responses. During tumor growth, tumor cells activate immune checkpoints, thereby evading the immune system's attack. Immune checkpoint inhibitors are used to prevent the activation of immune checkpoints, restoring the immune system's normal function of recognizing tumor cells, thereby attacking and killing tumor cells.

[0073] Active peptides

[0074] According to one aspect of the present invention, an active peptide for enhancing tumor immunotherapy is provided, wherein the amino acid sequence of the active peptide is RHSGSYLVTSV.

[0075] According to certain embodiments of the present invention, the nucleotide sequence of the active peptide is cgccattctgggtcttaccttgtgacatctgtt.

[0076] According to certain embodiments of the present invention, the amino acid sequence of the active peptide is an amino acid sequence in RHSGSYLVTSV that retains the same physiological activity after one or more amino acids are added, removed, or replaced.

[0077] According to certain embodiments of the present invention, the active peptide is a fusion peptide.

[0078] According to certain embodiments of the present invention, the N-terminus or C-terminus of the fusion peptide includes a membrane-penetrating peptide or a targeted membrane-penetrating peptide.

[0079] According to certain embodiments of the present invention, the membrane-penetrating peptide is any one of natural proteins, chimeric peptides, and artificially synthesized peptides.

[0080] According to certain embodiments of the present invention, the membrane-penetrating peptide is selected from any one of iRGD, RGD-4C, cRDG, NGR, TCP-1, pVEC, ANHP, DIV1, DV3, PEGA, HAP-1, HAP-2, F3, Pep42, TAT, R9, MPG, MPGΔNLS, Stearyl-R8, Transportan, and Pep1.

[0081] According to certain embodiments of the present invention, the N-terminus of the fusion peptide includes the membrane-penetrating peptide TAT.

[0082] According to certain embodiments of the present invention, the fusion peptide is TAT-APC11.

[0083] According to certain embodiments of the present invention, the TAT is GRKKRRQRRR.

[0084] According to certain embodiments of the present invention, the nucleotide sequence of the fusion peptide is: GRKKRRQRRRRHSGSYLVTSV.

[0085] According to certain embodiments of the present invention, the membrane-penetrating peptide or targeted membrane-penetrating peptide is connected to the active peptide by one of the following methods: encapsulation, electrostatic interaction, or covalent bonding.

[0086] According to certain embodiments of the present invention, the fusion peptide further includes a linker arm for connecting the membrane-penetrating peptide or the targeted membrane-penetrating peptide to the active peptide.

[0087] According to certain embodiments of the present invention, the linker arm is a short peptide with 1 to 6 amino acids, for example, a short peptide with 1, 2, 3, 4, 5 or 6 amino acids.

[0088] According to certain embodiments of the present invention, the active peptide further includes polyethylene glycol modification.

[0089] According to certain embodiments of the present invention, the polyethylene glycol is modified at the N-terminus or C-terminus of the active peptide.

[0090] According to certain embodiments of the present invention, the active peptide may further include other packaging and drug delivery forms containing the active peptide; preferably, the active peptide may be linked or encapsulated with nanomaterials.

[0091] interferon

[0092] The interferon (IFN) signaling pathway is a major component of innate immunity, playing a crucial role in host resistance to pathogens and anti-tumor activity. IFN-γ is the only member of the type II interferon family, generally secreted by activated T lymphocytes and maintained at low levels in normal tissues. As a cytokine with diverse biological functions, IFN-γ plays an important role in microbial infection and tumorigenesis and development. IFN-γ is an important activator of the transcription factor STAT1. Upon stimulation by IFN-γ, JAK1 kinase is activated, phosphorylating STAT1, forming a STAT1 dimer, which translocates into the nucleus to regulate the transcriptional activation of downstream target genes.

[0093] Pharmaceutical Compositions and Kits

[0094] The term "pharmaceutical composition" refers to a mixture containing a therapeutically effective amount of one or more of the said compounds, as well as their pharmaceutically acceptable tautomers, solvates, hydrates, or salts, and other pharmaceutically acceptable carriers. The purpose of preparing the said compounds into a pharmaceutical composition is to facilitate administration to a subject.

[0095] In this application, the terms "pharmacy kit" and "reagent kit" are used interchangeably. This application discloses a pharmaceutical kit containing a therapeutically effective amount of the therapeutic agent or pharmaceutical composition. According to some embodiments of this application, the pharmaceutical kit further contains one or more other therapeutic agents. According to some embodiments of this application, the pharmaceutical kit further includes instructions for use. According to some embodiments of this application, the pharmaceutical kit further includes a device for a corresponding route of administration, such as, but not limited to, a needle.

[0096] According to one aspect of the present invention, a pharmaceutical composition for treating tumors is provided, the pharmaceutical composition comprising the active peptide and a pharmaceutically acceptable carrier.

[0097] According to certain embodiments of the present invention, the pharmaceutical composition further includes other antitumor active ingredients.

[0098] According to certain embodiments of the present invention, the other antitumor active ingredients include immune checkpoint inhibitors.

[0099] According to certain embodiments of the present invention, the immune checkpoint inhibitor is selected from PD-1 inhibitors or PD-L1 inhibitors.

[0100] According to certain embodiments of the present invention, the PD-1 inhibitor is selected from PD-1 antibodies.

[0101] Method for preparing pharmaceutical compositions

[0102] According to one aspect of the present invention, the present invention provides a method for preparing the pharmaceutical composition, the method comprising mixing the active peptide with a pharmaceutically acceptable carrier.

[0103] Treatment methods and pharmaceutical applications

[0104] According to one aspect of the present invention, the present invention provides a method for treating tumors, the method being administered to a subject the active peptide or the pharmaceutical composition.

[0105] According to certain embodiments of the present invention, the tumor is selected from colorectal tumors, gastric cancer, breast cancer, melanoma, lung cancer, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

[0106] The use of the active peptide or the pharmaceutical composition in the preparation of a medicament for treating tumors.

[0107] According to certain embodiments of the present invention, the tumor is selected from colorectal tumors, gastric cancer, breast cancer, melanoma, lung cancer, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

[0108] Dosage form

[0109] The pharmaceutical compositions of the present invention can be prepared into any pharmaceutically permissible dosage form, including but not limited to tablets, oral preparations, granules, injections, liposomes, targeted drug delivery injections, pills, capsules, granules, powders, suppositories, powders, ointments, patches, injection solutions, solutions, suspensions, sprays, lotions, drops, liniments, etc. The pharmaceutical compositions can be prepared as dry powders and mixed with sterile water or buffer solutions to form a solution before administration. The pH of the buffer solution is typically 3-11, preferably 5-9, and more preferably 7-8.

[0110] The terms “administration,” “giving,” or “application” refer to the administration of a given dose of a compound or pharmaceutical composition to a subject via a suitable route of administration.

[0111] The term "route of administration" includes, but is not limited to, any route of administration known in the art, such as oral administration, intravenous administration, intrainhalation, sublingual administration, local administration, intramuscular administration, intraocular administration, transdermal absorption, parenteral administration, intraperitoneal administration, vaginal administration, buccal administration, and rectal administration. Those skilled in the art should understand that the route of administration depends on several factors, including the location of the disease, the age of the subject, the severity of the disease, and the composition of the pharmaceutical composition.

[0112] sequence

[0113] The amino acid sequence of the active peptide APC (SEQ ID NO: 1):

[0114] RHSGSYLVTSV

[0115] The nucleotide sequence of the active peptide APC (SEQ ID NO: 2):

[0116] cgccattctgggtcttaccttgtgacatctgtt

[0117] The amino acid sequence of the transmembrane peptide TAT (SEQ ID NO: 3):

[0118] GRKKRRQRRR

[0119] The amino acid sequence of the fusion peptide TAT-APC11 (SEQ ID NO: 4):

[0120] GRKKRRQRRRRHSGSYLVTSV

[0121] The core sequence of shAPC-1 (SEQ ID NO: 5):

[0122] gcCGAGTTAAGAAAGGGCAAA

[0123] The core sequence of shAPC-2 (SEQ ID NO: 6):

[0124] cgTGGATACTTTGTTACACTT

[0125] The sequence of TAT-APC5 (SEQ ID NO: 7):

[0126] GRKKRRQRRRLVTSV

[0127] The sequence of TAT-APC7 (SEQ ID NO: 8):

[0128] GRKKRRQRRRSYLVTSV

[0129] The sequence of TAT-APC9 (SEQ ID NO: 9):

[0130] GRKKRRQRRRSGSYLVTSV

[0131] The sequence of TAT-APC13 (SEQ ID NO: 10):

[0132] GRKKRRQRRRPKRHSGSYLVTSV

[0133] The sequence of TAT-APC15 (SEQ ID NO: 11):

[0134] GRKKRRQRRRQSPKRHSGSYLVTSV

[0135] The sequence of the protective peptide TAT-HA (SEQ ID NO: 12):

[0136] GRKKRRQRRRGDIMGEWGNEIFGAIAGFLG

[0137] In this application, when “about” is used to modify a numerical value, it means that the numerical value can fluctuate within the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0138] Unless otherwise stated in this application or obviously contradicted by the context, the terms “a,” “an,” “the,” “the,” and “at least one,” and similar designations used in the context of describing this application (including the claims) are to be interpreted to cover both the singular and plural. Unless otherwise stated in this application or obviously contradicted by the context, the terms “comprising,” “having,” “including,” and “containing” used in this application are to be interpreted as open-ended terms (i.e., “including but not limited to”). Unless otherwise stated in this application or obviously contradicted by the context, all methods described in this application may be performed in any suitable order as understood by those skilled in the art.

[0139] All patents, patent applications, and references cited in this application are incorporated herein by reference in their entirety, as if each reference were cited individually. In the event of any conflict between this application and the references provided herein, the content of this application shall prevail. Attached Figure Description

[0140] Figure 1 a) Real-time quantitative PCR was used to detect the expression level of APC in CT26 cells after APC-interfering lentivirus transfection. Figure 1b shows the immunoblotting analysis of the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in APC-deficient CT26 mouse colorectal cancer cells 2 hours after stimulation with 25 ng / ml IFNγ. (scramble: randomized control group, protected sequence from Gekkai; APC: Adenomatous polyposis coli gene; IFNγ: interferon gamma; STAT1: signal transducer and activator of transcription 1; p-STAT1: phosphorylated STAT1; IRF1: interferon regulatory factor 1)). (The core sequence of shAPC-1 is: gcCGAGTTAAGAAAGGGCAAA; the core sequence of shAPC-2 is cgTGGATACTTTGTTACACTT)

[0141] Figure 2 After stimulating APC-deficient CT26 cells with 50 ng / ml IFNγ for 12 hours, the expression of downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M was detected by real-time quantitative PCR. (scramble: randomized control group, protected sequence from Jikai Biotechnology; IFNγ: interferon gamma; APC: adenomatous polyposis coli gene; IRF1: interferon regulatory factor 1; LMP2: proteasome 20S subunit beta 9; TAP1: transporter 1 (ATP binding cassette subfamily B member); TAP2: transporter 2 (ATP binding cassette subfamily B member). H2-D1: Histocompatibility 2, D region locus 1; H2-K1: Histocompatibility 2, K region locus 1; B2M: Beta-2 microglobulin. Figure 3CT26 cells (transfected with shAPC-2 lentivirus) were stimulated with 50 ng / ml IFNγ and infused with the cell-penetrating peptide TAT (sequence: GRKKRRQRRR) containing active peptides of different lengths (5, 7, 9, 11, 13, and 15 amino acids at the terminal end of APCs as shown in the figure). Twelve hours later, the expression levels of downstream antigen-presenting related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M were detected by real-time quantitative PCR. (NC: negative control; IFNγ: interferon gamma; APC: adenomatous polyposis coli gene; IRF1: interferon regulatory factor 1; LMP2: proteasome 20S subunit beta 9; TAP1: transporter 1, ATP-binding cassette). TAP2: Transporter 2 (ATP binding cassette subfamily B member); H2-D1: Histocompatibility 2, D region locus 1; H2-K1: Histocompatibility 2, K region locus 1; B2M: Beta-2 microglobulin. (NC, negative control, represents the amino acid sequence containing only the transmembrane peptide: GRKKRRQRRR; TAT-APC5 sequence: GRKKRRQRRRLVTSV; TAT-APC7 sequence: GRKKRRQRRRSYLVTSV; TAT-APC9 sequence: GRKKRRQRRRSGSYLVTSV; TAT-APC11 sequence: GRKKRRQRRRRHSGSYLVTSV; TAT-APC13 sequence: GRKKRRQRRRPKRHSGSYLVTSV; TAT-APC15 sequence: GRKKRRQRRRQSPKRHSGSYLVTSV).

[0142] Figure 4a is a statistical graph showing the tumor growth curves of CT26 cells (transfected with shAPC-2 lentivirus) with different lengths of transmembrane peptides introduced into Balb / c mice. Figure 4 b is a statistical graph showing the tumor weight of CT26 cells (transfected with shAPC-2 lentivirus) with different lengths of active peptides after 16 days of growth in Balb / c mice. Figure 4 The statistical graph of the number of CD8 positive cells in tumor tissue of CT26 cells (transfected with shAPC-2 lentivirus) with different lengths of active peptides after 16 days of growth in Balb / c mice (NC: negative control; APC: adenomatous polyposis coli gene).

[0143] Figure 5 a is a structural diagram drawn after collecting co-crystallization diffraction data of the PDZ2 domain of the active peptide APC11 and PTPN13 using the beamline of a synchrotron radiation source. The diagram shows GSYLVTSV in the sequence, which is the 8 amino acids at the C-terminus of the APC protein. Figure 5 b shows the detailed interaction between the APC11 peptide and PDZ2 within the complex. The PDZ2 domain residues involved in binding PTPN13 are marked with dark gray bars, while the APC11 peptide is represented by light gray bars. The interacting water molecules are presented as spheres, and hydrogen bonds are represented by dashed lines (APC11: the active peptide consisting of the last 11 amino acids of the APC protein; PTPN13: protein tyrosine phosphatase non-receptor type 13).

[0144] Figure 6 To detect the binding constants of the active peptide APC11 (RHSGSYLVTSV) and its mutant APC11M (RHSGSYLVTSA) to the PDZ2 domain of PTPN13 using fluorescence polarization assays (APC11: the active peptide consisting of the last 11 amino acids of the APC protein; APC11M: the mutant peptide in the active peptide consisting of the last 11 amino acids of the APC protein where the last valine is mutated to alanine).

[0145] Figure 7To investigate the effects of the cell-penetrating peptide TAT-coupled APC11 (TAT-APC11) and its mutant TAT-APC11M on the binding of PDZ2 and STAT1 proteins of PTPN13 using GST fusion protein precipitation technology (input refers to the total protein mixture before the addition of immunoprecipitation reagent; GST: glutathionesulfhydryltransferase, STAT1: signal transducer and activator of transcription 1)). (The sequence of TAT-APC11 is: GRKKRRQRRRRHSGSYLVTSV; the protective peptide HA, with the sequence GRKKRRQRRRGDIMGEWGNEIFGAIAGFLG, can promote the release of the active peptide TAT-APC11 from the macropiniosome, thereby exerting a better effect).

[0146] Figure 8 This is the result of Western blot analysis of the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in CT26 cells (transfected with shAPC-2 lentivirus) treated with the transmembrane peptide TAT-conjugated APC11 (TAT-APC11) and its mutant TAT-APC11M, 2 hours after stimulation with 25 ng / ml IFNγ. (NC: negative control).

[0147] Figure 9 This study presents the results of quantitative real-time PCR (qPCR) analysis of the expression of downstream antigen-presenting molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M in CT26 cells (transfected with shAPC-2 lentivirus) after stimulation with 50 ng / ml IFNγ for 12 hours following transmembrane peptide delivery of the bioactive peptide APC11 or its mutant APC11M. (NC: negative control)

[0148] Figure 10To detect the mean fluorescence intensity (MFI) of tumor cells expressing major histocompatibility complex I (NC: negative control; MHC-I: major histocompatibility complex I; PE-H-2Kd / 2Dd: phycoerythrin-conjugated anti-H-2Kd / 2Dd antibody) after stimulation with 100 ng / ml IFNγ for 24 hours via flow cytometry (NC: negative control; MHC-I: major histocompatibility complex I; PE-H-2Kd / 2Dd: phycoerythrin-conjugated anti-H-2Kd / 2Dd antibody) after transmembrane peptide delivery of bioactive peptide APC11 or its mutant APC11M (transfected with shAPC-2 lentivirus)).

[0149] Figure 11 a is a statistical graph showing the tumor growth curve of CT26 cells (transfected with shAPC-2 lentivirus) in Balb / c mice after transfection with the membrane-penetrating peptide APC11 or the mutant APC11M. Figure 11 b is a statistical graph of tumor growth curves of CT26 cells (APC silenced) with active peptide APC11 or mutant APC11M in nude mice. Figure 11 c is a statistical graph showing the number of CD8-positive cells in tumor tissue of CT26 cells (APC-silenced) after 16 days of growth in Balb / c mice, detected by immunofluorescence, after the transmembrane peptide was introduced into the active peptide APC11 or the mutant APC11M. (NC: negative control)

[0150] Figure 12 a is a schematic diagram of a therapeutic experiment involving intraperitoneal injection of a polyethylene glycol-modified membrane-penetrating peptide linked to an active peptide APC11 (PEG-TAT-APC11) and / or a PD-1 antibody. Figure 12 b. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into Balb / c mice inoculated with CT26 (transfected with shAPC-2 lentivirus), and survival time was recorded. Figure 12 c. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into Balb / c mice inoculated with 4T1 cells, and survival time was recorded. Figure 12 d. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into C57BL / 6 mice inoculated with B16-F10 cells, and survival time was recorded. (isotype: isotype control, a negative control for PD1 antibody; PD-1: programmed cell death 1; NC: negative control) Detailed Implementation

[0151] The following specific examples further illustrate the content of the present invention, but the scope of protection of the present invention is not limited to these examples.

[0152] Example 1: IFNγ stimulation of APC-deficient mouse colorectal cancer cells CT26 reduced the phosphorylation of downstream STAT1 protein. Levels of oxidation and IRF1 expression

[0153] This embodiment uses Western blotting to detect the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in IFNγ-stimulated APC-deficient mouse colorectal cancer cells CT26. This embodiment found that silencing APC expression in CT26 cells inhibits STAT1 phosphorylation and downregulates the expression of IRF1, a downstream protein in the JAK1-STAT1 pathway.

[0154] CT26 cells were transfected with two lentiviruses interfering with APC and one control lentivirus. Cells were lysed with Trizol, and RNA was extracted from the three groups of cells using chloroform, isopropanol, and anhydrous ethanol. The RNA was reverse transcribed into complementary double-stranded cDNA using reverse transcriptase, and the efficiency of APC interference was detected by real-time quantitative PCR. (The core sequence of shAPC-1 is: gcCGAGTTAAGAAAGGGCAAA; the core sequence of shAPC-2 is cgTGGATACTTTGTTACACTT).

[0155] CT26 cells from the control group and two APC interference groups were cultured and stimulated with 25 ng / ml IFNγ for 0, 1, 2 and 4 hours. After cell collection and lysis, Western blotting was performed to detect the phosphorylation level of STAT1 protein and the expression level of IRF1 protein.

[0156] Figure 1 a) Real-time quantitative PCR was used to detect the expression level of APC in CT26 cells after transfection with interfering APC lentiviruses. The results showed that the expression of APC in CT26 cells from the two interfering lentiviruses was significantly lower than that in the control group. Figure 1 a). Therefore, both interfering lentiviruses can effectively inhibit APC expression, with lentivirus shAPC1 achieving an interference efficiency of 80% and lentivirus shAPC2 achieving an interference efficiency of 71%.

[0157] Figure 1 b shows the immunoblotting analysis of the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in IFNγ-stimulated APC-deficient mouse colorectal cancer cells CT26. Immunoblotting revealed that, compared with the control group, interference with APC significantly inhibited STAT1 phosphorylation and IRF1 expression in CT26 cells. The results indicate that silencing APC expression can inhibit the phosphorylation of IFNγ downstream molecules STAT1 and IRF1 expression.

[0158] The results of this embodiment show that APC plays an important role in IFNγ activation of downstream STAT1 and IRF1.

[0159] Example 2: Detection of STAT1 downstream antigen presentation-related molecules after IFNγ stimulation of APC-deficient CT26 cells. Express

[0160] This embodiment uses real-time quantitative PCR to detect the expression of STAT1 downstream antigen presentation-related molecules after IFNγ stimulation of APC-deficient CT26 cells.

[0161] CT26 cells containing the two APC interfering lentiviruses constructed in Example 1 and control cells were cultured. Cells were stimulated with 50 ng / ml IFNγ for 12 hours, and RNA was extracted. After reverse transcription into cDNA, the mRNA expression levels of STAT1 downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M were detected using real-time quantitative PCR. STAT1 can transcribe downstream IRF1 and synergistically promotes the transcription of downstream LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M. (The core sequence of shAPC-1 is: gcCGAGTTAAGAAAGGGCAAA, and the core sequence of shAPC-2 is cgTGGATACTTTGTTACACTT)

[0162] Figure 2 The results are from real-time quantitative PCR. Compared with the control group, the expression of downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M in APC-deficient CT26 cells after IFNγ stimulation of APC-deficient CT26 cells were compared with those in the control group.

[0163] The results of this embodiment show that silencing APC expression can inhibit the expression of IFNγ-induced antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M, thereby promoting the inhibition of antigen presentation ability of colorectal cancer cells and thus enabling immune escape. These results suggest that APC plays an important role in IFNγ-induced antigen presentation-related molecules.

[0164] Example 3: Detection of IFNγ-stimulated CT26 cells with transmembrane peptide TAT transdermal peptides of different lengths (APC silencing) Expression levels of post-antigen presentation-related molecules

[0165] In this embodiment, real-time quantitative PCR was used to detect the expression levels of antigen-presenting molecules in CT26 cells (APC silenced) after IFNγ stimulation via the transmembrane peptide TAT introduced into the cells with different lengths of active peptides. The transmembrane peptide's function is to penetrate the cell membrane.

[0166] Two APC-interfering CT26 cells constructed in Example 1 and a control group were cultured. A 25 μM protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control. In the treatment group, both a 25 μM protective peptide linked to TAT and a 50 μM peptide linked to different lengths of the C-terminus of the APC protein linked to TAT were added. After culturing for 4 hours, the cells were stimulated with 50 ng / ml IFNγ for 12 hours. The protective peptide, TAT-HA, with the sequence GRKKRRQRRRGDIMGEWGNEIFGAIAGFLG, can promote the release of the active peptide TAT-APC11 from the macropiniosome, thereby exerting a better effect.

[0167] Cells were collected and RNA was extracted from the cells using Example 2. After reverse transcription into cDNA, the mRNA expression levels of STAT1 downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M were detected using real-time quantitative PCR.

[0168] Figure 3 To detect the expression of downstream antigen-presenting related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M 12 hours after 50 ng / ml IFNγ stimulation via TAT infusion of CT26 cells (transfected with shAPC2 lentivirus) containing different lengths of active peptides, using real-time quantitative PCR. The results showed that the active peptide composed of the 11 terminal amino acids of the APC significantly enhanced the expression of IFNγ-induced antigen-presenting related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M.

[0169] The results of this embodiment show that the peptide APC11, composed of the 11 amino acids at the C-terminus of the APC protein, linked to the cell-penetrating peptide TAT, can promote the response to IFNγ in APC-silenced CT26 cells. Cells treated with APC11 showed significantly higher mRNA expression levels of antigen-presenting related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M compared to other groups. These results suggest that the 11 amino acids at the C-terminus of the APC protein, APC11, can significantly enhance the cellular response to IFNγ and promote the cell's antigen-presenting capacity.

[0170] Example 4: Different C-terminal peptide segments of APC protein were introduced into CT26 cells via transmembrane peptides (APC silencing), and then... (The sentence is incomplete and requires more context to translate accurately). Tumor growth curve in c mice

[0171] Using the method described in Example 1, APC expression in CT26 cells was interfered with via lentivirus. A protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control. In the treatment group, both the protective peptide linked to TAT and peptides of different lengths at the C-terminus of the APC protein linked to TAT were added simultaneously. After culturing for 4 hours, the cells were digested and centrifuged, resuspended in PBS, and counted. The final cell concentration was diluted to 1 × 10⁻⁶. 7 200 μL of cell suspension per Balb / c mouse was subcutaneously injected into the skin. On day 4 after subcutaneous tumor implantation, the tumor size was measured daily with calipers, and the tumor volume was calculated (volume = 0.5 × length × width × width). Mice were sacrificed on day 16, and the subcutaneous tumors were harvested, weighed, and recorded.

[0172] Figure 4 a is a statistical graph showing the tumor growth curves of CT26 cells (APC silenced) in Balb / c mice after transmembrane peptides were introduced into the cells with different lengths of active peptides. Figure 4 The results showed that when APC11 was introduced via the cell-penetrating peptide TAT, the tumor growth rate of APC-silenced CT26 cells in Balb / c mice was the slowest.

[0173] Figure 4 b is a statistical graph showing the tumor weight of CT26 cells (APC silenced) with different lengths of active peptides after 16 days of growth in Balb / c mice. Figure 4 The results showed that tumor cells introduced into APC11 via the cell-penetrating peptide TAT formed the smallest tumors in mice.

[0174] Figure 4 c is a statistical graph showing the number of CD8-positive cells in tumor tissue of CT26 cells (APC silenced) with different lengths of active peptides after 16 days of growth in Balb / c mice. Figure 4 The results showed that tumor cells introduced with the transmembrane peptide APC11 in vivo had the highest number of infiltrating T lymphocytes in the tumor tissue formed in mice.

[0175] The results of this embodiment suggest that the 11 amino acids at the C-terminus of the APC protein, APC11, can significantly promote the infiltration of CD8-positive T cells in tumor tissue and inhibit tumor growth.

[0176] Example 5: Diffraction data of co-crystallization of active peptides APC11 and PDZ2 collected by the beamline of a synchrotron radiation source were plotted. Structural diagram

[0177] The protein complex of the PDZ2 domain of PTPN13 protein and the C-terminal 11 amino acid residues of APC protein (APC11) was crystallized at 16 °C using a sitting drop vapor diffusion method.

[0178] An equal volume of reservoir solution containing 0.1 M citric acid (pH 3.5), 3 M NaCl, and 38 mg / ml PDZ2 protein was mixed in the presence of 5.5 mM APC11 peptide to obtain an APC11 peptide-bound PDZ2 cocrystal. Before flash freezing, 25% glycerol was added to the reservoir solution to cryoprotect the crystal. Diffraction data were acquired at the 19U1 beamline of the Shanghai Synchrotron Radiation Facility and processed using the HKL3000 program. Molecular substitutions were performed on the complex structure of PDZ2-APC11 using the Phaser37 program. Further modeling was conducted, and structural optimization was performed using Phenix40. The structural diagram was plotted using PyMol.

[0179] Figure 5 Figure a shows the structural diagram drawn after collecting diffraction data of co-crystallization of the active peptides APC11 and PDZ2 using a synchrotron radiation source beamline. The gray protein structure represents the PDZ2 domain of PTPN13, and the short rod represents the 8 amino acids at the C-terminus of the APC protein, with the sequence GSYLVTSV.

[0180] Figure 5 b shows the detailed interaction between the APC11 peptide and PDZ2 within the complex. The PDZ2 residues involved in the binding are marked with dark gray bars, while the eight amino acids GSYLVTSV of the APC11 peptide that can be displayed on the front of the figure are represented by light gray bars. The interacting water molecules are presented as spheres, and hydrogen bonds are represented by dashed lines.

[0181] The results of this embodiment suggest that the last three amino acids of the active peptide APC11 can be inserted between the α-helix and β-sheet of the PDZ2 domain of the PTPN13 protein. In particular, the terminal valine has a strong hydrophobic interaction with S21 of PDZ2 and plays a key role in the binding of the two.

[0182] Example 6: Fluorescence polarization assay to detect the PDZ2 structure of active peptide APC11 and its mutant APC11M and PTPN13 Associative constant of a domain

[0183] In this embodiment, fluorescence polarization assays were used to detect the binding constants of the active peptide APC11 and its mutant APC11M with the PDZ2 domain of PTPN13.

[0184] The 11 amino acids at the C-terminus of the APC protein (APC11) and the N-terminus of the mutant peptide APC11M are linked to FITC fluorescence, respectively. The concentration of the FITC-labeled peptide is maintained at 1.7 nM. The PDZ2 domain of the PTPN13 protein is diluted 2-fold in binding buffer containing 20 mM Tris (pH 6.8) and 150 mM NaCl. Fluorescence polarization signals are recorded using a black 384-well plate reader on a Synergy H1 microplate reader, and the binding constant Kd is calculated using a GraphPad Prism 9 curve fitting method.

[0185] Figure 6 The binding constants of the active peptide APC11 and its mutant APC11M (a mutant peptide in the last 11 amino acids of the active peptide of APC protein with the last valine mutated to alanine) to the PDZ2 domain of PTPN13 were determined using fluorescence polarization assays. The results showed that the 11 amino acids at the C-terminus of APC11 could bind to the PDZ2 domain of PTPN13, while the C-terminal valine of APC11 mutated to alanine could not bind to PDZ2.

[0186] The results of this embodiment suggest that the last valine in the 11 amino acids at the C-terminus of the APC protein, APC11, plays a key role in its binding to PTPN13.

[0187] Example 7: TAT-coupled APC11 (TAT-APC11) and its mutant TAT-APC11M on PTPN13 and STAT1 proteins The effect of white combination

[0188] In this embodiment, the GST fusion protein sedimentation technique was used to detect the effect of cell-penetrating peptide TAT coupled with APC11 (TAT-APC11) and its mutant TAT-APC11M on the binding of PTPN13 to STAT1 protein.

[0189] 20 μg of TAT-APC11 or its mutant TAT-APC11M peptide was added to 500 μl of binding buffer containing recombinant GST-STAT 1 (20 μg) and HA-PDZ2 (20 μg), and incubated with GST agarose beads at 4 °C for 12 h. After incubation, the protein-binding magnetic beads were washed with washing buffer, and the protein-binding magnetic beads were mixed with loading buffer for denaturation. The results were then analyzed by Western blotting using anti-GST and anti-HA antibodies.

[0190] Figure 7 The effects of the cell-penetrating peptide TAT-conjugated APC11 (TAT-APC11) and its mutant TAT-APC11M on the binding of PDZ2 and STAT1 proteins of PTPN13 were investigated using GST fusion protein sedimentation technology. The results showed that the active peptide APC11, composed of the 11 amino acids at the C-terminus of the APC protein, could reduce the binding of PDZ2 and STAT1 proteins of PTPN13, while the mutant APC11M, which mutates the last valine of this peptide to alanine, had no effect.

[0191] The results of this embodiment suggest that the active peptide APC11, composed of 11 amino acids at the C-terminus of the APC protein, can effectively inhibit the binding of PTPN13 to the STAT1 protein.

[0192] Example 8: CT26 cells after transfusion of the cell-penetrating peptide TAT into the 11 amino acids APC11 at the C-terminus of the APC protein (APC silencing) Phosphorylation levels of downstream STAT1 protein and expression of IRF1 under IFNγ stimulation.

[0193] In this embodiment, Western blotting was used to detect the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in CT26 cells (APC silenced) after the 11 amino acids APC11 at the C-terminus of the APC protein were introduced via the cell-penetrating peptide TAT.

[0194] Three cell groups were used: a control group, a treatment group, and a mutant group. The expression of APC in CT26 cells was interfered with using the method described in Example 1 via lentivirus. A protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control group. In the treatment group, both the protective peptide linked to TAT and APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT) were added. A mutant group was also established: the culture medium contained both the protective peptide linked to TAT and a mutant APC11M (with the terminal valine mutated to alanine) of APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT).

[0195] After culturing the three groups of cells for 2 hours, they were stimulated for 2 hours with IFNγ at concentration gradients of 0, 5 ng / ml, 25 ng / ml, and 50 ng / ml as shown in the figure. The cells were then collected, lysed, and subjected to Western blotting experiments to detect the phosphorylation level of STAT1 protein and the expression level of IRF1 protein.

[0196] Figure 8 To detect the phosphorylation level of downstream STAT1 protein and the expression of IRF1 in CT26 cells (APC silenced) after IFNγ stimulation by immunoblotting with transmembrane peptides containing active peptides of different lengths.

[0197] The results showed that, compared with the control group, the addition of the cell-penetrating peptide TAT-linked APC11, consisting of the 11 amino acids at the C-terminus of the APC protein, significantly increased the phosphorylation level of STAT1 and the expression of IRF1 in the treatment group. However, in the mutant group, the peptide APC11M, which mutated the terminal valine to alanine, did not increase STAT1 phosphorylation or IRF1 expression.

[0198] The results of this embodiment suggest that the 11 amino acids at the C-terminus of the APC protein, APC11, can enhance the cellular response to IFNγ and promote the activation of the STAT1 signaling pathway.

[0199] Example 9: CT26 cells after transfusion of the cell-penetrating peptide TAT into the 11 amino acids APC11 at the C-terminus of the APC protein. (APC silencing) Expression of antigen presentation-related molecules under IFNγ stimulation

[0200] In this embodiment, quantitative real-time PCR was used to detect the expression of antigen presentation-related molecules in CT26 cells (APC silenced) after the 11 amino acids APC11 at the C-terminus of the APC protein were introduced via the cell-penetrating peptide TAT.

[0201] Three cell groups were used: a control group, a treatment group, and a mutant group. Using the method described in Example 1, lentivirus was used to interfere with APC expression in CT26 cells. A protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control group. In the treatment group, both the protective peptide linked to TAT and APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT) were added. A mutant group was also established: the culture medium was supplemented with the protective peptide linked to TAT and a mutant APC11M (with the terminal valine mutated to alanine) of APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT).

[0202] After culturing the three groups of cells for 2 hours, they were stimulated with 25 ng / ml IFNγ for 12 hours. Cells were collected and RNA was extracted from the cells. After reverse transcription into cDNA, the mRNA expression levels of STAT1 downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1 and B2M were detected by real-time quantitative PCR.

[0203] Figure 9 To detect the expression of downstream antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M in CT26 cells (APC silencing) after IFNγ stimulation by quantitative real-time PCR using transmembrane peptide-transmitted bioactive peptide APC11 or its mutant APC11M.

[0204] The results showed that, compared with the control group, the expression of IFNγ-induced antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M at the mRNA level was significantly increased when APC11 was introduced via the cell-penetrating peptide TAT in the treatment group; however, the expression of the corresponding molecules could not be increased when the mutant APC11M was introduced via TAT.

[0205] The results of this embodiment show that the active peptide APC11, which is 11 amino acids at the C-terminus of the APC protein, can significantly increase the expression of IFNγ-induced antigen presentation-related molecules IRF1, LMP2, TAP1, TAP2, H2-D1, H2K1, and B2M, while its mutant APC11M cannot.

[0206] Example 10: CT26 cells after transfusion of the cell-penetrating peptide TAT into the 11 amino acids APC11 at the C-terminus of the APC protein (APC silencing) Expression of major histocompatibility antigen class I in tumor cells under IFNγ stimulation.

[0207] In this embodiment, flow cytometry was used to detect the expression of major histocompatibility antigen class I in CT26 cells (APC silenced) after the 11 amino acids APC11 at the C-terminus of the APC protein were introduced via the cell-penetrating peptide TAT.

[0208] Three cell groups were used: a control group, a treatment group, and a mutant group. Using the method described in Example 1, lentivirus was used to interfere with APC expression in CT26 cells. A protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control group. In the treatment group, both the protective peptide linked to TAT and APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT) were added. A mutant group was also established: the culture medium was supplemented with the protective peptide linked to TAT and a mutant APC11M (with the terminal valine mutated to alanine) of APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT).

[0209] After culturing the three cell groups for 2 hours, stimulation with 100 ng / ml IFNγ was performed for 24 hours. Cells were digested and washed, then resuspended in staining buffer (PBS containing 0.1% BSA). Anti-H-2kb / 2Db Class I antibody or isotype control antibody was added, and staining continued on ice for 40 minutes. After washing, flow cytometry samples were collected on an LSR Tortessa X-20 and analyzed using FlowJo software. CT26 cells were gated by staining with isotype control antibody.

[0210] Figure 10 To detect the expression of major class I histocompatibility antigens in CT26 cells (APC silenced) after IFNγ stimulation by flow cytometry, which were infused with the transmembrane peptide TAT and contained the active peptide APC11 or its mutant APC11M.

[0211] The results showed that the active peptide APC11 could significantly increase the expression of IFNγ-induced major class I histocompatibility antigens, while its mutant APC11M could not.

[0212] The results of this embodiment show that the 11 amino acids at the C-terminus of the APC protein, APC11, can enhance the cellular response to IFNγ and promote the cellular antigen presentation capacity.

[0213] Example 11: CT26 cells after transmembrane peptide TAT was introduced into the 11 amino acids APC11 at the C-terminus of the APC protein. Tumor growth in Balb / c mice (APC silencing)

[0214] Three cell groups were used: a control group, a treatment group, and a mutant group. Using the method described in Example 1, lentivirus was used to interfere with APC expression in CT26 cells. A protective peptide linked to the cell-penetrating peptide TAT was added to the culture medium as a control. In the treatment group, both the protective peptide linked to TAT and APC11 (the C-terminal 11 amino acids of the APC protein linked to TAT) were added. A mutant group was also established: the culture medium contained both the protective peptide linked to TAT and a mutant APC11M (with the terminal valine mutated to alanine).

[0215] After culturing the three groups of cells for 4 hours, the cells were digested, centrifuged, resuspended in PBS, and counted. The final cell concentration was diluted to 1×10⁻⁶. 7 Cells / mL were subcutaneously injected into 8-week-old Balb / c mice and nude mice with 200 μL of mouse cell suspension per mouse. On the 4th day after subcutaneous tumor implantation, the size of the tumor was measured daily with calipers, and the tumor volume was calculated (volume = 0.5 × length × width × width). Mice were sacrificed at the corresponding time points, the subcutaneous tumors were collected, weighed, and the data were recorded.

[0216] Figure 11 Figure a shows the tumor growth curve statistics of CT26 cells (APC silencing) introduced with transmembrane peptide TAT into active peptide APC11 or mutant APC11M in Balb / c mice. The results showed that, compared with the control and mutant groups, the growth rate of APC-silencing CT26 cells introduced with transmembrane peptide TAT into APC11 in the treatment group was significantly inhibited, and was significantly slower than that in the control and mutant groups, while the mutant APC11M had no effect.

[0217] Figure 11 b represents the growth rate among the three groups in nude mice. Statistical graph of tumor growth curves of CT26 cells (APC silenced) with active peptide APC11 or mutant APC11M in nude mice. The results show that the introduction of APC11 into tumor cells via the transmembrane peptide had no effect on the growth rate in immunodeficient nude mice.

[0218] Figure 11 c is a statistical graph showing the number of CD8-positive cells in tumor tissue of CT26 cells (APC-silenced) introduced with the cell-penetrating peptide TAT linked to APC11 or the mutant APC11M after 16 days of growth in Balb / c mice, as detected by immunofluorescence. Subcutaneous tumors from Balb / c mice were frozen sections, and immunofluorescence staining with anti-CD8 antibody was performed to detect the expression of CD8-positive T cells. The results were observed and photographed using a laser confocal microscope. Immunofluorescence results showed that tumor cells treated with the cell-penetrating peptide TAT linked to APC11 in vivo exhibited a significantly increased number of CD8-positive T lymphocytes infiltrating the tumor tissue, significantly higher than the control and mutant groups. Cells introduced with the mutant APC11M had no effect.

[0219] The results of this embodiment show that the 11 amino acids at the C-terminus of the APC protein, APC11, can significantly promote the infiltration of CD8-positive T cells in tumor tissue and inhibit tumor growth, with the C-terminal valine of APC11 playing the most important role.

[0220] Example 12: Polyethylene glycol-modified transmembrane peptide and linked to 11 amino acids at the C-terminus of APC protein APC11 (PEG- Effect of TAT-APC11 on the survival rate of tumor-bearing mice

[0221] Using the method described in Example 1, APC expression in CT26 cells was interfered with by lentivirus. CT26 cells with silenced APC, or mouse breast cancer cell line 4T1 cells, or B mouse melanoma B16-F10 cells were digested, centrifuged, resuspended in PBS, and then counted. The final cell concentration was diluted to 1×10⁻⁶. 7 Cells were injected intraperitoneally at a concentration of 1 / mL into each Balb / c mouse. On day 1 post-implantation, each mouse received an intraperitoneal injection of 200 μg of polyethylene glycol-modified transmembrane peptide linked to the active peptide APC11 (PEG-TAT-APC11), repeated every 2 days for a total of 8 injections. On day 2 post-implantation, each mouse received an intraperitoneal injection of 200 μg of anti-PD-1 antibody, repeated every 3 days for a total of 5 injections. The survival time of the mice was observed and recorded. Figure 12 a).

[0222] Figure 12 a is a schematic diagram of a therapeutic experiment involving intraperitoneal injection of a polyethylene glycol-modified membrane-penetrating peptide linked to an active peptide APC11 (PEG-TAT-APC11) and / or a PD-1 antibody.

[0223] Figure 12 b. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into Balb / c mice inoculated with CT26 (APC silenced) and survival time was recorded.

[0224] The results showed that the survival time of Balb / c mice was significantly prolonged under the action of PEG-TAT-APC11, while PD-1 antibody treatment did not improve the survival time of mice. Figure 12 The results in b showed that the survival time of mice was further improved when both were used in combination.

[0225] Figure 12 c. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into Balb / c mice inoculated with 4T1 cells, and survival time was recorded.

[0226] The results showed that the survival time of Balb / c mice was significantly prolonged under the action of PEG-TAT-APC11; PD-1 antibody treatment also improved the survival time of mice. Figure 12 The results showed that the survival time of mice was further improved when both were used in combination.

[0227] Figure 12 d. PEG-TAT-APC11 and / or PD-1 antibodies were injected intraperitoneally into C57BL / 6 mice inoculated with B16-F10 cells and survival time was recorded.

[0228] The results showed that the survival time of C57BL / 6 mice was significantly prolonged under the action of PEG-TAT-APC11; PD-1 antibody treatment also improved the survival time of mice. Figure 12 The results showed that the survival time of mice was further improved when both were used in combination.

[0229] The results of this embodiment show that the 11 amino acids at the C-terminus of the APC protein, APC11, have a therapeutic effect on tumors. It also exhibits a synergistic effect when used in combination with PD-1.

[0230] Through the above research, this invention reveals that the absence of APC can promote the binding of PTPN13 to STAT1, inhibiting the STAT1 signaling pathway and thus promoting tumor immune escape. Furthermore, the 11-amino acid terminus peptide of APC can inhibit the effect of PTPN13 dephosphorylase on STAT1, enhance the transcriptional activity of antigen-presenting molecules in the STAT1 signaling pathway, and thus promote the infiltration of CD8-positive T cells in tumors. The APC11 peptide linked with a transmembrane peptide can inhibit tumor growth and improve survival time in mice. These studies suggest that the 11-amino acid terminus peptide of APC has promising applications in anti-tumor therapy.

Claims

1. An active peptide for enhancing tumor immunotherapy, wherein the amino acid sequence of the active peptide is as follows: (1)RHSGSYLVTSV; or (2) is an amino acid sequence that still has the same physiological activity after adding, deleting, or replacing one or more amino acids in (1).

2. The active peptide according to claim 1, characterized in that, The active peptide is a fusion peptide; the N-terminus or C-terminus of the fusion peptide includes a membrane-penetrating peptide or a targeted membrane-penetrating peptide.

3. The active peptide as described in claim 2, characterized in that, The membrane-penetrating peptide is any one of natural proteins, chimeric peptides, and artificially synthesized peptides; the membrane-penetrating peptide is selected from any one of iRGD, RGD-4C, cRDG, NGR, TCP-1, pVEC, ANHP, DIV1, DV3, PEGA, HAP-1, HAP-2, F3, Pep42, TAT, R9, MPG, MPGΔNLS, Stearyl-R8, Transportan, and Pep1.

4. The active peptide according to claim 2, characterized in that, The membrane-penetrating peptide or the targeted membrane-penetrating peptide is connected to the active peptide by one of the following methods: encapsulation, electrostatic interaction, or covalent bonding; the fusion peptide further includes a linker arm, which is used to connect the membrane-penetrating peptide or the targeted membrane-penetrating peptide to the active peptide; wherein the linker arm is a short peptide with 1 to 6 amino acids.

5. The active peptide according to any one of claims 2-4, characterized in that, The active peptide further includes polyethylene glycol modification; the polyethylene glycol modification is performed on the N-terminus or C-terminus of the active peptide.

6. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises the active peptide of any one of claims 1-4 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes other antitumor active ingredients.

8. The pharmaceutical composition according to claim 7, characterized in that, The other antitumor active ingredients include immune checkpoint inhibitors; the immune checkpoint inhibitors are selected from PD-1 inhibitors or PD-L1 inhibitors, wherein the PD-1 inhibitors are selected from PD-1 antibodies.

9. The use of the active peptide as described in any one of claims 1-5 or the pharmaceutical composition as described in any one of claims 6-8 in the preparation of a medicament for treating tumors.

10. The application as described in claim 9, wherein the tumor is selected from colorectal tumors, gastric cancer, breast cancer, melanoma, lung cancer, liver cancer, uterine tumors, leukemia, lymphoma, multiple myeloma, etc.

Images