A polypeptide composition with enhanced anti-tumor immune effect and application thereof

Abstract

The application discloses a polypeptide composition with enhanced anti-tumor immune effect and application thereof, and belongs to the technical field of anti-tumor adjuvant drugs. The polypeptide composition comprises ZW1 and ZW2; the amino acid sequence of the ZW1 is shown as SEQ ID NO. 1, and the amino acid sequence of the ZW2 is shown as SEQ ID NO. 2; and the use amount of the ZW1 and the ZW2 is 1:1. The polypeptide composition constructed by the application is completely composed of common amino acids, is simple to prepare, is low in price, is low in immunogenicity, is good in biological safety, can be used as a good T cell activator, can enhance the anti-tumor immune effect, can significantly enhance the drug efficacy of an immune checkpoint inhibitor on a tumor, can be applied to the field of tumor immunotherapy to improve the response rate of cancer immunotherapy population, and can expand the tumor patient population benefiting from cancer immunotherapy (immune checkpoint inhibitor).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-tumor adjuvant drugs, in particular to a polypeptide composition with enhanced anti-tumor immune effect and application thereof. BACKGROUND

[0002] Malignant tumor is a fatal and difficult-to-treat major disease. In recent years, tumor immunotherapy based on the normalizing mechanism of the body's immunity has been gradually applied in clinical practice, opening up a new era of clinical treatment of tumors. The current 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, etc., and 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, and these inhibitory protein molecules are immune checkpoints. Immune checkpoint inhibitors (ICIs) remove the body's immune suppression by blocking the collection of immune checkpoints and their ligands, and re-activate 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 show primary or secondary drug resistance. Although there are more than a thousand clinical studies exploring combination therapy strategies for ICIs for tumors, there is still no proven effective strategy, and it is still a big problem to develop highly efficient synergistic combination therapy for ICIs anti-tumor treatment to achieve effective response of anti-tumor immunotherapy.

[0004] In recent years, it has been found that small molecule polypeptides have the characteristics of easy synthesis, easy modification, low cost, low toxicity, low immunogenicity, and low drug resistance. Some epitope polypeptides expressed by symbiotic microorganisms are found to be able to induce cytotoxic T cell response activation through "molecular mimicry", thereby effectively enhancing the anti-tumor response of PD-1 / PD-L1 blocking therapy. Therefore, the polypeptide-based immunotherapy is expected to enhance the therapeutic effect of ICIs and solve the problem of low response rate of clinical ICIs. SUMMARY

[0005] The purpose of the present application is to provide a polypeptide composition with enhanced anti-tumor immune effect and its application, so as to solve the problems existing in the prior art. The polypeptide composition constructed by the present application can enhance the anti-tumor immune effect and can also significantly enhance the efficacy of immune checkpoint inhibitors on tumors.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a polypeptide composition with enhanced anti-tumor immune effect, which comprises 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 amount of ZW1 and ZW2 is 1:1.

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

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

[0012] In the preparation of a synergist of an immune checkpoint inhibitor for tumor treatment;

[0013] The combination of an immune checkpoint inhibitor in the preparation of a drug for treating tumors.

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

[0015] Optionally, the tumor comprises colon cancer.

[0016] The present application also provides an anti-tumor immune effect enhancer, which comprises the polypeptide composition and a pharmaceutically acceptable excipient.

[0017] The present application also provides a synergist of an immune checkpoint inhibitor for tumor treatment, which comprises the polypeptide composition and a pharmaceutically acceptable excipient.

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

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

[0020] Optionally, the administration is parenteral.

[0021] The application also provides a medicine for treating tumors, comprising the polypeptide composition in combination with an immune checkpoint inhibitor.

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

[0023] The application discloses the following technical effects:

[0024] The polypeptide composition provided by the application has the following advantages: having an anti-tumor immunoregulatory effect, having low immunogenicity, having low toxic side effects, being capable of being used for tumor treatment, being simple to prepare and low in cost, and having good application prospects.

[0025] The polypeptide drug combination constructed by the application is completely composed of common amino acids, is simple to prepare, low in price, low in immunogenicity, good in biological safety, can be used as a good T cell activator, enhances the anti-tumor immune effect, and is thus applied to the field of biological medicines; the polypeptide composition constructed by the application in combination with an immune checkpoint inhibitor can enhance the anti-tumor immune response, significantly enhance the efficacy of the immune checkpoint inhibitor on various tumors, and is higher in safety, and can be applied to the field of tumor immunotherapy to improve the response rate of the population treated by cancer immunotherapy, and expand the population of tumor patients benefiting from cancer immunotherapy (immune checkpoint inhibitor). BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0027] Figure 1 The chromatogram is identified for the polypeptide ZW1;

[0028] Figure 2 The mass spectrum is identified for the polypeptide ZW1;

[0029] Figure 3 The chromatogram is identified for the polypeptide ZW2;

[0030] Figure 4 The mass spectrum is identified for the polypeptide ZW2;

[0031] Figure 5 Flow chart for Example 2;

[0032] Figure 6 Detection results of polypeptide ZW1 and ZW2 composition promoting mouse spleen lymphocyte proliferation; A: C57BL / 6J mice; B: Balb / c mice;

[0033] Figure 7 Detection results of polypeptide ZW1 and ZW2 composition promoting mouse spleen lymphocyte activation; A: C57BL / 6J mice; B: Balb / c mice;

[0034] Figure 8 Detection results of polypeptide ZW1 and ZW2 composition promoting mouse spleen lymphocyte promoting tumor cell apoptosis; A: MC38 cells; B: CT26 cells;

[0035] Figure 9 Flow chart for Example 3;

[0036] Figure 10 HE staining results of main organs of mice in each administration group of MC38 model;

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

[0038] Figure 12 Tumor weight histogram of mice in each administration group; A: MC38 cells; B: CT26 cells;

[0039] Figure 13 Flow cytometry results of immune cell activation in mouse tumor tissue (day 18); A: MC38 CD4 + 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: CT26 CD8 + IFN-γ + TNF-α + T cells. DETAILED DESCRIPTION

[0040] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0041] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only examples of the various embodiments of the application. Other embodiments can be employed without departing from the scope of the application. Accordingly, the phrase "some embodiments" as used herein does not refer to the same embodiment, unless otherwise indicated.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0043] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0044] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0045] Example 1 Synthesis of polypeptides

[0046] The amino acid sequences of the polypeptide ZW1 and the polypeptide ZW2 are shown in Table 1, and both of the polypeptides are synthesized by Shanghai Nuoyou Biotechnology Co., Ltd. using solid-phase synthesis method. The polypeptide composition provided by the present application is composed of common amino acids, and is simple to prepare, inexpensive, and 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 chromatography conditions of ZW1 are shown in Table 2, the mass spectrometry conditions are shown in Table 3, the chromatography conditions of ZW2 are shown in Table 4, and the mass spectrometry conditions are shown in Table 5. The HPLC detection wavelength was 220 nm, and the final polypeptide product purity was >95%, and the polypeptide structure was identified by ESI-MS. The results are shown in Figures 1-4 . wherein Figure 1 is the identification chromatogram of ZW1, Figure 2 is the mass spectrum of ZW1, with a peak time of 11.068 min and a molecular weight of 1709.93; Figure 3 is the identification chromatogram of ZW2, Figure 4 is the mass spectrum of ZW2, with a peak time of 11.393 min and a molecular weight of 1475.7.

[0050] Table 2 Identification chromatography conditions of ZW1

[0051]

[0052]

[0053] Table 3 Identification mass spectrometry conditions of ZW1

[0054]

[0055] Table 4 Identification chromatography conditions of ZW2

[0056]

[0057] Table 5 Identification mass spectrometry conditions of ZW2

[0058]

[0059]

[0060] Example 2 Enhancement of the anti-tumor immune effect of the polypeptide composition

[0061] I. Experimental methods

[0062] 1. Experimental materials

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

[0064] (2) Tumor cell line: murine colon cancer cell line (MC38, ATCC; CT26, ATCC).

[0065] (3) Test sample: polypeptides ZW1 and ZW2.

[0066] 2. Experimental steps

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

[0068] (1) Mouse spleen lymphocyte isolation: After the mice were decapitated, they were soaked in 75% ethanol for 2 min. The spleen tissue was removed in a clean bench, and mouse lymphocyte isolation medium was used to obtain the spleen lymphocyte;

[0069] (2) Preparation of effector cells: Resuspend the mouse spleen lymphocytes in 1640 complete medium (containing 10% heat-inactivated FBS, 1% double antibody, recombinant IL-2 (20 IU / mL), β-mercaptoethanol (50 μM)), count and inoculate into 24-well plates (4 x 10 6 cells / well), and add 20 μg ZW1 and 20 μg ZW2. Induce stimulation for 48 hours to form effector cells;

[0070] (3) Preparation of target cells: Resuscitate the mouse colon cancer cells MC38 and CT26, and normally culture and pass them. Label them with CFSE to obtain the target cells.

[0071] (4) Detection of the ability of polypeptides to promote the proliferation of mouse spleen lymphocytes: Resuspend the mouse spleen lymphocytes in 1640 complete medium, count and inoculate into 96-well plates (1 x 10 6 cells / well), and add 10 μg ZW1 and 10 μg ZW2 to stimulate lymphocyte proliferation. Stimulate once every 2 days for a total of three times. Collect the cells on the 6th day, and use multi-color flow cytometry to evaluate lymphocyte proliferation.

[0072] (5) Detection of the activation level of spleen lymphocytes: Collect the effector cells induced and stimulated by 20 μg ZW1 and 20 μg ZW2 for 48 hours, and use multi-color flow cytometry to evaluate the activation level of the effector cells.

[0073] (6) Detection of the apoptosis proportion of target cells: The effector cells and target cells are co-cultured at an effector-to-target ratio of 10:1 at 37°C for 24 hours, and then the cells are collected. After iodinated pyridine staining, the proportion of apoptotic cells is detected using a flow cytometer.

[0074] II. Experimental results

[0075] Figure 6 The detection results of the polypeptide ZW1 and ZW2 composition promoting the proliferation of mouse spleen lymphocytes showed that, in the C57BL / 6J mouse-derived spleen lymphocytes, compared with the control group (PBS), the polypeptide ZW1 and ZW2 composition (ZWpep) promoted the proliferation of CD3 + , CD4 + and CD8 +T cells showed a significant pro-proliferative effect (p<0.05); similarly, in Balb / c mouse-derived spleen lymphocytes, compared with the control group, the combination of peptides ZW1 and ZW2 significantly increased CD3+. + (p<0.001), CD4 + (p<0.05) and CD8 + (p<0.01) T cells also showed a significant proliferative effect, proving that the combination of peptides ZW1 and ZW2 can effectively promote the proliferation of T lymphocytes.

[0076] Figure 7 The results of the detection of the activation of mouse spleen lymphocytes by the combination of peptides ZW1 and ZW2 showed that, compared with the control group, stimulation with the combination of peptides ZW1 and ZW2 effectively increased CD4+ in spleen lymphocytes derived from C57BL / 6J mice and Balb / c mice. + IFN-γ + TNF-α + and CD8 + IFN-γ + TNF-α + T cells were used to demonstrate that the combination of peptides ZW1 and ZW2 can effectively induce T lymphocyte activation.

[0077] Figure 8 The results show that stimulation of mouse splenic lymphocytes with the combination of peptides ZW1 and ZW2 promotes tumor cell apoptosis. Compared with splenic lymphocytes treated with PBS (Ctrl), splenic lymphocytes activated by the combination of peptides ZW1 and ZW2 (ZWpep) induced more apoptosis in MC38 and CT26 mouse colon cancer cells, indicating that the combination of peptides ZW1 and ZW2 can effectively enhance the anti-tumor immune effect.

[0078] Example 3: Therapeutic effect of peptide composition combined with immune checkpoint inhibitor 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: mouse colon cancer cell lines (MC38, ATCC; CT26, ATCC).

[0083] (3) Test samples: peptides ZW1 and ZW2.

[0084] (4) Immune checkpoint inhibitor: PD-1 mAb (aPD-1), clone number RPM1-14, reagent 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 procedure

[0091] The experimental procedure is shown in Table 7, and is as follows. Figure 9

[0092] (1) Subcutaneous inoculation of tumor cells: 5x10 5 / each for MC38 cell line, and 5x10 5 / each for CT26 cell line.

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

[0094] (3) Intraperitoneal injection of IgG or aPD-1 at 200 μg / each on days 7, 10, 13, and 16, respectively.

[0095] (4) Measurement of tumor size on days 7, 10, 13, 16, and 18, respectively, and calculation of tumor volume.

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

[0097] (5) Euthanasia of mice on day 18, removal of tumor tissue for photography and weighing, and HE staining of heart, liver, spleen, lung, kidney, and other tissues to determine organ damage;

[0098] (6) Measurement of tumor volume of mice, measurement of tumor weight of mice at the end time point, and evaluation of tumor tissue immune cell infiltration by polychromatic flow staining for efficacy evaluation;

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

[0100] II. Experimental results

[0101] Figure 10 ​HE staining results of major organs in mice of the MC38 model treatment groups show that no tissue damage was found in mice in the ZW1 and ZW2 combination treatment group (ZWpep) and the combination therapy group (αPD-1+ZWpep), demonstrating the safety of subcutaneous administration of the ZW1 and ZW2 combination.

[0102] Figure 11 The curves showing the changes in tumor volume in mice of each treatment group are shown. Figure 12 The results show that in both MC38 and CT26 colorectal cancer mouse models, the combination therapy group (αPD-1+ZWpep) exhibited significant (p<0.0001) tumor reduction compared to the placebo group (IgG) and the single-drug immune checkpoint inhibitor group (αPD-1), demonstrating that the combination of peptides ZW1 and ZW2 can enhance the antitumor effect of PD-1 monoclonal antibody.

[0103] like Figure 13 As shown, it is the experimental endpoint MC38 ( Figure 13 AB) and CT26 Figure 13 Multicolor flow cytometry staining results of tumor tissues in MC38 and CT26 mouse models showed that, compared with the single-drug immune checkpoint inhibitor group (αPD-1), the combination therapy group (αPD-1+ZWpep) had increased CD8+ in tumor tissues. + IFN-γ + TNF-α + T cells and CD4 + IFN-γ + TNF-α + T cells were used to confirm that the combination of peptides ZW1 and ZW2 enhanced the anti-tumor immune response to PD-1 monoclonal antibodies. Compared to the placebo group (IgG), tumor tissue in the group treated with the combination of peptides ZW1 and ZW2 (IgG + ZWpep) showed increased CD8+. + IFN-γ + TNF-α + T cells and CD4 + IFN-γ + TNF-α + T cells confirmed the regulatory effect of subcutaneous injection of the peptide ZW1 and ZW2 combination on systemic immunity.

[0104] 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 polypeptide composition with enhanced anti-tumor immune effects, characterized in that, The polypeptide composition includes ZW1 and ZW2; 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.

2. The polypeptide composition according to claim 1, characterized in that, The ratio of ZW1 to ZW2 is 1:

1.

3. The use of the polypeptide composition according to claim 1 in any of the following: Application in the preparation of antitumor immune effect enhancers; Application in the preparation of potentiators for immune checkpoint inhibitors in tumor therapy; Application of combined immune checkpoint inhibitors in the preparation of drugs for treating tumors; The immune checkpoint inhibitor is a PD-1 monoclonal antibody; The tumor is colon cancer.

4. An antitumor immune effect enhancer, characterized in that, It comprises the polypeptide composition of claim 1 and pharmaceutically acceptable excipients.

5. A potentiator for immune checkpoint inhibitors in tumor treatment, characterized in that, The composition comprises the polypeptide composition of claim 1 and pharmaceutically acceptable excipients; The immune checkpoint inhibitors include PD-1 monoclonal antibodies.

6. The enhancer as described in claim 4 or the synergist as described in claim 5, characterized in that, The excipients include at least one of the following: diluent, filler, excipient, binder, humectant, disintegrant, absorption promoter, surfactant, adsorbent carrier, lubricant, and flavoring agent.

7. The enhancer as described in claim 4 or the synergist as described in claim 5, characterized in that, Administer the medication via non-gastrointestinal routes.

8. A drug for treating tumors, characterized in that, It comprises the polypeptide composition of claim 1 in combination with an immune checkpoint inhibitor; The immune checkpoint inhibitors include PD-1 monoclonal antibodies.

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