Peptides targeting H4K16la and their application in cancer treatment

Abstract

This invention discloses peptides targeting H4K16la and their application in tumor treatment, belonging to the field of biomedical technology. The peptides capable of recognizing and binding to H4K16la-modified regions include peptides with amino acid sequences as shown in any one of SEQ ID NO. 1-3. The peptides targeting H4K16la include peptides with amino acid sequences as shown in any one of SEQ ID NO. 6-8. This invention demonstrates for the first time that the three short peptides targeting H4K16la shown in SEQ ID NO. 6-8 can restore M1 polarization and CD8 by downregulating CCN1. + This study significantly enhances T cell function and sensitizes PD-1 antibody therapy, thereby improving the efficacy of tumor treatment. It is applicable to drug-resistant solid tumors with high H4K16la / CCN1 expression, laying a theoretical foundation and providing technical support for the development of novel tumor immunotherapy drugs and overcoming immune checkpoint resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to peptides targeting H4K16la and their application in the treatment of tumors. Background Technology

[0002] Malignant tumors are major intractable diseases that seriously threaten human life and health and hinder the development of public health. Their incidence and mortality rates remain high year after year, making them a core challenge that urgently needs to be addressed in the global medical and health field. In the clinical treatment system for tumors, traditional treatments such as surgery, chemotherapy, and radiotherapy can, to some extent, eliminate tumor lesions and inhibit tumor proliferation. However, they have many limitations, including high invasiveness, strong side effects, inability to completely eliminate micrometastases, and a high risk of inducing tumor recurrence and metastasis. Their effectiveness is particularly limited for advanced and metastatic malignant tumors. With the rapid development of tumor immunology and molecular biology, tumor immunotherapy, with its core advantages of precise targeting, high specificity, and good tolerability, has broken through the technical bottlenecks of traditional tumor treatments, becoming the fourth-generation core technology for tumor treatment after surgery, chemotherapy, and radiotherapy, providing a new direction for the radical cure and long-term prognosis improvement of malignant tumors.

[0003] Immune checkpoint blockade (ICB) therapy, especially anti-PD-1 / PD-L1 antibodies, reverses tumor-mediated immunosuppression by specifically blocking the PD-1 / PD-L1 immunosuppressive pathway on the surface of tumor cells and immune cells. This reactivates the tumor recognition and killing functions of the body's own T lymphocytes, thereby inhibiting tumor proliferation and eliminating tumor cells. ICB therapy has achieved clinical success in various malignant tumors. However, a large amount of clinical data confirms that this therapy generally faces the challenge of drug resistance, with most patients developing primary or acquired resistance. The reason for this lies in the presence of immunosuppressive cells in the tumor microenvironment (TME), such as M2 tumor-associated macrophages (TAMs) and depleted CD8 cells. + T cells have severely limited their clinical application and efficacy improvement.

[0004] Recent studies have revealed histone lactylation (such as H4K16la) as a novel epigenetic modification that can promote tumor immune escape by regulating the expression of metabolism-related genes. CCN1 (Cyr61) is a key downstream effector molecule of H4K16la, which can inhibit macrophage M1 polarization and CD8+. + T cell effector function. The H4K16la-CCN1 signaling axis may be a key novel pathway regulating the tumor immunosuppressive microenvironment and mediating primary and acquired resistance to ICBs, making it an ideal drug target for overcoming tumor immune resistance and improving the clinical efficacy of ICBs. However, there are currently no reports of small molecule or short peptide drugs targeting the H4K16la-CCN1 axis. Summary of the Invention

[0005] The purpose of this invention is to provide peptides targeting H4K16la and their application in tumor treatment, thereby addressing the problems existing in the prior art. This invention develops three short peptides targeting H4K16la and demonstrates for the first time that these short peptides can restore M1 polarization and CD8 by downregulating CCN1. + This study significantly enhances T cell function and sensitizes PD-1 antibody therapy, thereby improving the efficacy of tumor treatment. It is applicable to drug-resistant solid tumors with high H4K16la / CCN1 expression, laying a theoretical foundation and providing technical support for the development of novel tumor immunotherapy drugs and overcoming immune checkpoint resistance.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a peptide capable of recognizing and binding to the H4K16la modified region, comprising peptides with amino acid sequences as shown in any one of SEQ ID NO. 1-3.

[0007] The present invention also provides the use of the above-mentioned peptide in the preparation of products targeting H4K16la.

[0008] The present invention also provides a peptide targeting H4K16la, comprising peptides with amino acid sequences as shown in any one of SEQ ID NO. 6-8.

[0009] The present invention also provides the application of the above-mentioned peptide in the preparation of immune checkpoint blockade therapy sensitizers, wherein the immune checkpoint blockade therapy sensitizers have the effect of improving the therapeutic effect of immune checkpoint blockade.

[0010] The present invention also provides an immune checkpoint blockade therapy sensitizer, wherein the active ingredient of the immune checkpoint blockade therapy sensitizer includes the above-mentioned peptide.

[0011] The present invention also provides the use of the above-mentioned peptide in the preparation of antitumor drugs.

[0012] Optionally, the tumor includes lung cancer and colon cancer.

[0013] The present invention also provides an antitumor drug, wherein the active ingredient of the drug includes the above-mentioned peptide.

[0014] Furthermore, the active ingredient of the drug includes the above-mentioned peptide and PD-1 antibody composition.

[0015] The present invention discloses the following technical effects: This invention develops three peptides targeting H4K16la, whose amino acid sequences are shown in SEQ ID NO.6-8. They consist of a TAT cell-penetrating peptide, a peptide that can target and bind to the H4K16la modified region, and a C-terminal nuclear localization / stabilizing peptide.

[0016] Experiments have confirmed that the peptides of this invention can be delivered into tumor cells across the membrane, and by inhibiting H4K16la modification levels and downregulating CCN1 expression, they restore macrophage polarization to the M1 type and restore CD8. + This effectively reverses PD-1 antibody resistance and significantly enhances immune checkpoint blockade therapy by improving the IFN-γ secretion function of T cells.

[0017] This invention demonstrates for the first time that the three short peptides targeting H4K16la shown in SEQ ID NO. 6-8 can restore M1 polarization and CD8 by downregulating CCN1. + This study significantly enhances T cell function and sensitizes PD-1 antibody therapy, thereby improving the efficacy of tumor treatment. It is applicable to drug-resistant solid tumors with high H4K16la / CCN1 expression, laying a theoretical foundation and providing technical support for the development of novel tumor immunotherapy drugs and overcoming immune checkpoint resistance. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Design of short peptide structures and three candidate sequences; where A is the structure of the short peptide; and B is the sequence of the three candidate peptides. Figure 2 The results are shown in the cellular level experiments for three short peptides. A represents the Western Blot results of downregulating H4K16la and CCN1 levels in CMT167 cells at 80 μM; B represents the quantitative results of A; C represents the Western Blot results of downregulating H4K16la and CCN1 levels in MC38 cells at 80 μM; D represents the quantitative results of C; E represents the Western Blot results of downregulating H4K16la and CCN1 levels in CMT167 cells at different concentrations of Pep#1 peptide, with peptide concentrations increasing from 20 μM, 40 μM, and 80 μM; F represents the Western Blot results of downregulating H4K16la and CCN1 levels in MC38 cells at different concentrations of Pep#1 peptide, with peptide concentrations increasing from 20 μM, 40 μM, and 80 μM. Figure 3 The results are from in vivo anti-tumor experiments; where A is the flowchart of the in vivo anti-tumor experiment; B is a representative image of the tumors in each group of mice; C is the curve of tumor volume change in each group of mice; and D is the curve of tumor weight change in each group of mice. Figure 4 These are the results of flow cytometry analysis of immune cells; where A represents CD8+ in tumor tissues of mice in each group. + T cell flow cytometry analysis results; B represents CD45 in live tumor cells from each group of mice. + The results of cell proportions; C represents CD8+ in viable tumor tissue cells from each group of mice. + Results of T cell proportions; D represents TNF-α in each group of mice. + CD8 + The result of the mean fluorescence intensity of T cells; E represents the IFN-γ of mice in each group. + CD8 + The mean fluorescence intensity of T cells; F represents the Gzmb of mice in each group. + CD8 + The result of the mean fluorescence intensity of T cells; G represents the MHC II of mice in each group. + The results of the average fluorescence intensity of M1 macrophages; H represents the M1 / M2 ratio of each group of mice. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0026] This invention provides three peptides targeting H4K16la, whose structures, from N-terminus to C-terminus, sequentially comprise a TAT cell-penetrating peptide, an H4K16-targeting core peptide, and a C-terminal nuclear localization / stabilizing peptide, as shown in the following sequences: H4K16 targets the following three core peptides: Pep#1: GLKGGAKRHR (SEQ ID NO.1); Pep#2: GKGGAKRHRKV (SEQ ID NO.2); Pep #3: GGAKRHRVLR (SEQ ID NO. 3).

[0027] TAT cell membrane penetration peptide sequence: RKKRRQRRR (SEQ ID NO.4); C-terminal nuclear localization / stable sequence: PKKKKRV (SEQ ID NO.5); Peptide sequences targeting H4K16la include the following three types: Pep#1: RKKRRQRRR - GLKGGAKRHR - PKKKKRV (SEQ ID NO.6); Pep#2: RKKRRQRRR - GKGGAKRHRKV - PKKKKRV (SEQ ID NO.7); Pep #3: RKKRRQRRR - GGAKRHRVLR - PKKKKRV (SEQ ID NO. 8).

[0028] The present invention illustrates the specific application effects of the above-mentioned short peptides through the following embodiments: Example 1: Synthesis of Peptides Tripeptides as shown in SEQ ID NO. 6-8 were synthesized using a solid-phase synthesis method. The C-terminus was amidated, and the N-terminus was linked to 5-FAM for cellular uptake tracking. The HPLC purity of the samples was determined to be >95%, and they were prepared for future use.

[0029] Example 2: Cellular-level experiment on the downregulation of H4K16la and CCN1 by peptides Log-phase CMT167 (lung cancer) and MC38 (colon cancer) cells were randomly divided into a control group (Ctrl), Pep#1 group, Pep#2 group, and Pep#3 group. The control group was cultured in blank 10% FBS medium, while Pep#1 to Pep#3 groups were cultured in 10% FBS medium containing 20 μM, 40 μM, and 80 μM of the corresponding peptides, respectively. After 48 hours of culture, the cells were lysed, and the levels of H4K16la and CCN1 proteins were detected using Western blotting.

[0030] The results are as follows Figure 2 As shown in the AF, peptides Pep#1, Pep#2, and Pep#3 all significantly reduced the levels of H4K16la and CCN1 proteins, with Pep#1 showing the strongest effect in a dose-dependent manner.

[0031] Example 3: In vivo antitumor experiment of peptide combined with PD-1 antibody according to Figure 3 The experimental procedure shown in A involved selecting 6-week-old female C57BL / 6 mice and subcutaneously inoculating them with 1×10⁻⁶ mice. 6 CMT167 cells were used to construct an animal tumor model. On day 10 post-inoculation, mice were randomly divided into Pep, PD1, and Pep+PD1 groups, with healthy mice serving as the Ctrl group. The Ctrl group received an equal volume of PBS; the Pep group received Pep#1 20 mg / kg / day via intraperitoneal injection; the PD1 group received PD-1 antibody 200 μg / mouse via intraperitoneal injection on days 15, 17, and 19; and the Pep+PD1 group received both Pep#1 and PD-1 antibody via intraperitoneal injection at the same dosages as the Pep and PD1 groups. Mice were sacrificed on day 30, and tumor volume and weight were measured.

[0032] The results are as follows Figure 3 As shown in the BD diagram, the tumor inhibition rate of the Pep+PD1 group was significantly higher than that of the single-drug group (p<0.001).

[0033] Flow cytometry was used to further detect tumor-infiltrating immune cells in each group of mice.

[0034] The results are as follows Figure 4 As shown, it can be seen that the Pep+PD1 group has CD45 + CD8 in cells + The highest proportion of T cells was observed in the Pep+PD1 group; CD8 + The IFN-γ positivity rate in T cells was significantly higher in the Pep+PD1 group than in the PD1 monotherapy group; CD8 in the Pep+PD1 group +The TNF-α positivity rate in T cells was significantly higher in the Pep+PD1 group than in the PD1 monotherapy group; CD8 in the Pep+PD1 group + The positive rate of Gzmb in T cells was significantly higher than that in the PD1 monotherapy group; M1 type (MHC II) macrophages were present in the Pep+PD1 group. + The proportion has increased significantly.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A peptide capable of recognizing and binding to the H4K16la modified region, characterized in that, Including peptides with amino acid sequences as shown in any one of SEQ ID NO. 1-3.

2. The use of the peptide according to claim 1 in the preparation of products targeting H4K16la.

3. A peptide targeting H4K16la, characterized in that, Including peptides with amino acid sequences as shown in any of SEQ ID NO. 6-8.

4. The use of the peptide according to claim 3 in the preparation of an immune checkpoint blockade therapy sensitizer, characterized in that, The immune checkpoint blockade therapy sensitizer has the effect of improving the efficacy of immune checkpoint blockade therapy.

5. An immune checkpoint blockade therapy sensitizer, characterized in that, The active ingredient of the immune checkpoint blockade therapy sensitizer includes the peptide described in claim 3.

6. The use of the peptide according to claim 3 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The tumors include lung cancer and colon cancer.

8. An antitumor drug, characterized in that, The active ingredient of the drug includes the peptide described in claim 3.

9. The antitumor drug according to claim 8, characterized in that, The active ingredient of the drug includes the combination of the peptide and PD-1 antibody as described in claim 3.

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