Immunoregulatory polypeptide and application thereof
The polypeptide AYTYR and its homologous peptide were obtained by enzymatically lyzing the fungal immunomodulatory protein, and reversed and modified and modified. The polypeptide rk5 was prepared for tumor treatment, which solved the side effects of existing tumor immunotherapy drugs and achieved a safe and effective tumor suppression effect.
Patent Information
- Application Number
- CN202510333980.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-07-04
AI Technical Summary
The existing tumor immunotherapy drugs have immune-related side effects during use, and the pharmacodynamic substances of oral fungal immunomodulatory proteins are unclear. It is of great significance to develop safe and effective tumor immunotherapy drugs.
Polypeptide AYTYR and its homologous peptides were obtained by enzymatically lysing fungal immunomodulatory proteins, and polypeptides rk5 and PS-rk were obtained by reverse flip and modification, which enhances the anti-enzymatic lysis ability and targets the tumor site. Polypeptide probes were prepared for tumor diagnosis and treatment.
The peptide rk5 significantly inhibits tumor growth in the mouse CT26 colorectal cancer model, and its therapeutic effect is comparable to that of PD-L1 antibodies. It provides a candidate for tumor immunodiagnosis and treatment, and has good application prospects and economic value.
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Figure CN120248061A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and specifically, relates to an immunomodulatory polypeptide and its application. Background Art
[0002] Although tumor immunotherapy has significant efficacy, in many cancer patients, due to the overactivation of the immune system, a series of immune-related side effects will be triggered, such as autoimmune diseases and cytokine release syndrome, etc. Therefore, it is crucial to find safe and effective tumor immunotherapy drugs. Traditional Chinese medicine is a drug prepared from natural products such as animals and plants through different methods of processing. Especially many traditional Chinese medicines that are both edible and medicinal have been found in long-term practice to be able to be used as food ingredients for cooking and can also regulate the human immune system. These drugs usually have low toxicity and side effects. Therefore, it is of great value to develop highly effective and low-toxic tumor immunotherapy drugs based on this.
[0003] Fungal traditional Chinese medicines such as Ganoderma lucidum and Flammulina velutipes contain a large number of protein components. Oral fungal immunomodulatory protein (FIP) has been proven to have high anti-tumor activity. Past studies have generally focused on the anti-tumor activity of the FIP protein itself. However, during the actual anti-tumor process of oral proteins, due to the presence of digestive enzymes in the body, the absorbed substance by the body is not the complete protein. Therefore, the active pharmaceutical ingredient of oral fungal immunomodulatory protein (FIP) is still unclear. Therefore, providing a polypeptide with better anti-enzymolysis and immune activity is of great significance for developing tumor immunotherapy drugs applicable to treatment routes such as oral administration and injection. Summary of the Invention
[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide a polypeptide and a fusion polypeptide.
[0005] The second purpose of the present invention is to provide a probe containing the above polypeptide.
[0006] The third purpose of the present invention is to provide the application of the probe of the above polypeptide in the preparation of reagents for tumor diagnosis or tracer imaging.
[0007] The fourth purpose of the present invention is to provide the application of the above polypeptide, fusion polypeptide and polypeptide probe in the preparation of drugs for treating tumors or immune diseases.
[0008] The fifth purpose of the present invention is to provide a drug containing the above polypeptide.
[0009] The above purposes of the present invention are achieved by the following technical solutions: The present invention provides a polypeptide, and the amino acid sequence of the polypeptide is B-Y-J-Y-R, wherein B-Y-J-Y-R is B-Tyr-J-Tyr-Arg, and B and J independently selected from any amino acid.
[0010] Since trypsin and chymotrypsin are two major digestive enzymes in the intestine, trypsin can enzymatically hydrolyze peptide chains at the carboxyl terminus of arginine (Arg, R) and lysine (Lys, K); chymotrypsin enzymatically hydrolyzes peptide segments at the sites of aromatic amino acids tyrosine (Tyr, Y), tryptophan (Trp, W) and phenylalanine (Phe, F). In order to explore the pharmacodynamic substances of orally administered fungal immunomodulatory protein (FIP), trypsin and chymotrypsin were used to enzymatically hydrolyze the fungal immunomodulatory protein LZ-8 in Ganoderma lucidum, and it was found that the obtained polypeptide AYTyr had good immunomodulatory properties. Through FIP homologous sequence alignment, it was found that the peptide segment KR5 from Flammulina velutipes FIP-fve and some homologous peptide segments from other different fungal sources all had better immunomodulatory properties. These peptide segments with immunomodulatory properties all had the characteristics of B-Tyr-J-Tyr-Arg, and B and J could be independently selected from any amino acid. Further studies have shown that after reverse inversion and modification of KR5, the obtained polypeptides rk5 (rysyk) and ps-rk (prdsfmsrysyk) have the same immunostimulatory effect as KR5 (the activity remains unchanged), both are superior to the Flammulina velutipes immunomodulatory protein FIP-fve, and rk5 can target tumor sites and significantly inhibit the growth of CT26 tumors.
[0011] Furthermore, the B is any one of E, P, Q, K, namely Glu, Pro, Gln or Lys; J is any one of T, K, S, namely Thr, Lys or Ser.
[0012] Furthermore, the amino acid sequence of the polypeptide is one of AYTyr (SEQ ID No.1), EYTyr (SEQ ID No.2), AYQyr (SEQ ID No.3), KYSyr (SEQ ID No.4), PYKYR (SEQ ID No.5), RYKYR (SEQ ID No.6) or QYKYR (SEQ ID No.7).
[0013] Furthermore, the configuration of each amino acid in the polypeptide is independently selected from D-configuration or L-configuration.
[0014] The present invention provides a polypeptide, which is a polypeptide obtained by reverse inversion of the above polypeptide.
[0015] Furthermore, the amino acid sequence of the polypeptide is rysyk (SEQ ID No.8). The reverse inversion strategy is to change the natural amino acids of the polypeptide to D-configuration amino acids to enhance its anti-enzymatic hydrolysis ability; among them, reverse inversion is to reverse the C-terminus and N-terminus of the amino acid, and the amino acid becomes D-configuration.
[0016] The present invention provides a fusion polypeptide, which is obtained by modifying the above-mentioned polypeptide or the polypeptide prepared by the reverse sequence flipping strategy at the N-terminus or C-terminus.
[0017] Furthermore, the modification is a protein affinity peptide modification.
[0018] Furthermore, the protein affinity peptide is an albumin affinity peptide.
[0019] Even further, the modification is to perform albumin affinity peptide modification at the N-terminus of the polypeptide.
[0020] Preferably, the amino acid sequence of the fusion polypeptide is prdsfmsrysyk (SEQ ID No.9).
[0021] When the amino acid sequence of the polypeptide is known, the above-mentioned polypeptide can be obtained by conventional polypeptide synthesis technical means in the art, such as liquid-phase synthesis or solid-phase synthesis in chemical synthesis methods, fermentation method or enzymatic synthesis method in biosynthesis methods.
[0022] As a preferably implementable embodiment, the preparation method of the above-mentioned polypeptide is as follows: S1. Mix resin, N,N-dimethylformamide and dichloromethane, swell and wash. S2. Add an amino acid to the mixture in step S1 for a condensation reaction to obtain a resin-crosslinked peptide segment. S3. Cut the resin-crosslinked peptide segment and precipitate it to obtain the product.
[0023] Specifically, step S1 is to add 0.2 mM Rink resin to 1.5 mL DMF (N,N-dimethylformamide) and 1.5 mL DCM (dichloromethane) to swell the resin for 30 min. Pour the swollen resin into a synthesizer, suck out the swelling solution, add 2 mL of deprotection solution (piperidine:DMF = 1:4), and shake on a shaker for 10 min to obtain the product.
[0024] Specifically, the washing in step S1 is to wash with DMF three times, wash with DCM three times, and then wash with DMF three times, and the washing time for each time is 2 min.
[0025] Specifically, step S3 is to cut the peptide chain from the resin with trifluoroacetic acid solution, add ice-cold diethyl ether to precipitate the polypeptide, and purify the polypeptide by HPLC after drying.
[0026] The present invention labels the polypeptide rk5 with FITC and intraperitoneally injects it into mice, and observes the distribution of the polypeptide in various organs of the mice, and finds that the polypeptide rk5 can target the tumor site in the body of the mice.
[0027] Therefore, the present invention provides a polypeptide probe, and the general structural formula of the polypeptide probe is M 标 -L 连 -R 多肽 ; wherein, M 标 is a photo-labeling or radionuclide complexing group, L 连 is a linking group, and R 多肽 is a monomer, dimer or multimer of the above-mentioned polypeptide or fusion polypeptide.
[0028] The present invention provides the application of the above-mentioned polypeptide probe in the preparation of a reagent for tumor diagnosis or tracer imaging.
[0029] In the mouse CT26 colorectal cancer model, rk5 can significantly inhibit tumor growth, and the therapeutic effect is equivalent to that of the PD-L1 antibody. Therefore, the present invention also provides the application of the above-mentioned polypeptide, fusion polypeptide or polypeptide probe in the preparation of a drug for treating tumors or immune diseases.
[0030] The present invention also provides a drug, and the drug comprises the above-mentioned polypeptide or fusion polypeptide.
[0031] Furthermore, the drug further comprises a pharmaceutically acceptable excipient.
[0032] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an immunomodulatory polypeptide and its application. The present invention discovers that the peptide segment KR5 obtained by enzymatic hydrolysis of the Flammulina velutipes immunomodulatory protein FIP-fve and its partially homologous peptide segments from different fungal sources all have good immunomodulatory properties, and its amino acid sequence is B-Y-J-Y-R. After reverse sequence flipping and modification transformation of KR5, the immunostimulatory effects of the obtained D-configured polypeptide and modified polypeptide are unchanged compared with KR5, and both are superior to the Flammulina velutipes immunomodulatory protein FIP-fve. In the mouse CT26 colorectal cancer model, rk5 can significantly inhibit tumor growth, and the therapeutic effect is equivalent to that of the PD-L1 antibody. The FIP active polypeptide provided by the present invention provides a theoretical basis for exploring the pharmacodynamic substances and action mechanisms of FIP, provides candidate drugs for tumor immunodiagnosis and treatment, etc., and has good application prospects and high economic value. Description of the Drawings
[0033] Figure 1 For monitoring the immunomodulatory activities of FIP and KR5 homologous polypeptides by co-culture experiments of peripheral blood mononuclear cells.
[0034] Figure 2 For monitoring the immunomodulatory activities of reverse sequence flipping and ps-modified polypeptides by co-culture experiments of peripheral blood mononuclear cells.
[0035] Figure 3 For monitoring the in vivo distribution of polypeptides by mouse imaging.
[0036] Figure 4 To detect the in vivo anti-tumor activity of the polypeptide in a CT26 xenograft tumor model. Detailed implementation manners
[0037] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.
[0038] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.
[0039] Example 1 Synthesis of polypeptide 1. Synthesis of polypeptide First, the present invention uses trypsin and chymotrypsin (the polypeptide sequences obtained by simultaneous enzymatic hydrolysis of the two enzymes are relatively numerous, and most of them have only two or three amino acids. The activity of the particularly short polypeptides is relatively limited. Therefore, the present invention first uses trypsin hydrolysis, and polypeptides with more than 15 amino acids are then hydrolyzed with chymotrypsin) to perform peptide chain enzymatic hydrolysis on Ganoderma immunomodulatory proteins LZ-8, LZ-9, FIP-gmi, etc., to obtain polypeptide chains with no more than 15 amino acids, and obtain the polypeptide AYTYR (SEQ ID No.1) with good immunomodulatory characteristics among them.
[0040] Synthesis method of polypeptide: (1) Accurately weigh 0.2 mM Rink resin, add 1.5 mL DMF (N,N-dimethylformamide) and 1.5 mL DCM (dichloromethane) to swell the resin for 30 min. Pour the swollen resin into the synthesizer, pump out the swelling solution, add 2 mL of deprotection solution (piperidine:DMF = 1:4), and shake on a shaker for 10 min.
[0041] (2) Wash with DMF three times, wash with DCM three times, and then wash with DMF three times. The washing time for each time is 2 min. After successful ninhydrin test, amino acids can be added for condensation reaction. After 30 minutes, wash and perform ninhydrin test. If all the amino acids are added to the resin, the resin should be colorless at this time. Otherwise, addition needs to be performed again.
[0042] (3) After all the amino acids are added according to the above method, cut the peptide chain from the resin with trifluoroacetic acid solution, add ice ether to precipitate the polypeptide, and purify the polypeptide by HPLC after drying.
[0043] Mass spectrometry identification shows that the synthesized polypeptide sequence has an amino acid sequence of AYTYR.
[0044] 2. Synthesis of homologous peptide segments Fungal traditional Chinese medicines such as Flammulina velutipes contain fungal immunomodulatory proteins (FIPs), and there is more than 70% homology among them. In order to explore the peptide segments with immunological activity in other types of immunomodulatory proteins, the present invention compares the homologous peptide segments of AYTYR in FIPs from different species sources, and the comparison results are shown in Table 1 below.
[0045] Synthesize homologous polypeptides according to the above experimental method and identify them by mass spectrometry.
[0046]
[0047] Example 2 Detection of immunological activity of polypeptides 1. Peripheral blood mononuclear cell co-culture experiment Separate human peripheral blood mononuclear cells using lymphocyte separation medium and place them in PBS (pH 7.2) for later use. After centrifuging the cells (2000 rpm, 4 °C, 10 min), resuspend them in RPMI 1640 medium (containing 10% FBS) and adjust the cell concentration to 4×10 5 Cells per milliliter, take 500 μL and spread it on a 48-well plate.
[0048] Set up an unstimulated group and a stimulated group. The stimulated group is added with anti-human CD3 with a final concentration of 1 μg / mL and anti-human CD28 functional antibody with a concentration of 0.5 μg / mL, and the unstimulated group is added with the corresponding volume of PBS (pH 7.2). On the basis of the above stimulation wells, add polypeptides with a concentration of 20 μM, mix well and place them in a cell culture incubator for a total culture time of 72 h. Detect the immunomodulatory activity of the peptide segments.
[0049] Collect the cells after centrifugation and detect the IFN-γ secretion of CD8 + T cells by intracellular cytokine staining (flow cytometer).
[0050] The experimental results are as Figure 1 shown. Among them, AYTYR (SEQ ID No.1), EYTYR (SEQ ID No.2), AYQYR (SEQ ID No.3), KYSYR (SEQ ID No.4), PYKYR (SEQ ID No.5), RYKYR (SEQ ID No.6) or QYKYR (SEQ ID No.7) all have good immunomodulatory properties. Since KYSYR is derived from Flammulina velutipes, an edible fungus with a large production and low price in China, the present invention focuses on researching and modifying the polypeptide KYSYR and provides a medicine and food homologous drug with relatively high safety and expected to reduce the side effects of tumor immunotherapy.
[0051] Example 3 Synthesis and immunological activity detection of polypeptides rk5 and ps-rk Since KR5 is a natural amino acid, it cannot resist in vivo enzymatic degradation after administration. The D-configured amino acid is a non-natural amino acid and can well resist enzymatic degradation. Therefore, the reverse sequence inversion strategy is adopted to change the natural amino acid of KR5 into the D-configured amino acid, so as to enhance its anti-enzymatic degradation ability.
[0052] Since polypeptides of five amino acids are easily excreted by the kidneys and have a short half-life, albumin affinity peptide ps is used to modify rk5, which may prolong its half-life.
[0053] As the above polypeptide synthesis method, polypeptides KR5, rk5 and ps-rk are prepared. Among them, rk5 is obtained by modification using the reverse sequence inversion strategy, and its amino acid sequence is rysyk (SEQ ID No.8); ps-rk is obtained by modification using the albumin affinity peptide modification strategy on the basis of rk5, and its amino acid sequence is prdsfmsrysyk (SEQ ID No.9). The immunological activity detection method is the same as above.
[0054] The results are as Figure 2 shown. From the results, the immunological activation effects of polypeptides rk5 and ps-rk are unchanged compared with KR5, indicating that the modification does not affect the activity of KR5, and the immunological activities of KR5, rk5 and ps-rk are all superior to the immunomodulatory protein FIP-fve of Flammulina velutipes.
[0055] Example 4 Distribution of polypeptides in mice Polypeptide rk5 is labeled with FITC and intraperitoneally injected into mice (wild-type C57BL / 6 mice, purchased from Zhuhai Besttone Biotechnology Co., Ltd.). The injection dose is 100 μg / mouse. After 12 h of injecting the polypeptide, the heart, liver, spleen, lung, kidney, brain, lymph nodes and tumor tissues of the mice are taken, and a small animal imager is used to image each organ of the mice to observe the distribution of the polypeptide in each organ of the mice.
[0056] The results are as Figure 3 shown. Polypeptide rk5 can target the tumor site in vivo, and the therapeutic effect at the tumor site is significantly prolonged.
[0057] Example 5 Study on the in vivo anti-tumor activity of orally administered polypeptides BALB / c mice with subcutaneous tumors on the right back are used. Two weeks after tumor inoculation, the mice are randomly grouped, and each group of mice is continuously administered for two weeks (rk5: 2 mg / kg, qd , i.p. ; PD-L1 antibody: 12.5 mg / kg, q3d , i.p. ), and the tumor volume and body weight changes of the tumor-bearing mice are recorded.
[0058] The anti-tumor results of orally administered polypeptides are as Figure 4As shown, after administration of the polypeptide rk5, it can significantly inhibit the growth of CT26 tumors, and the therapeutic effect is comparable to that of the PD-L1 antibody.
[0059] In summary, the present invention discovers that the peptide segment KR5 enzymatically hydrolyzed from the Flammulina velutipes immunomodulatory protein FIP-fve and its partially homologous peptide segments from different fungal sources all have good immunomodulatory properties. After reverse sequence flipping and albumin affinity peptide modification of KR5, the obtained D-configured polypeptide rk5 and ps-rk have the same immunostimulatory effect (unchanged activity) as KR5, and both are superior to the Flammulina velutipes immunomodulatory protein FIP-fve. In the mouse CT26 colorectal cancer model, rk5 can significantly inhibit tumor growth, and the therapeutic effect is comparable to that of the PD-L1 antibody. The FIP active polypeptide provided by the present invention provides a theoretical basis for exploring the pharmacodynamic substances and action mechanisms of FIP, provides candidate drugs for tumor immunodiagnosis and treatment, and has good application prospects and high economic value.
Claims
1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is B-Y-J-Y-R, wherein B and J are independently selected from any amino acid.
2. A polypeptide, characterized in that, The polypeptide is the polypeptide obtained by reversing and inverting the polypeptide described in claim 1.
3. A fusion polypeptide, characterized in that, The fusion polypeptide is obtained by modifying the polypeptide described in any one of claims 1 or 2 at the N-terminus or C-terminus.
4. The polypeptide according to claim 1, wherein B is selected from any one of E, P, Q, and K; J is selected from any one of T, K, and S.
5. The polypeptide according to claim 1, wherein The configuration of each amino acid in the polypeptide is independently selected from the D configuration or the L configuration.
6. A polypeptide probe, characterized in that, The general structural formula of the polypeptide probe is M 标 -L 连 -R 多肽 ; Among them, M 标 is a light label or a radionuclide complexing group, L 连 is a linking group, R 多肽 is a monomer, dimer or multimer of the polypeptide or fusion polypeptide according to any one of claims 1 to 5.
7. Use of the polypeptide probe described in claim 6 in the preparation of a reagent for tumor diagnosis or tracer imaging.
8. Use of the polypeptide or fusion polypeptide described in any one of claims 1 to 5 or the polypeptide probe described in claim 6 in the preparation of a drug for treating tumors or immune diseases.
9. A drug, characterized in that, The drug comprises the polypeptide or fusion polypeptide described in any one of claims 1 to 5.
10. The drug according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.