Cascade response self-assembly polypeptide for remodeling tumor cell antigen composition, bioactive solution and application thereof

Abstract

The invention provides a cascade response self-assembly polypeptide for remodeling tumor cell antigen composition, a bioactive solution of the cascade response self-assembly polypeptide and application of the cascade response self-assembly polypeptide. The polypeptide sequentially comprises a hydrophobic end-capping group, an alkaline phosphatase response self-assembly polypeptide sequence, a reduced glutathione response sequence and a T cell epitope peptide sequence. The polypeptide can respond to high-expression alkaline phosphatase in a tumor microenvironment to generate self-assembly and promote efficient internalization of cells; then, the antigen peptide is released under the action of reductive glutathione in tumor cells, and the antigen complex is given to the tumor cells through a main histocompatibility complex I-type molecular antigen presentation pathway. In addition, the specific hydrophobic end-capping group can up-regulate expression of I-type molecules of main histocompatibility complexes of tumor cells, enhance antigen presentation and remarkably enhance the recognition and killing efficiency of antigen-specific T cells on the tumor cells. Combined adoptive immunity and immune checkpoint inhibitor therapy is suitable for combined immunotherapy of solid tumors.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to a cascade-response self-assembling polypeptide for reshaping the composition of tumor cell antigens, its bioactive solution, and its applications. Background Technology

[0002] The specific interaction between the polypeptide-major histocompatibility complex class I molecules (antigen complexes, i.e., pMHC-I) on the tumor cell membrane and their homologous T cell receptors (TCRs) is the core basis for activating adaptive immunity and achieving tumor clearance. However, the immune escape problem caused by the loss of expression of tumor cell surface antigens and antigen expression heterogeneity significantly affects the recognition and clearance of tumors by T lymphocytes.

[0003] Currently, there are two main strategies to address these challenges: (1) using chemotherapy to upregulate the expression of major histocompatibility complex class I (MHC-I) molecules in tumors, thereby enhancing endogenous antigen presentation, and combining this with immune checkpoint blockade therapy (ICB); and (2) precisely delivering exogenous synthetic antigens to the tumor site, conferring pMHC-I to tumor cells on demand to guide T cells in immune attack. However, the effectiveness of antigen presentation is highly dependent on the tumor cells' own MHC-I expression level, and antigen-specific T cells have low efficiency in effectively recognizing pMHC-I.

[0004] To address the aforementioned issues, this study proposes a cascade-response peptide self-assembly system. This system not only upregulates MHC-I molecule expression in tumor cells and enhances antigen presentation levels, but also expands the types of antigens presented based on this technology. By utilizing an endogenous antigen presentation pathway, tumor cells efficiently display pre-designed T-cell antigens, ensuring that the antigens are effectively recognized by T cells and enhancing their ability to eliminate tumor cells.

[0005] Invention Patent Content In view of this, the purpose of the present invention is to provide a cascade-response self-assembling polypeptide for reshaping the composition of tumor cell antigens, its bioactive solution and its application, so as to solve or alleviate the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a cascade-response self-assembly polypeptide for reshaping the composition of tumor cell antigens, comprising a hydrophobic end-capping group, an alkaline phosphatase-responsive self-assembly polypeptide sequence, a reduced glutathione-responsive sequence, and a T-cell antigen epitope peptide sequence connected in sequence; wherein the hydrophobic end-capping groups are Nap and CPT, the amino acid sequence of the alkaline phosphatase-responsive self-assembly polypeptide sequence is GFFpYE, the reduced glutathione-responsive sequence is a disulfide bond, and the amino acid sequence of the T-cell antigen epitope peptide sequence is SIINFEKL.

[0007] Preferably, the hydrophobic end-capping groups are 2-naphthaleneacetic acid and camptothecin, respectively.

[0008] Preferably, the structural formula of the cascade-responsive self-assembling peptide based on the hydrophobic end-capping group of 2-naphthaleneacetic acid is:

[0009] Preferably, the structural formula of the camptothecin-based cascade-response self-assembling peptide based on hydrophobic end-capping groups is:

[0010] A bioactive solution for a cascade-response self-assembling polypeptide for remodeling tumor cell antigen composition, comprising the aforementioned cascade-response self-assembling polypeptide for remodeling tumor cell antigen composition, using 1× phosphate buffer as a solvent.

[0011] Preferably, the pH of the 1× phosphate buffer solution is 7.0~7.4.

[0012] Preferably, the concentration of the cascade response self-assembling polypeptide composed of the remodeled tumor cell antigen in 1× phosphate buffer is as follows: when the hydrophobic end-capping group is 2-naphthaleneacetic acid, the concentration is 2.0 mg / (0.9~1.1 mL) 1× phosphate buffer; when the hydrophobic end-capping group is camptothecin, the concentration is 2.4 mg / (0.9~1.1 mL) 1× phosphate buffer.

[0013] This invention provides the application of the above-described cascade-response self-assembling peptides that reshape the composition of tumor cell antigens or the above-described bioactive solutions in the preparation of peptides that reshape the composition of tumor cell antigens.

[0014] Preferably, the polypeptide is used to enhance antigen presentation levels and improve antigen-specific T cells to efficiently kill mouse colon cancer cells.

[0015] This invention also provides the application of the above-mentioned cascade response self-assembling peptides that reshape the composition of tumor cell antigens or the above-mentioned bioactive solutions in enhancing the expression of MHC-I molecules in tumor cells and enhancing antigen-specific T cells to efficiently kill tumors.

[0016] Compared with existing technologies, the present invention has the following beneficial effects: The cascade-response self-assembling peptides that reshape the composition of tumor cell antigens provided by the present invention can respond to the high expression of alkaline phosphatase (ALP) in tumors to self-assemble into nanofibers, enhancing cell internalization; subsequently, they respond to the release of antigen peptides by reduced glutathione (GSH) in tumor cells, effectively utilizing the intracellular MHC-I-related antigen presentation pathway to endow tumor cells with pMHC-I. Furthermore, the peptide CPSA constructed using CPT as a hydrophobic end-cap can upregulate the expression of MHC-I in tumor cells, enhancing antigen presentation levels and significantly improving antigen specificity CD8.+ T lymphocytes (extracted from OT-1 mice) recognize and kill tumor cells, and the peptides have good biocompatibility.

[0017] The above overview is for illustrative purposes only and is not intended to be limiting in any way. Further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description, in addition to the illustrative aspects, embodiments, and features described above. Attached Figure Description

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

[0019] Figure 1 The chemical structure and high-resolution mass spectrum of the self-assembled polypeptide PSA composed of reconstructed tumor cell antigens synthesized in Example 1 of this invention are shown below. Figure 2 The chemical structure and high-resolution mass spectrum of the self-assembled polypeptide CPSA, which is composed of reconstructed tumor cell antigens, synthesized in Example 2 of this invention; Figure 3 The chemical structure and high-resolution mass spectrum of the polypeptide Ag synthesized in Comparative Example 1 are shown. Figure 4 The chemical structure and high-resolution mass spectrum of the polypeptide YSA synthesized in Comparative Example 1 of this invention are shown below. Figure 5 The chemical structure and high-resolution mass spectrum of the polypeptide YGA synthesized in Comparative Example 1 of this invention are shown below. Figure 6 The left image shows the flow cytometry diagram of pMHC-I conferred by PSA to MC38 colon cancer cells in Experiment Example 1 of this invention, and the right image shows the decrease of pMHC-I on the cell membrane over time. Figure 7 This is a comparison chart of the results of the PSA group and the comparative groups in the LDH release level test in Experimental Example 2 of the present invention; Figure 8 This is a transmission electron microscope image of the CPSA solution of the polypeptide synthesized in Example 2, taken using a transmission electron microscope in Experimental Example 3 of the present invention. Figure 9 The left image shows the flow cytometry plot (left image) and the flow cytometry fluorescence intensity statistics plot (right image) of CPSA enhancing MHC-I expression in MC38 colon cancer cells in Experiment Example 4 of this invention. Figure 10The left image shows the flow cytometry plot (left image) and the flow cytometry fluorescence intensity statistics plot (right image) of CPSA-conferred pMHC-I in MC38 colon cancer cells in Experiment Example 5 of this invention. Figure 11 This is a comparison chart of the results of the CPSA group and the control group in the LDH release level test in Experiment Example 6 of this invention; Figure 12 This is a graph showing the tumor volume growth curves of mice in each treatment group in Experiment Example 7 of this invention. Figure 13 This is a statistical chart of tumor inhibition rates in each treatment group at the treatment endpoint in Experiment Example 7 of this invention; Figure 14 This is a graph showing the weight monitoring records of mice in each treatment group during the treatment period in Experiment Example 7 of this invention. Detailed Implementation

[0020] The invention will be more readily understood by referring to the following detailed description of preferred embodiments and included examples. Unless otherwise specified, 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 invention pertains. In case of conflict, the definitions in this specification shall prevail.

[0021] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0022] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0023] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0024] The singular form includes the plural objects of discussion unless the context clearly indicates otherwise. "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event occurs and the possibility that the event does not occur.

[0025] Approximate terms used in the specification and claims to modify quantities indicate that the invention is not limited to that specific quantity, but also includes acceptable modifications close to that quantity that do not alter the relevant essential function. Correspondingly, the use of "about," "approximately," etc., to modify a numerical value means that the invention is not limited to that precise value. In some instances, approximate terms may correspond to the precision of the instrument used to measure the value. In this application's specification and claims, scope definitions can be combined and / or interchanged, unless otherwise stated, these scopes include all subscopes contained therein.

[0026] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of this invention do not impose any limitation on the quantity (i.e., number of times) of the elements or components. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers to the singular form.

[0027] Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials and test strains used in the following examples were purchased from commercial channels.

[0029] This invention provides a cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition, characterized in that it comprises, in sequence, a hydrophobic end-capping group, an alkaline phosphatase-responsive self-assembling polypeptide sequence, a reduced glutathione-responsive sequence, and a T-cell antigen epitope peptide sequence; wherein the hydrophobic end-capping groups are Nap and CPT, the amino acid sequence of the alkaline phosphatase-responsive self-assembling polypeptide sequence is GFFpYE, the reduced glutathione-responsive sequence is a disulfide bond, and the amino acid sequence of the T-cell antigen epitope peptide sequence is SIINFEKL.

[0030] In this invention, the hydrophobic end-capping groups are 2-naphthaleneacetic acid and camptothecin, respectively. The cascade-response self-assembling polypeptides that reshape tumor cell antigens are all L-configured amino acids; Furthermore, the structural formula of the cascade-responsive self-assembling peptide based on the hydrophobic end-capping group of 2-naphthaleneacetic acid is as follows:

[0031] Furthermore, the structural formula of the camptothecin-based cascade-response self-assembling peptide based on the hydrophobic end-capping group is as follows:

[0032] The present invention does not have any particular limitation on the preparation method of the cascade response self-assembly polypeptide that reshapes the composition of tumor cell antigens. Conventional preparation methods in the art can be used. In the specific implementation of the present invention, the Fmoc-short peptide solid-phase synthesis method is preferred.

[0033] The present invention provides a bioactive solution for a cascade-response self-assembly polypeptide for reshaping the composition of tumor cell antigens, comprising the above-mentioned cascade-response self-assembly polypeptide for reshaping the composition of tumor cell antigens, using 1× phosphate buffer as a solvent.

[0034] Specifically, the concentration of the cascade-response self-assembling polypeptide that remodels the tumor cell antigen composition in 1× phosphate buffer is: When the hydrophobic end-capping group is 2-naphthaleneacetic acid, the concentration is 2.0 mg / (0.9~1.1 mL) 1× phosphate buffer; When the hydrophobic end-capping group is camptothecin, the concentration is 2.4 mg / (0.9~1.1 mL) 1× phosphate buffer.

[0035] The pH of the 1× phosphate buffer solution is 7.0~7.4.

[0036] In this invention, the bioactive solution is preferably obtained by mixing the cascade-response self-assembling polypeptide composed of the remodeled tumor cell antigen with the 1× phosphate buffer.

[0037] This invention provides the application of the above-described cascade-response self-assembling peptides that reshape the composition of tumor cell antigens or the above-described bioactive solutions in the preparation of peptides that reshape the composition of tumor cell antigens.

[0038] Specifically, the polypeptide is used to enhance antigen presentation levels and improve antigen-specific T cells to efficiently kill mouse colon cancer cells.

[0039] This invention also provides the application of the above-mentioned cascade response self-assembling peptides that reshape the composition of tumor cell antigens or the above-mentioned bioactive solutions in enhancing the expression of MHC-I molecules in tumor cells and enhancing antigen-specific T cells to efficiently kill tumors.

[0040] In this invention, the tumor cells are preferably mouse colon cancer cells, and in the specific implementation of this invention, the mouse colon cancer tumor cells are preferably MC38 cells.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] The sources of the formulations involved in the embodiments of the present invention are as follows: 2-Cl-Trt resin was purchased from Jier Biochemical (Shanghai) Co., Ltd., with a degree of substitution of 1.158 mmol / g; Fmoc amino acids (including phosphorylated tyrosine) were all purchased from Jier Biochemical (Shanghai) Co., Ltd., with a purity of over 98%. N,N-Diisopropylethylamine (DIPEA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of over 99%. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) was purchased from Tianjin Xiens Biochemical Technology Co., Ltd., with a purity of 98%. Trifluoroacetic acid (TFA) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of 99%. Triisopropylsilane (TIS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of 99%; 2-naphthaleneacetic acid (Nap) was purchased from Tianjin Xinsheng Biochemical Technology Co., Ltd., with a purity of 98%. Camptothecin (CPT) was purchased from Tianjin Xiens Biochemical Technology Co., Ltd., with a purity of 98%. Anhydrous dichloromethane (DCM) was purchased from Tianjin Chemical Reagent Company; N,N-dimethylformamide (DMF) was purchased from Tianjin Chemical Reagent Company; Chromatographically pure methanol was purchased from Tianjin Concord Technology Co., Ltd. Piperidine was purchased from Tianjin Chemical Reagent Company; The cell culture medium (Dulbecco's Modified Eagle Medium, hereinafter referred to as DMEM) was purchased from ThermoFisher Scientific and was sterile. Fetal bovine serum was purchased from Thermo Fisher Scientific and was sterile. Flow cytometry antibodies were purchased from PE H-2Kb bound to SIINFEKL and Alexa Fluor® 647 anti-mouse H-2Kb antibody from Biolegend (USA). aPD-L1 antibody was purchased from Bio X Cell (USA). C57BL / 6J mice, 6-8 weeks old, female, purchased from Vital River Biotechnology Co., Ltd. CD8 + T cells were extracted from OT-1 transgenic mice and purchased from The Jackson Laboratory (USA).

[0043] The device involved in this embodiment of the invention is: The high-performance liquid chromatograph (HPLC) was manufactured by Lumtech, Germany. The high-performance liquid chromatography-mass spectrometry system was a Shimadzu LC-MS 2020 from Japan. The electronic balance is from Arturious, Germany, model BS124S; The transmission electron microscope (TEM) was a Tecnai G2 F20 system; The freeze dryer is from Beijing Yatai Kelong, model LGJ-1-50; The laser confocal microscope is a Leica TCS SP8 from Germany. Flow cytometer, BD (USA), model LSR Fortessa; High-resolution mass spectrometry, Agilent Technologies, USA, model Agilent 6520 Q-TOF LC / MS; Example 1 (Synthesis of self-assembled peptide Nap-GFFpY-ss-SIINKEKL): The Nap-terminated peptide, abbreviated as PSA, was synthesized using the standard Fmoc-amino acid solid-phase synthesis method. The specific steps are as follows: S1. Weigh 0.5 mmol of 2-Cl-Trt resin into a solid-phase synthesis tube, add 15 mL of anhydrous dichloromethane (DCM), and place it on a shaker and shake for 10 minutes to allow the 2-Cl-Trt resin to fully swell. S2. Use a syringe bulb to completely extrude anhydrous dichloromethane (DCM) from a solid-phase synthesizer containing 2-Cl-Trt resin; S3. Dissolve 1 mmol of Fmoc-protected leucine (Fmoc-Leu-OH) in 10 mL of anhydrous dichloromethane (DCM), add 2 mmol of N,N-diisopropylethylamine (DIPEA), and after complete dissolution, add it to a solid-phase synthesis tube and place it on a shaker to react at room temperature for 1 hour. S4. Remove the reaction solution from the solid-phase synthesis tube with a rubber bulb, then wash with 10 mL of anhydrous dichloromethane (DCM) for 1 minute each time, for a total of 3 washes. Add 16 mL of a prepared solution with a volume ratio of anhydrous dichloromethane (DCM): N,N-diisopropylethylamine (DIPEA): methanol of 17:1:2, and react at room temperature for 15 minutes. S5. Remove the reaction solution from the solid-phase synthesis tube using a rubber bulb. First, wash the resin with anhydrous dichloromethane (DCM), using 10 mL of DCM each time for 1 minute, for a total of 3 washes. Then, wash the resin with N,N-dimethylformamide (DMF), using 10 mL of DMF each time for 1 minute, for a total of 3 washes. Next, add 15 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine, react at room temperature for 30 minutes, and then wash the resin with N,N-dimethylformamide (DMF), using 10 mL of DMF each time for 1 minute, for a total of 5 washes. S6. Weigh 1 mmol of the second Fmoc-protected amino acid K (Fmoc-Lys (Boc)-OH), 1 mmol of HATU, and 2 mmol of DIPEA. Dissolve them in 10 mL of N,N-dimethylformamide (DMF). Add the dissolved amino acid solution to a solid-phase synthesis tube and place it on a shaker to react at room temperature for 1 hour. S7. Repeat steps S5 and S6, adding Fmoc-amino acid, Fmoc-ss, and the capping group Nap sequentially; the synthesis steps of Fmoc-ss are as described in Zhan, J. et al. Tandem molecular self‐assembly in liver cancer cells. Angew. Chem. Int. Ed. 57, 1813–1816 (2018). Then wash five times with N,N-dimethylformamide (DMF) before proceeding to the next step. S8. Wash with N,N-dimethylformamide (DMF), using 10 mL of N,N-dimethylformamide (DMF) each time for 1 minute, for a total of 5 washes. After washing 5 times with N,N-dimethylformamide (DMF), proceed to the next reaction step. S9. Prepare 10 mL of cleavage buffer with a ratio of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% H2O (volume fraction). Add the buffer to the solid-phase synthesizer described above and react at room temperature for 1 hour. Cut the product off the 2-Cl-Trt resin, remove the solvent using a rotary evaporator, and precipitate with diethyl ether to obtain the crude self-assembled peptide. Subsequently, separate and purify the peptide using high-performance liquid chromatography (HPLC) to obtain the self-assembled peptide PSA with reconstituted tumor cell antigen composition of Example 1. Detect the self-assembled peptide PSA with reconstituted tumor cell antigen composition obtained in Example 1 by LC-MS. The experimental results are shown in [Figure 1]. Figure 1 .

[0044] Example 2 (Synthesis of CPT-GFFpY-ss-SIINKEKL): CPT-terminated self-assembled peptides were prepared according to the Fmoc-short peptide solid-phase synthesis method in Example 1. The Nap terminator in step S7 was replaced with CPT. The specific synthetic steps for attaching the CPT terminator to the N-terminus of the peptide are described in Zhang, Z. et al. Dual-enzyme-instructed peptide self-assembly to boost immunogenic celldeath by coordinating intracellular calcium overload and chemotherapy. ACSNano. 19, 488–503 (2025). The resulting CPT-terminated peptide is abbreviated as CPSA. Experimental results are shown in […]. Figure 2 .

[0045] Comparative Example 1 (Synthesis of polypeptides Ag, YSA, and YGA): The polypeptide of Comparative Example 1 was prepared according to the Fmoc-short peptide solid-phase synthesis method of Example 1. The amino acid sequences of the polypeptides were SIINFEKL, Nap-GFFYE-ss-SIINFEKL, and Nap-GFFYE-GSG-SIINFEKL, respectively. The abbreviations of the obtained polypeptides were Ag, YSA, and YGA, respectively. The polypeptides obtained in Comparative Example 1 were detected by high performance liquid chromatography-mass spectrometry. The chemical structure and molecular weight of Ag are shown in the figure. Figure 3 The chemical structure and molecular weight results of YSA are shown in [the table below]. Figure 4 The chemical structure and molecular weight of YGA are shown in [the table below]. Figure 5 .

[0046] Experimental Example 1 (PSA Antigen Presentation Level Assay at the Cellular Level) 1.1 Experimental Grouping: There are a total of 5 groups: PSA, YSA, YGA, Ag, and Blank.

[0047] 1.2 Cell Culture and Treatment: Cells are arranged at 1×10 5 Colon cancer cells (MC38) were seeded in 12-well plates at a specific density. After overnight cell adhesion, 1 mL of PSA (50 μM), a polypeptide composed of remodeled tumor cell antigens from Example 1, and 1 mL of YSA (50 μM), YGA (50 μM), and Ag (50 μM), a polypeptide composed of remodeled tumor cell antigens from Comparative Example 1, were added. Cells were incubated for 24 hours. MC38 cells without polypeptide treatment served as the Blank group. After 24 hours of incubation, the Ag group and PSA group were washed twice with PBS, replaced with fresh DMEM medium, and incubated for 12, 24, and 48 hours as reserve samples for antigen display stability testing.

[0048] 1.3 Preparation of polypeptide solutions: Weigh 2.1 mg of PSA, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the peptide, thus obtaining a 1 mM peptide stock solution PSA. Then dilute it to 50 μM with cell culture medium.

[0049] 1.4 Flow cytometry detection: After incubation, the cells were washed twice with PBS, and then stained with the PE H-2K antibody by flow cytometry. b The bound to SIINFEKL was stained on ice for 30 minutes, washed three times with PBS, and finally the proportion of antigen-positive tumor cells was determined by flow cytometry.

[0050] 1.5 Experimental Results: like Figure 6 As shown in the left figure, MC38 tumor cells, after being incubated with PSA, YSA, YGA, and Ag for 24 h, respectively, generated H-2K. b Significant differences were observed in the antigen complex. Because the Ag group peptides directly loaded the peptide antigen Ag onto the unloaded MHC-I molecule on the tumor cell membrane, the highest levels of H-2K were generated. bAntigen complexes. Among the three groups of self-assembling peptides PSA, YSA, and YGA, PSA, which exhibits ALP-responsive self-assembly and GSH-responsive antigen release, can present antigens to tumor cells more efficiently. This cascade response strategy can effectively reshape the antigenic composition of tumor cells. Figure 6 The right figure shows that after washing with PBS and replacing the culture medium with fresh medium, the antigen expression of tumor cells in the Ag group and PSA group was tested at 12h, 24h, and 48h. Although the Ag group was able to generate the highest level of H-2K, b Antigen complexes, but 12 hours after changing to fresh culture medium, H-2K on the MC38 cell membrane... b The level of antigen complexes decreased rapidly, and H-2K generated between the Ag group and the PSA group... b The levels of antigen complexes were similar.

[0051] Experimental Example 2 (LDH Release Level Test of PSA): 1.1 Experimental Grouping: There were a total of 6 groups: PSA+T cells, YSA+T cells, YGA+T cells, PBS+T cells, Ag+T cells, and Blank cells.

[0052] 1.2 Cell Culture and Treatment: According to 7×10 3 Mouse colon cancer MC38 cells were seeded in 96-well plates at a specific density. After the cells adhered overnight, the medium was replaced with 0.1 mL of medium containing 50 μM peptides PSA, YSA, YGA, and Ag, and the cells were incubated for another 12 hours.

[0053] 1.3 Preparation of polypeptide solutions: Weigh 2.1 mg of PSA, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the peptide, thus obtaining a 1 mM peptide stock solution PSA. Then dilute it to 50 μM with cell culture medium.

[0054] 1.4 T cell co-culture and LDH detection: After incubation, wash the cells twice with PBS, then add 7 × 10⁶ cells to each well. 4 An activated CD8 + T lymphocytes (derived from OT-1 mice) were co-cultured for 36 hours. The Blank group consisted only of MC38 cells, lacking CD8. + T lymphocytes. 96-well plates were centrifuged at 1200 rpm for 5 minutes. The cell supernatant was collected, and the LDH release level in each group was measured using an LDH kit.

[0055] 1.5 Test Results: like Figure 7 As shown, the PSA and Ag groups had the highest LDH release levels, which could mediate OT-1 CD8. + T cells effectively killed MC38 cells.

[0056] 1.6 Experimental Conclusions: The results indicate that the cascade-response self-assembling peptide PSA provided by this invention can effectively promote the killing effect of antigen-specific T cells on tumor cells.

[0057] Experiment Example 3 (Transmission Electron Microscopy Experiment of CPSA): 1.1 Sample preparation: Weigh 2.4 mg of the self-assembled polypeptide CPSA prepared in Example 2, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the polypeptide and obtain 1 mM polypeptide stock solution CPSA.

[0058] 1.2 Sample processing and grouping: Take 10 μL of the polypeptide stock solution and add it to a carbon-supported copper grid. Hold for 180 seconds, blot dry with filter paper, then add 10 μL of 5% phosphotungstic acid for negative staining for 120 seconds. Blot dry with filter paper and allow to air dry at room temperature. This is the CPSA group.

[0059] Add ALP solution to the polypeptide stock solution at a final concentration of 1 U / mL, and incubate in a 37℃ water bath for 6 hours to obtain CPSA+ALP; add GSH solution to the sample at a final concentration of 5 mM, and incubate in a 37℃ water bath for 24 hours to obtain CPSA+ALP+GSH; both groups of samples were negatively stained using the same method as above.

[0060] 1.3 Morphological observation: The microstructure of each group of samples was observed using a JEM100CXII transmission electron microscope (120 kV, magnification ×28000).

[0061] 1.4 Experimental Results: The results are as follows Figure 8 As shown, the polypeptide mother liquor CPSA prepared in Example 2 is a sparse short nanofiber; after self-assembly in response to ALP, CPSA+ALP becomes a dense cross-linked nanofiber network; after GSH is added to release the antigen, CPSA+ALP+GSH becomes a discrete long nanofiber.

[0062] 1.5 Experimental Conclusions: The above results indicate that the CPSA mother liquor, which remodels the composition of tumor cell antigens, has pre-assembly properties under physiological conditions. It self-assembles into nanofibers in response to ALP catalysis, and the morphology of the fibers is further altered after GSH reduction.

[0063] Experiment Example 4 (CPSA-regulated MHC-I expression in tumor cells): 1.1 Experimental Grouping: There are a total of 5 groups: CPSA, CPT, PSA, Ag, and Control.

[0064] 1.2 Cell Culture and Treatment: According to 2×10 5 Mouse colon cancer MC38 cells were seeded in 6-well plates at a certain density. After the cells adhered overnight, they were incubated for 12 hours in 1 mL of DMEM medium containing CPSA (50 μM), CPT (50 μM), PSA (50 μM), and Ag (50 μM). Untreated MC38 cells were used as the control group.

[0065] 1.3 Preparation of polypeptide solutions: Weigh 2.4 mg of the polypeptide CPSA prepared in Example 2, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the polypeptide and obtain 1 mM polypeptide stock solution CPSA. Then dilute it to 50 μM with DMEM cell culture medium.

[0066] 1.4 Flow cytometry detection: After incubation, the cells were washed twice with PBS, and then stained with the Alexa Fluor® 647 anti-mouse H-2K antibody by flow cytometry. b The antibody was stained on ice for 30 minutes, washed three times with PBS, and then H-2K on the cell membrane was measured by flow cytometry. b Level of expression.

[0067] 1.5 Test Results: like Figure 9 As shown, compared with PSA, CPSA significantly increases H-2K on the cell membrane. b Expression levels of H-2K on the MC38 cell membrane after CPSA treatment b The level of expression improved by approximately 1.81 times.

[0068] 1.6 Experimental Conclusions: These results indicate that the cascade-response self-assembling peptide CPSA, with CPT as the hydrophobic terminator, can effectively upregulate the expression level of MHC-I molecules in tumor cells.

[0069] Experimental Example 5 (Antigen Presentation Level Assay of CPSA at the Cellular Level): 1.1 Experimental Grouping: There are a total of 5 groups: CPSA, CPT, PSA, Ag, and Control.

[0070] 1.2 Cell Culture and Treatment: According to 1×10 5 The colon cancer cells MC38 were seeded in 6-well plates at a certain density. After the cells adhered overnight, 1 mL of DMEM medium containing CPSA (50 μM), CPT (50 μM), PSA (50 μM), and Ag (50 μM) was replaced. Untreated cells were used as Controls. Each group of cells was incubated in the corresponding medium for 12 hours.

[0071] 1.3 Preparation of polypeptide solutions: Weigh 2.4 mg of the polypeptide CPSA prepared in Example 2, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the polypeptide and obtain 1 mM polypeptide stock solution CPSA. Then dilute it to 50 μM with DMEM cell culture medium.

[0072] 1.4 Flow cytometry detection: After incubation, the cells were washed twice with PBS, and then stained with the PE H-2K antibody by flow cytometry. b The bound to SIINFEKL was stained on ice for 30 minutes, washed three times with PBS, and the antigen presentation efficiency of each group was determined by flow cytometry.

[0073] 1.5 Experimental Results: like Figure 10 As shown, CPSA significantly improves H-2K performance compared to PSA. b - The level of antigen complex generation is increased by approximately 14.94 times, which can effectively endow tumor cells with antigen complex targets.

[0074] 1.6 Experimental Conclusions: These results demonstrate that the cascade-responsive self-assembling peptide CPSA, with CPT as the hydrophobic terminator, significantly enhances the generation of tumor cell antigen complexes, showcasing great potential for customizing antigen complex targets for tumor cells.

[0075] Experimental Example 6 (LDH Release Level Test of CPSA): 1.1 Experimental Grouping Five groups were set up: CPSA+T cells, CPT+T cells, PSA+T cells, PBS+T cells, and Blank group.

[0076] 1.2 Cell culture and treatment; According to 7×10 3 The mouse colon cancer cell line MC38 was seeded in 96-well plates at a certain density. After the cells adhered overnight, they were incubated in 0.1 mL of DMEM medium containing 50 μM compounds CPSA, CPT and PSA for 12 hours. 1.3 Preparation of polypeptide solutions: Weigh 2.4 mg of the polypeptide CPSA prepared in Example 2, add 1 mL of 1× phosphate buffer, and add sodium carbonate (1M) solution dropwise (2 μL each time) to adjust the pH to 7.4 to completely dissolve the polypeptide and obtain 1 mM polypeptide stock solution CPSA. Then dilute it to 50 μM with DMEM cell culture medium.

[0077] 1.4 T cell co-culture and LDH detection: After incubation, wash twice with PBS, then add 7×10⁻⁶ of the activated solution to each well. 4 CD8 + T lymphocytes (derived from OT-1 mice) were co-cultured for 36 hours. The Blank group consisted only of MC38 cells, lacking CD8. + T lymphocytes. 96-well plates were centrifuged at 1200 rpm for 5 minutes. The cell supernatant was collected, and the LDH release level in each group was measured using an LDH kit.

[0078] 1.5 Experimental Results: like Figure 11 As shown, the CPSA+T cell group exhibited the best killing effect on MC38 tumor cells, superior to the PSA+T cell group, indicating that CPSA enhances H-2K. b The level of antigen complex generation can significantly enhance the effect of antigen-specific T lymphocytes in killing tumor cells.

[0079] 1.6 Experimental Conclusions: This result indicates that CPSA enhances H-2K b While increasing the level of antigen complex generation, it can significantly enhance the killing effect of antigen-specific T lymphocytes on tumor cells.

[0080] Experiment Example 7 (Animal Tumor Model Experiment): 1.1 Establishment of animal models: Female C57BL / 6J mice aged 6-8 weeks were selected, and 100 μL of a solution containing 2×10⁻⁶ ppm was subcutaneously injected into the right shoulder and back of each mouse.6 A mouse subcutaneous colon cancer tumor model was constructed using DMEM culture medium containing MC38 cells.

[0081] 1.2 Experimental grouping and drug administration: When the mouse tumor grows to approximately 100 mm 3 Mice were randomly divided into 7 groups of 5 mice each. The drug administration groups and dosages were as follows: PSA + aPD-L1 group: PSA (10.85 mg / Kg) was administered via tail vein injection + aPD-L1 (1.00 mg / Kg) was administered via intraperitoneal injection; CPSA + aPD-L1 group: CPSA (11.92 mg / Kg) was administered via tail vein injection + aPD-L1 (1.00 mg / Kg) was administered via intraperitoneal injection; CPSA group: CPSA (11.92 mg / Kg) was administered via tail vein injection; PSA group: PSA (10.85 mg / Kg) was administered via tail vein injection; aPD-L1 group: aPD-L1 (1.00 mg / Kg) was administered intraperitoneally; CPT group: CPT (1.81 mg / Kg) was administered via tail vein injection; PBS group: 100 μL PBS was injected via the tail vein; The peptide drug was administered on days 1, 3, 5, and 7, while the aPD-L1 antibody was administered on days 1, 4, and 7. The administration volume for each group was 100 μL.

[0082] 1.3 Adoptive T-cell reinfusion: 24 hours after the initial administration, 10 million in vitro activated CD8 molecules were injected via tail vein. + T lymphocytes (extracted from OT-1 mice) 1.4 Observation and Measurement Indicators: Tumor volume and body weight changes in mice were recorded every two days. Tumor volume was calculated using the formula: length × width. 2 / 2. The mice were euthanized on day 15, and the tumors were obtained by dissection.

[0083] 1.5 Tumor suppression effect: Tumor volume growth curve and tumor inhibition rate at the treatment endpoint are as follows: Figure 12 and Figure 13 As shown.

[0084] Tumor volume growth curve as shown Figure 12 As shown in the figure, the tumor inhibition rate at the treatment endpoint is... Figure 13As shown in the figure. Tumor inhibition results showed that the tumor inhibition rates of the CPSA+aPD-L1 group, PSA+aPD-L1 group, CPSA group, PSA group, aPD-L1 group, and CPT group were 74.5 ± 13.6%, 60.6 ± 14.6%, 50.3 ± 15.9%, 57.2 ± 14.6%, 48.3 ± 9.5%, and 0.0 ± 32.5%, respectively. 1.6 Results Analysis: Although CPSA can significantly enhance H-2K in tumor cells b - Antigen complex levels, while the tumor suppression efficiencies of the CPSA monotherapy group and the PSA monotherapy group were similar. After combination therapy with immune checkpoint inhibitors, the CPSA + aPD-L1 combination therapy group showed the best tumor suppression effect, with a tumor suppression rate superior to the PSA + aPD-L1 group.

[0085] 1.7 Biocompatibility assessment: The curves of body weight change in mice in each group during treatment are as follows: Figure 14 As shown, all mice showed a steady increase in body weight during the drug treatment period, indicating that the constructed nanomaterials have good biocompatibility.

[0086] The experimental results above demonstrate that the cascade-response self-assembly polypeptide for reconstructing tumor cell antigen composition provided by this invention can respond to the enzymatic self-assembly of tumor cells with high ALP expression, enhancing cell internalization. Subsequently, under the action of GSH within the tumor cells, the antigen peptide is released, effectively utilizing the intracellular MHC-I-related antigen presentation pathway to confer pMHC-I to the tumor cells. Furthermore, CPT, as a hydrophobic terminator, can upregulate MHC-I expression in tumor cells, enhancing antigen presentation levels and significantly improving antigen specificity (CD8). + T lymphocytes (extracted from OT-1 mice) recognize and kill tumor cells. In a mouse colon cancer model, when the peptide CPSA is combined with adoptive T cell infusion and used in conjunction with immune checkpoint inhibitor therapy, it shows a significant trend of enhanced tumor suppression and is suitable for combined immunotherapy of solid tumors with low MHC-I expression.

[0087] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A cascade-response self-assembling polypeptide for remodeling the composition of tumor cell antigens, characterized in that: It comprises, in sequence, a hydrophobic capping group, an alkaline phosphatase-responsive self-assembled polypeptide sequence, a reduced glutathione-responsive sequence, and a T-cell antigen epitope peptide sequence; the hydrophobic capping groups are Nap and CPT, respectively; the amino acid sequence of the alkaline phosphatase-responsive self-assembled polypeptide sequence is GFFpYE; the reduced glutathione-responsive sequence is a disulfide bond; and the amino acid sequence of the T-cell antigen epitope peptide sequence is SIINFEKL.

2. The cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition according to claim 1, characterized in that: The hydrophobic end-capping groups are 2-naphthaleneacetic acid and camptothecin, respectively.

3. The cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition according to claim 2, characterized in that, The structural formula of the self-assembled polypeptide based on the cascade response of hydrophobic end-capping groups to 2-naphthaleneacetic acid is: 。 4. The cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition according to claim 2, characterized in that, The structural formula of camptothecin-based cascade-responsive self-assembling peptides based on hydrophobic end-capping groups is:

5. A bioactive solution for remodeling a cascade-response self-assembling polypeptide of tumor cell antigen composition, characterized in that: The cascade-response self-assembling polypeptide comprising the remodeling of tumor cell antigen composition as described in any one of claims 1 to 4, using 1× phosphate buffer as a solvent.

6. The bioactive solution for a cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition according to claim 5, characterized in that: The pH of the 1× phosphate buffer solution is 7.0~7.

4.

7. The bioactive solution for a cascade-response self-assembling polypeptide for reshaping tumor cell antigen composition according to claim 5, characterized in that: The concentration of the cascade peptide that remodels the tumor cell antigen composition in 1× phosphate buffer is as follows: when the hydrophobic end-capping group is 2-naphthaleneacetic acid, the concentration is 2.0 mg / (0.9~1.1 mL) 1× phosphate buffer; when the hydrophobic end-capping group is camptothecin, the concentration is 2.4 mg / (0.9~1.1 mL) 1× phosphate buffer.

8. The use of the cascade-response self-assembling polypeptide remodeling the composition of tumor cell antigens as described in any one of claims 1 to 3, or the bioactive solution as described in any one of claims 5 to 7, in the preparation of polypeptides remodeling the composition of tumor cell antigens.

9. The application according to claim 8, characterized in that, The peptide is used to enhance antigen presentation levels and improve antigen-specific T cells to efficiently kill mouse colon cancer cells.

10. The cascade-response self-assembling polypeptide that reshapes the composition of tumor cell antigens according to any one of claims 1 to 4, or the bioactive solution according to any one of claims 5 to 7, is used to enhance the expression of MHC-I molecules in tumor cells and enhance antigen-specific T cells to efficiently kill tumors.

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