Polypeptide composition capable of enhancing anti-tumor immune effect and application of polypeptide composition

Abstract

The invention discloses a polypeptide composition capable of enhancing an anti-tumor immune effect and application of the polypeptide composition, and belongs to the technical field of anti-tumor auxiliary drugs. The polypeptide composition comprises ZW1 and ZW2; the amino acid sequence of the ZW1 is as shown in SEQ ID NO. 1, and the amino acid sequence of the ZW2 is as shown in SEQ ID NO. 2; the dosage ratio of the ZW1 to the ZW2 is 1: 1. The polypeptide composition constructed by the invention is completely composed of common amino acids, is simple to prepare, low in price, low in immunogenicity and good in biological safety, can be used as a good T cell activator to enhance the anti-tumor immune effect, can also significantly enhance the drug effect of an immune checkpoint inhibitor on tumors, and has broad application prospects. The compound can be applied to the field of tumor immunotherapy to improve the response rate of cancer immunotherapy crowds and expand cancer patient crowds benefited by cancer immunotherapy (immunotherapy checkpoint inhibitors).

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor adjuvant drugs, and particularly to a polypeptide composition having enhanced anti-tumor immune effects and its application. Background Art

[0002] Malignant tumors are a class of fatal and difficult-to-treat major diseases. In recent years, tumor immunotherapy based on the mechanism of normalizing the body's immunity has been gradually applied clinically, opening a new era of tumor clinical treatment. Currently, the main tumor immunotherapy methods include immune checkpoint therapy, adoptive cell immunotherapy, non-specific immune stimulation therapy, chimeric antigen receptor-T cells, chimeric antigen receptor-natural killer cells, T cell receptor-T cells, and tumor vaccines. Immune checkpoint therapy is one of the more effective methods in tumor immunotherapy.

[0003] T cells highly express a variety of inhibitory protein molecules including Programmed cell death protein 1 (PD-1), Programmed cell death Ligand 1 (PD-L1), Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), Lymphocyte activation gene-3 (LAG-3), etc., thereby effectively inhibiting T cell activation and triggering tumor immune escape. These inhibitory protein molecules are immune checkpoints. Immune checkpoint inhibitors (ICIs) relieve the body's immune suppression by blocking the binding of immune checkpoints to their ligands, and reactivate the host immune system to fight tumors. However, only about 30% of patients show definite clinical efficacy to ICIs treatment, and about 60-70% of patients develop primary or secondary drug resistance. Although more than a thousand clinical studies have explored combination treatment strategies for ICIs in tumors, no effective strategy has been proven. Developing an efficient synergistic combination therapy for ICIs anti-tumor treatment to achieve an effective response to anti-tumor immunotherapy remains a major problem.

[0004] In recent years, studies have found that small molecule polypeptides have the characteristics of being easy to synthesize, easy to modify, low cost, low toxicity, low immunogenicity, and not easy to develop drug resistance. Some epitope polypeptides expressed by symbiotic microorganisms have been found to activate cytotoxic T cell responses through "molecular mimicry", thus effectively enhancing the anti-tumor response of PD-1 / PD-L1 blockade therapy. Therefore, polypeptide-based immunotherapy combinations are expected to enhance the therapeutic efficacy of ICIs and solve the problem of low response rates of clinical ICIs. Summary of the Invention

[0005] The object of the present invention is to provide a polypeptide composition with enhanced anti-tumor immune effects and its application to solve the problems existing in the above-mentioned prior art. The polypeptide composition constructed by the present invention can enhance anti-tumor immune effects and can also significantly enhance the efficacy of immune checkpoint inhibitors against tumors.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polypeptide composition with enhanced anti-tumor immune effects, and the polypeptide composition includes ZW1 and ZW2;

[0008] The amino acid sequence of ZW1 is shown in SEQ ID NO.1, and the amino acid sequence of ZW2 is shown in SEQ ID NO.2.

[0009] Optionally, the dosage of ZW1 and ZW2 is 1:1.

[0010] The present invention also provides the application of the polypeptide composition in any of the following:

[0011] Application in the preparation of an anti-tumor immune effect enhancer;

[0012] Application in the preparation of a synergist for immune checkpoint inhibitors in tumor treatment;

[0013] Application of the combination of immune checkpoint inhibitors in the preparation of drugs for treating tumors.

[0014] Optionally, the immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

[0015] Optionally, the tumor includes colon cancer.

[0016] The present invention also provides an anti-tumor immune effect enhancer, which contains the polypeptide composition and pharmaceutically acceptable excipients.

[0017] The present invention also provides a synergist for immune checkpoint inhibitors in tumor treatment, which contains the polypeptide composition and pharmaceutically acceptable excipients;

[0018] The immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

[0019] Optionally, the excipient includes at least one of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, and a flavoring agent.

[0020] Optionally, it is administered parenterally.

[0021] The present invention also provides a drug for treating tumors, comprising the polypeptide composition in combination with an immune checkpoint inhibitor;

[0022] The immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

[0023] The present invention discloses the following technical effects:

[0024] The polypeptide composition provided by the present invention has the following advantages: having an anti-tumor immune regulation effect; having low immunogenicity; having low toxicity and side effects; being capable of being used for tumor treatment; being simple to prepare and having low cost; and having good application prospects.

[0025] The polypeptide drug combination constructed by the present invention is completely composed of common amino acids, is simple to prepare, inexpensive, has low immunogenicity, and good biosafety. It can be used as a good T cell activator to enhance the anti-tumor immune effect, and thus can be applied to the biomedical field. The polypeptide composition constructed by the present invention in combination with an immune checkpoint inhibitor can enhance the anti-tumor immune response, significantly enhance the efficacy of the immune checkpoint inhibitor against various tumors, and has higher safety. It can be applied to the field of tumor immunotherapy to improve the response rate of cancer immunotherapy patients and expand the population of cancer patients who benefit from cancer immunotherapy (immune checkpoint inhibitors). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is the identification chromatogram of polypeptide ZW1;

[0028] Figure 2 It is the identification mass spectrum of polypeptide ZW1;

[0029] Figure 3 It is the identification chromatogram of polypeptide ZW2;

[0030] Figure 4 It is the identification mass spectrum of polypeptide ZW2;

[0031] Figure 5 It is the flowchart of Example 2;

[0032] Figure 6 It is the test result of the polypeptide ZW1 and ZW2 composition promoting the proliferation of mouse spleen lymphocytes; A: C57BL / 6J mice; B: Balb / c mice;

[0033] Figure 7 It is the test result of the polypeptide ZW1 and ZW2 composition promoting the activation of mouse spleen lymphocytes; A: C57BL / 6J mice; B: Balb / c mice;

[0034] Figure 8 It is the test result of mouse spleen lymphocytes promoting tumor cell apoptosis after being stimulated by the polypeptide ZW1 and ZW2 composition; A: MC38 cells; B: CT26 cells;

[0035] Figure 9 It is the flowchart of Example 3;

[0036] Figure 10 It is the HE staining result of the main organs of mice in each administration group of the MC38 model;

[0037] Figure 11 It is the tumor volume change curve of mice in each administration group; A: MC38 cells; B: CT26 cells;

[0038] Figure 12 It is the statistical chart of tumor weight of mice in each administration group; A: MC38 cells; B: CT26 cells;

[0039] Figure 13 It is the flow cytometry staining result of immune cell activation (on the 18th day) in mouse tumor tissues; A: MC38CD4 + IFN-γ + TNF-α + T cells; B: MC38 CD8 + IFN-γ + TNF-α + T cells; C: CT26 CD4 + IFN-γ + TNF-α + T cells; D: CT26CD8 + IFN-γ + TNF-α + T cells. Detailed implementation manners

[0040] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0044] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0045] Example 1 Synthesis of Polypeptides

[0046] The amino acid sequences of polypeptide ZW1 and polypeptide ZW2 are shown in Table 1. Both polypeptides were synthesized by Shanghai Nuoyou Biotechnology Co., Ltd. using the solid-phase synthesis method. The polypeptide composition provided by the present invention is completely composed of common amino acids, is simple to prepare, inexpensive, has low immunogenicity, and good biosafety.

[0047] Table 1 Polypeptide Amino Acid Sequence Table

[0048]

[0049] The polypeptides were identified by chromatography (HPLC) and mass spectrometry (ESI-MS). The chromatographic conditions for ZW1 are shown in Table 2, and the mass spectrometric conditions are shown in Table 3. The chromatographic conditions for ZW2 are shown in Table 4, and the mass spectrometric conditions are shown in Table 5. The HPLC detection wavelength was 220 nm, and the purity of the final polypeptide product was >95%. The polypeptide structure was identified by ESI-MS. The results are shown in Figures 1-4 . Among them Figure 1 is the identification chromatogram of ZW1, Figure 2 is the mass spectrum of ZW1, the peak emergence time is 11.068 min, and the molecular weight is: 1709.93; Figure 3 is the identification chromatogram of ZW2, Figure 4 is the mass spectrum of ZW2, the peak emergence time is 11.393 min, and the molecular weight is: 1475.7.

[0050] Table 2 Identification Chromatographic Conditions of ZW1

[0051]

[0052]

[0053] Table 3 Identification Mass Spectrometric Conditions of ZW1

[0054]

[0055] Table 4 Identification Chromatographic Conditions of ZW2

[0056]

[0057] Table 5 Identification Mass Spectrometric Conditions of ZW2

[0058]

[0059]

[0060] Example 2 Enhancement Effect of Polypeptide Composition on Antitumor Immune Effect

[0061] I. Experimental Method

[0062] 1. Experimental Materials

[0063] (1) Mouse Strains: 6-week-old female C57BL / 6J mice and 6-week-old female Balb / c mice.

[0064] (2) Tumor Cell Lines: Mouse colon cancer cell lines (MC38, ATCC; CT26, ATCC).

[0065] (3) Test Samples: Polypeptides ZW1 and ZW2.

[0066] 2. Experimental Procedures

[0067] The experimental procedure is as Figure 5 shown below.

[0068] (1) Isolation of mouse spleen lymphocytes: After the mice were sacrificed by cervical dislocation, they were soaked in 75% ethanol for 2 min. After removing the mouse spleen tissue in a laminar flow hood, mouse lymphocyte separation medium was used to obtain spleen lymphocytes;

[0069] (2) Preparation of effector cells: Mouse spleen lymphocytes were resuspended with RPMI 1640 complete medium (containing 10% heat-inactivated FBS, 1% penicillin-streptomycin, recombinant IL-2 (20 IU / mL), β-mercaptoethanol (50 μM)). After counting, they were seeded into 24-well plates (4×10 6 cells / well), and 20 μg of ZW1 and 20 μg of ZW2 were added. After inducing and stimulating for 48 hours, effector cells were formed;

[0070] (3) Preparation of target cells: Mouse colon cancer cells MC38 and CT26 were resuscitated, cultured and passaged normally, and labeled with CFSE, which were used as target cells.

[0071] (4) Detection of the ability of polypeptides to promote the proliferation of mouse spleen lymphocytes: Mouse spleen lymphocytes were resuspended with RPMI 1640 complete medium. After counting, they were seeded into 96-well plates (1×10 6 cells / well), and 10 μg of ZW1 and 10 μg of ZW2 were added to stimulate lymphocyte proliferation. Stimulation was performed once every 2 days for a total of three times. Cells were collected on the 6th day, and the proliferation of lymphocytes was evaluated by multi-color flow cytometry staining.

[0072] (5) Detection of the activation level of spleen lymphocytes: Effector cells induced and stimulated with 20 μg of ZW1 and 20 μg of ZW2 for 48 hours were collected, and the activation level of effector cells was evaluated by multi-color flow cytometry staining.

[0073] (6) Detection of the apoptosis ratio of target cells: Effector cells and target cells were co-cultured at an effector-to-target ratio of 10:1 at 37 °C for 24 hours, then the cells were collected. After staining with propidium iodide, the apoptosis ratio of the cells was detected by flow cytometry.

[0074] II. Experimental Results

[0075] Figure 6 The detection results of the promotion of the proliferation of mouse spleen lymphocytes by the polypeptide ZW1 and ZW2 combination. In spleen lymphocytes derived from C57BL / 6J mice, compared with the control group (PBS), the polypeptide ZW1 and ZW2 combination (ZWpep) had an effect on CD3 + , CD4 + and CD8 +T cells showed significant proliferative effects (p < 0.05); similarly, in spleen lymphocytes from Balb / c mice, compared with the control group, the combination of polypeptides ZW1 and ZW2 had significant proliferative effects on CD3 + (p < 0.001), CD4 + (p < 0.05), and CD8 + T cells (p < 0.01), demonstrating that the combination of polypeptides ZW1 and ZW2 could effectively promote the proliferation of T lymphocytes.

[0076] Figure 7 The detection results of the promotion of the activation of mouse spleen lymphocytes by the combination of polypeptides ZW1 and ZW2. Compared with the control group, the combination of polypeptides ZW1 and ZW2 effectively increased CD4 + IFN-γ + TNF-α + and CD8 + IFN-γ + TNF-α + T cells in spleen lymphocytes from C57BL / 6J mice and Balb / c mice, demonstrating that the combination of polypeptides ZW1 and ZW2 could effectively induce the activation of T lymphocytes.

[0077] Figure 8 The detection results of the promotion of tumor cell apoptosis by mouse spleen lymphocytes after stimulation with the combination of polypeptides ZW1 and ZW2. Compared with spleen lymphocytes treated with PBS (Ctrl), spleen lymphocytes activated by the combination of polypeptides ZW1 and ZW2 (ZWpep) induced more apoptosis of MC38 and CT26 murine colon cancer cells, indicating that the combination of polypeptides ZW1 and ZW2 could effectively enhance the anti-tumor immune effect.

[0078] Example 3 Therapeutic effects of the polypeptide composition combined with immune checkpoint inhibitors on tumors

[0079] I. Experimental methods

[0080] 1. Experimental materials

[0081] (1) Mouse strains: 6-week-old female C57BL / 6J mice and 6-week-old female Balb / c mice.

[0082] (2) Tumor cell lines: murine colon cancer cell lines (MC38, ATCC; CT26, ATCC).

[0083] (3) Test samples: polypeptides ZW1, ZW2.

[0084] (4) Immune checkpoint inhibitor: anti-PD-1 monoclonal antibody (αPD-1), clone number RPM1-14, the reagent was purchased from BioXCell, USA.

[0085] 2. Experimental grouping

[0086] The experimental grouping is shown in Table 6.

[0087] Table 6 Experimental grouping of mice

[0088]

[0089]

[0090] 3. Experimental procedures

[0091] The experimental process is as Figure 9 shown below.

[0092] (1) Subcutaneous inoculation of tumor cells: 5×10 5 / mouse of MC38 cell line, 5×10 5 / mouse of CT26 cell line.

[0093] (2) Subcutaneous injection of the polypeptide ZW1 and ZW2 composition (ZWpep) on days 6, 9, and 12 respectively, 100 μg / mouse for each polypeptide.

[0094] (3) Intraperitoneal injection of IgG or αPD-1 on days 7, 10, 13, and16 respectively, 200 μg / mouse.

[0095] (4) Measure the tumor size on days 7, 10, 13, 16, and 18 respectively, and calculate the tumor volume.

[0096] Tumor volume = (tumor width diameter × tumor width diameter × tumor length diameter) × 1 / 2

[0097] (5) Euthanize the mice on day 18, take out the tumor tissue for photographing and weighing, and take tissues such as the heart, liver, spleen, lung, and kidney for HE staining to clarify the organ damage situation;

[0098] (6) Evaluate the efficacy by measuring the tumor volume of mice, measuring the weight of mice tumors at the terminal time point, and evaluating the infiltration of immune cells in tumor tissues by multi-color flow cytometry staining;

[0099] (7) Observe and evaluate whether the polypeptide composition causes organ damage by HE staining of tissue sections of mouse heart, liver, spleen, lung, kidney, etc. for safety evaluation.

[0100] II. Experimental results

[0101] Figure 10The results of HE staining of the major organs of mice in each administration group of the MC38 model are shown. It can be seen that no tissue damage was found in the mice of the polypeptide ZW1 and ZW2 combination administration group (ZWpep) and the combination therapy group (αPD-1+ZWpep), which proves the safety of the polypeptide ZW1 and ZW2 combination by subcutaneous administration.

[0102] Figure 11 The tumor volume change curves of mice in each administration group are as follows. Figure 12 The statistical chart of tumor weights of mice in each administration group is shown. The results show that in both the MC38 and CT26 colorectal cancer mouse models, compared with the placebo treatment group (IgG) and the single-agent immune checkpoint inhibitor group (αPD-1), the combination therapy group (αPD-1+ZWpep) showed significant (p<0.0001) tumor reduction, which proves that the polypeptide ZW1 and ZW2 combination can enhance the anti-tumor effect of the anti-PD-1 monoclonal antibody.

[0103] As Figure 13 shown, it is the multi-color flow cytometry staining results of tumor tissues of mice at the experimental endpoint in MC38 ( Figure 13 A-B) and CT26 ( Figure 13 C-D). The results show that in both the MC38 and CT26 mouse tumor models, compared with the single-agent immune checkpoint inhibitor group (αPD-1), the combination therapy group (αPD-1+ZWpep) had increased CD8 + IFN-γ + TNF-α + T cells and CD4 + IFN-γ + TNF-α + T cells, which confirmed that the polypeptide ZW1 and ZW2 combination can enhance the anti-tumor immune response of the anti-PD-1 monoclonal antibody. Compared with the placebo treatment group (IgG), the tumor tissue of the polypeptide ZW1 and ZW2 combination administration group (IgG+ZWpep) had increased CD8 + IFN-γ + TNF-α + T cells and CD4 + IFN-γ + TNF-α + T cells, which confirmed the regulatory effect of subcutaneous injection of the polypeptide ZW1 and ZW2 combination on systemic immunity.

[0104] The above-described embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide composition with enhanced anti-tumor immune effect, characterized in that, The polypeptide composition comprises ZW1 and ZW2; The amino acid sequence of ZW1 is shown as SEQ ID NO.1, and the amino acid sequence of ZW2 is shown as SEQ ID NO.

2.

2. The polypeptide composition according to claim 1, wherein The dosage of ZW1 and ZW2 is 1:

1.

3. The application of the polypeptide composition according to claim 1 in any one of the following: The application in the preparation of an anti-tumor immune effect enhancer; The application in the preparation of a synergist for immune checkpoint inhibitors in tumor treatment; The application in the preparation of a drug for treating tumors in combination with an immune checkpoint inhibitor.

4. The application according to claim 3, wherein The immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

5. The application according to claim 3, wherein The tumor includes colon cancer.

6. An anti-tumor immune effect enhancer, characterized in that, Comprising the polypeptide composition according to claim 1 and a pharmaceutically acceptable excipient.

7. A synergist for immune checkpoint inhibitors in tumor treatment, characterized in that, Comprising the polypeptide composition according to claim 1 and a pharmaceutically acceptable excipient; The immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

8. The enhancer according to claim 6 or the synergist according to claim 7, characterized in that The excipient includes at least one of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, and a flavoring agent.

9. The enhancer according to claim 6 or the synergist according to claim 7, characterized in that, Administered by a parenteral route.

10. A drug for treating tumors, characterized in that, Comprising the polypeptide composition according to claim 1 in combination with an immune checkpoint inhibitor; The immune checkpoint inhibitor includes a PD-1 monoclonal antibody.

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