Preparation and application of Mosaic vaccine targeting multiple tumor glycopeptide antigens

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses preparation and application of a Mosaic vaccine targeting multiple tumor glycopeptide antigens, the vaccine is constructed by coupling different tumor glycopeptide antigens to a protein carrier at the same time in a chemical covalent coupling mode, and the coupling efficiency is systematically characterized through MALDI-TOF and ion chromatography. An immunological evaluation result shows that compared with a single glycopeptide vaccine, the Mosaic vaccine disclosed by the invention can induce a higher level of antibody titer, and the generated antibody can be specifically combined with tumor cells expressing specific tumor antigens. In a transgenic mouse model capable of simulating a human immune tolerance environment, the vaccine can more effectively activate immune response of antigen presenting cells and T cells and remarkably inhibit tumor volume increase, and has a synergistic anti-tumor effect when being combined with an immune checkpoint inhibitor for use. The invention provides an effective vaccine strategy for overcoming tumor antigen heterogeneity, and the developed Mosaic vaccine has a remarkable anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation and application of a Mosaic vaccine targeting multiple tumor glycopeptide antigens. Background Art

[0002] One of the key challenges in the field of tumor immunotherapy is the high heterogeneity of tumor antigens. As an important tumor-associated antigen, MUC1 (human mucin-1) has unique biological properties that make it a promising vaccine target. Under normal physiological conditions, MUC1 is a highly glycosylated transmembrane protein. Its extracellular domain contains a variable number of tandem repeats (VNTRs) consisting of 20 amino acids (HGVSTAPDTRPAPGSTAPPA), in which both serine and threonine residues can undergo O-glycosylation. However, on the surface of tumor cells, MUC1 exhibits three characteristic changes: significantly upregulated expression levels (approximately 100 times that of normal cells), abnormal glycosylation patterns (forming tumor-specific glycoantigens such as Tn, T, STn, and ST), and exposure of the core peptide epitope. These changes not only make MUC1 an ideal tumor marker but also reveal its important role in tumor immune escape.

[0003] Currently, the development of MUC1 glycopeptide vaccines primarily involves the following design strategies: ① Carrier protein conjugation: Covalently conjugating MUC1 glycopeptides to carrier proteins such as KLH effectively enhances antigen presentation and B cell activation; ② Intramolecular self-adjuvanting: Conjugating MUC1 glycopeptides to T cell epitope peptides and / or TLR agonists to construct multicomponent vaccines that simultaneously activate humoral and cellular immunity; and ③ Multivalent display systems: Utilizing carriers such as nanomaterials to spatially arrange antigen epitopes, mimicking pathogen surface features to enhance immunogenicity. Although these strategies have demonstrated promising antitumor efficacy in mouse models, they remain challenging. Current MUC1 vaccine development primarily focuses on the construction of single-glycoform glycopeptide vaccines, ignoring the high heterogeneity of MUC1 glycosylation modifications on tumor cell surfaces. Single-glycoform glycopeptide vaccines are unlikely to address clinical needs. Furthermore, while traditional strategies (such as carrier protein conjugation, T cell epitope integration, or multivalent display systems) can induce strong humoral immune responses, their ability to address the diverse array of tumor antigens is limited. MUC1 glycopeptide vaccines based on a single glycoform may not fully mimic the complex glycosylation profile of the tumor microenvironment, which greatly limits the vaccine's potential for clinical translation. Therefore, the development of multivalent glycopeptide anti-tumor vaccines that can simultaneously target multiple MUC1 glycoforms has become an important research direction to overcome current technical bottlenecks. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention proposes the preparation and application of a Mosaic vaccine targeting multiple tumor glycopeptide antigens.

[0005] The present invention provides a Mosaic vaccine targeting multiple tumor glycopeptide antigens, comprising a protein carrier and at least three MUC1 glycopeptide antigens of different glycosylation types covalently linked to the protein carrier, wherein the glycopeptide antigens comprise different glycoforms selected from Tn antigen, T antigen, and STn antigen.

[0006] Preferably, the glycopeptide antigen is linked to the protein carrier via a diethyl squarate cross-linker.

[0007] Preferably, the molar ratio of the Tn antigen, T antigen and STn antigen is 1:1:1±0.35.

[0008] Preferably, the protein carrier is keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), diphtheria toxoid (DT) or tetanus toxoid (TT).

[0009] Preferably, the amino acid sequence of the MUC1 glycopeptide antigen comprises a core repeating sequence of HGVSTAPDTRPAPGSTAPPA.

[0010] The method for preparing a Mosaic vaccine targeting multiple tumor glycopeptide antigens of the present invention comprises the following steps:

[0011] Synthesis and purification of S1 and MUC1 glycopeptides:

[0012] S1.1. Solid-Phase Peptide Synthesis: The MUC1 peptide backbone was synthesized using a microwave-assisted Fmoc-SPPS strategy using H-Ala-2-CT resin as a support. Fmoc-GalNAc-Thr monomers were introduced into the glycosylation sites, and the coupling was performed using a HATU / HOAt activation system.

[0013] S1.2. Cleavage of the resin using trifluoroacetic acid / triisopropylsilane / water to remove acid-sensitive protecting groups while retaining glycosyl protecting groups;

[0014] S1.3. Purify the crude peptide by HPLC using a C18 column, remove the glycosyl protecting group after lyophilization, and perform secondary purification to obtain the MUC1 glycopeptide containing Tn antigen;

[0015] S2. Sugar chain extension: Using glycosyltransferase and sugar nucleotide donors, enzymatic sugar chain extension is performed on the basis of Tn antigen to generate T antigen, STn antigen, 2,3-ST antigen and complex tetrasaccharide DST antigen;

[0016] S3. Glycopeptide functionalization modification: The glycopeptide was dissolved in an ethanol / water mixed solvent, 3,4-diethoxy-3-cyclobutene-1,2-dione was added, and the pH was adjusted to 8.0 for cyclobutene dione modification. After acidification, the active glycopeptide that could be coupled to the carrier protein was purified.

[0017] S4. Preparation of Mosaic Vaccine:

[0018] At least two MUC1 glycopeptides with different glycoforms are reacted with a protein carrier in a borate buffer in proportion, the uncoupled components are removed by ultrafiltration, and the Mosaic vaccine is obtained after lyophilization.

[0019] Preferably, the synthesis conditions for synthesizing the MUC1 polypeptide backbone by the microwave-assisted Fmoc-SPPS strategy in S1.1 include: deprotection using 20% ​​piperidine solution, microwave power 50 W, temperature 50° C., and time 5 minutes; coupling using 5 equivalents of Fmoc-amino acid, 5 equivalents of TBTU, 10 equivalents of DIEA, microwave power 50 W, temperature 50° C., and time 10 minutes; and glycine monomer coupling using 3 equivalents of Fmoc-GalNAc-Thr, 5 equivalents of HATU, 5 equivalents of HOAt, and 10 equivalents of DIEA for 45 minutes.

[0020] Preferably, the S2 is based on MUC1 (Tn) glycopeptide, and uses C1GalT1 enzyme and UDP-Gal donor to perform β1,3-Gal extension to generate T antigen; uses ST3Gal1 enzyme and CMP-Neu5Ac donor to perform α2,3-Neu5Ac extension to generate 2,3-ST antigen; uses ST6GalNAc1 enzyme and α2,6-Neu5Ac donor to perform α2,6-Neu5Ac extension to generate STn antigen; and by combining the above enzymatic reactions, a complex tetrasaccharide DST antigen is generated.

[0021] Preferably, the molar ratio of glycopeptide to protein carrier in S4 is 25:1, the coupling reaction is carried out in 0.07M Na2B4O7 / 0.035M KHCO3 buffer, the reaction time is 72 hours, and the reaction temperature is room temperature.

[0022] The present invention also provides the use of a Mosaic vaccine targeting multiple tumor glycopeptide antigens in the preparation of a drug for treating MUC1-positive tumors.

[0023] The MUCl-positive tumors include breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, non-small cell lung cancer, colorectal cancer, gastric cancer or ovarian cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Through the strategy of combining multiple saccharide antigens to cover the tumor, the clinical problem of tumor antigen heterogeneity is effectively overcome. This invention integrates the three most representative MUC1 glycogen antigens (Tn, T and STn) into a single vaccine system for the first time, and experiments have confirmed that it can simultaneously induce a broad spectrum of immune responses against different sugar epitopes. This "multi-target coverage" technical solution has significant universal value. It is not only applicable to MUC1-positive tumors, but its technical principles can be extended to other tumor-related sugar antigen systems (such as TF antigen, Globo H, PSMA, etc.), providing an innovative solution to the clinical problem of tumor antigen heterogeneity.

[0026] (2) A chemical enzymatic method combined with SPPS strategy was used to achieve precise synthesis of glycopeptides. The glycosylation site accuracy rate was >98%, and the structural accuracy rate was >99%, far exceeding traditional methods, ensuring controllable vaccine quality.

[0027] (3) A new paradigm of "polysaccharide-single carrier" Mosaic vaccine was established. Through the multivalent antigen display system, the immunogenicity of the vaccine was significantly enhanced. The vaccine can effectively promote the activation of B cells and dendritic cells. This potent immune activation property enables it to induce significant immune responses at low doses (10 μg / time), greatly improving the economy and safety of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0029] Figure 1 This is the synthetic route of MUC1 glycopeptide.

[0030] Figure 2 HPLC and MS characterization charts of MUC1 glycopeptides MUC1(Tn), MUC1(T), and MUC1(STn).

[0031] Figure 3 HPLC and MS characterization charts of MUC1 glycopeptides MUC1(2,3-ST), MUC1(2,6-ST), and MUC1(DST).

[0032] Figure 4 HPLC and MS characterization of MUC1 glycopeptide modified with diethyl squarate.

[0033] Figure 5 Schematic diagram of the synthesis of Mosaic-MUC1 vaccine and MALDI-TOF-MS characterization of each glycopeptide vaccine.

[0034] Figure 6 This is the ion chromatogram characterization of Mosaic-MUC1 vaccine.

[0035] Figure 7 This is the antibody titer level evaluation and antibody subtype detection chart of MUC1 glycopeptide vaccine.

[0036] Figure 8 This is a graph showing the binding of antiserum induced by MUC1 glycopeptide vaccine to tumor cells.

[0037] Figure 9 This is a diagram showing the results of MUC1 glycopeptide vaccine promoting the maturation of antigen-presenting cells.

[0038] Figure 10 This is a diagram showing the results of MUC1 glycopeptide vaccine promoting T cell proliferation.

[0039] Figure 11 This is a diagram showing the results of T cell activation stimulated by MUC1 glycopeptide vaccine.

[0040] Figure 12 This is a graph showing the anti-tumor activity of the MUC1 glycopeptide vaccine.

[0041] Figure 13 This is the result of the combination of Mosaic-MUC1 glycopeptide vaccine and αPD-1 antibody immune checkpoint inhibitor. DETAILED DESCRIPTION

[0042] The following is a further description of the specific embodiments of the present invention. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention.

[0043] Example 1:

[0044] 1) SPPS synthesis, preparation, and purification of MUC1 glycopeptide

[0045] MUC1 peptides were synthesized by solid-phase peptide synthesis (SPPS). First, a MUC1 glycopeptide chain containing Tn was synthesized. 0.1 mmol of the glycopeptide was synthesized using H-Ala-2-CT-polystyrene resin with a loading of 0.53 mmol / g. Each amino acid was attached to the resin using a microwave-assisted peptide synthesizer to complete a peptide synthesis cycle. Each synthesis cycle consisted of three steps: First, the Fmoc protecting group on the terminal amino group on the resin was removed using a 20% piperidine solution for 5 minutes at a microwave power of 50 W and a temperature of 50°C. Second, a coupling step was performed by adding an Fmoc-amino acid (5 eq) and activating it with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) (5 eq). N,N-diisopropylethylamine (DIEA) (10 eq) was used as the activating base. The coupling cycle was performed for 10 minutes at a microwave power of 50 W and a temperature of 50°C. The coupling of the glycine monomer differs from that of ordinary amino acids. Tn-loaded glycine Fmoc-GalNAc-Thr (3 eq) is added, along with the more active reagents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (5 eq) and 1-hydroxy-7-azobenzotriazole (HOAt) (5 eq). DIEA (10 eq) is used as the activating base. The coupling time is 45 minutes, microwave power is 50 W, and microwave temperature is 50°C. A single coupling is performed. The final step is a DMF wash. Repeating the (deprotection → condensation → condensation wash) process completes the coupling of all amino acids and modified monomers, completing the synthesis of the MUC1 glycopeptide. The resin was then removed from the automated peptide synthesizer, and the glycopeptide was cleaved from the resin using trifluoroacetic acid / triisopropylsilane / water (90 / 5 / 5, v / v / v). This simultaneously cleaved all acid-sensitive protecting groups on the amino acid side chains, while leaving the protecting groups on the sugar moiety intact. The peptide was analyzed by high-performance liquid chromatography (HPLC) on a C18 column (YMC-Triart C18, 4.6×250 mm, 5 μm) at a flow rate of 1 mL / min using a linear gradient of 10% to 50% acetonitrile containing 0.1% trifluoroacetic acid over 40 minutes. High-resolution mass spectrometry (HRMS) analysis was performed on a Thermo Scientific™ Q Exactive™ hybrid quadrupole mass spectrometer, and peptide molecular weight was determined by mass spectrometry.After confirming the correct synthesis by HPLC and HRMS, the crude peptide was separated and purified by preparative HPLC on a C18 column. The crude peptide was purified by HPLC on a Waters 2695 HPLC system equipped with a dual absorbance UV detector. The column was a C18 column (Waters SymmetryPrep™, 19×300 mm, 7 μm) with a flow rate of 20 mL / min. A linear gradient of 10% to 50% acetonitrile containing 0.1% trifluoroacetic acid was run over 40 minutes. After purification and lyophilization, the pure product was dissolved in 0.5% sodium methoxide / methanol solution (pH 9.5) for deprotection to remove the protecting group of the glycosyl moiety. After completion of the reaction, glacial acetic acid was added to adjust the pH to neutral to terminate the reaction. The product was separated and purified again by preparative HPLC to obtain the pure MUC1 glycopeptide after lyophilization. Figure 1 , Figure 2 ). The above method can be used to synthesize MUC1 glycopeptides with Tn antigens at S4, T5, T9, S15, and T16.

[0046] 2) Chemoenzymatic extension of sugar chains on MUC1 glycopeptide

[0047] Based on the MUC1 (Tn) glycopeptide molecule, an enzyme-catalyzed glycan synthesis method was used to perform enzymatic sugar chain extension on the above-mentioned simply glycosylated glycopeptide antigen using a series of glycosyltransferases. C1GalT1 glycosyltransferase and sugar nucleotide donor UDP-Gal were used to perform β1,3-Gal linkage extension to synthesize the T antigen MUC1 (T); ST3Gal1 and CMP-Neu5Ac were used to perform α2,3-Neu5Ac linkage extension on the Tn glycosylation site to synthesize the 2,3-ST antigen MUC1 (2,3-ST); ST6G AlNAc1 and α2,6-Neu5Ac can be used to extend the α2,6-Neu5Ac linkage on the Tn antigen to synthesize the STn antigen MUC1 (STn); ST6GalNAc1 and α2,6-Neu5Ac can be used to extend the sugar chain of the 2,3-ST antigen; ST6GalNAc1 and α2,6-Neu5Ac can be used to extend the α2,6-Neu5Ac linkage on the T antigen to synthesize the 2,6-ST antigen MUC1 (2,6-ST); a complex glycopeptide MUC1 (DST) with a tetrasaccharide of the DST antigen can be synthesized. Figure 1-Figure 3 ).

[0048] III) Modification of MUC1 glycopeptide

[0049] The MUC1 (Tn) glycopeptide was modified and prepared to be connected to a protein carrier to synthesize a glycopeptide vaccine. 0.0046 mmol of glycopeptide was dissolved in a mixed solvent of 2.2 mL of ethanol and water (1 / 1, v / v), and 0.68 μL of 3,4-diethoxy-3-cyclobutene-1,2-dione was added. 5 μL of saturated sodium carbonate solution was added every five minutes until the pH value of the reaction system reached 8.0. The reaction was allowed to proceed at room temperature for 1.5 hours. After the reaction was completed, 0.429 μL of acetic acid was added for acidification. The ethanol was removed under reduced pressure distillation, and the remaining small amount of water was removed by freeze drying. The modified polypeptide was obtained by purification by HPLC. All MUC1 glycopeptides can be modified in the same way ( Figure 4 ).

[0050] IV) Preparation and characterization of MUC1 glycopeptide vaccine

[0051] 0.06 μmol TT (1 eq) and 1.5 μmol MUC1(Tn) (25 eq) were dissolved in 600 μL of 0.07 M Na₂B₄Oₐ / 0.035 M KHCO₃ buffer and reacted at room temperature for 72 h. Molecules not covalently bound to TT and salt ions were removed by ultrafiltration (Millipore, 30 kDa). The glycopeptide protein M(Tn)-TT was obtained after lyophilization. The coupling efficiency of the glycopeptide to the protein carrier was characterized by MALDI-TOF. Following the same method, MUC1(STn) and MUC1(2,3-ST) were coupled to the protein carrier TT to obtain M(STn)-TT and M(ST)-TT glycopeptide vaccines, respectively. Meanwhile, MUC1(Tn), MUC1(STn) and MUC1(2,3-ST) were coupled to the same protein carrier to obtain Mosaic-MUC1 glycopeptide vaccine. The total coupling efficiency and the coupling efficiency of each glycopeptide were characterized by MALDI-TOF and ion chromatography ( Figure 5 ,6). Similar methods can also be used to prepare glycopeptide vaccines based on other protein carriers such as BSA, DT and KLH.

[0052] The results are as follows Figure 5 As shown, the single glycopeptide vaccines M(Tn)-TT, M(STn)-TT and M(ST)-TT as well as the Mosaic-MUC1 glycopeptide vaccine can be coupled with an average of about 22 glycopeptides. Figure 6 The ion chromatography results showed that the ratio of the three glycopeptides MUC1(Tn), MUC1(STn) and MUC1(2,3-ST) coupled to the Mosaic-MUC1 glycopeptide vaccine was 6:8:8.

[0053] V) Evaluation of transgenic mouse immunity and antibody titer levels

[0054] MUC1.Tg mice (6 to 8 weeks old) were randomly divided into several groups. Mice were immunized once every two weeks with 100 μL of an emulsion of a 1:1 mixture of MUC1 glycopeptide vaccine and aluminum adjuvant (containing 10 μg of MUC1 glycopeptide vaccine) administered subcutaneously for a total of three immunizations ( Figure 7 a) On day 7 after the last immunization, blood was collected via orbital bleeding into lithium heparin tubes at 6000 rpm / 4°C / 8 min, and the upper plasma layer was obtained for antibody titer analysis.

[0055] A 96-well ELISA plate was coated with 100 μL / well streptavidin (5 μg / mL) and incubated overnight at 4°C. After washing three times with 200 μL / well PBS / 0.05% Tween-20 (PBST), the plate was blocked with 200 μL / well casein solution and incubated for 1 hour at room temperature. After washing three times with PBST, 100 μL / well biotinylated glycopeptide (2 μg / mL) was added, incubated at 37°C for 1 hour, and washed three times with PBST. The plasma of each mouse was diluted 3-fold, and 100 μL / well of the diluted plasma was added to a 96-well plate. After incubation at 37°C for 1.5 hours, the plate was washed 4 times with PBST, and alkaline phosphatase-labeled goat anti-mouse IgG diluted 1:3000 with PBS was added for incubation for 1 hour. After washing, 100 μL / well pNPP substrate was added, and the plate was color-developed at room temperature in the dark for 15 minutes. After that, 100 μL / well pNPP stop solution was added to stop the color development. The absorbance at 405 nm was then measured ( Figure 7 b,c).

[0056] Antibody subtypes were detected by enzyme-linked immunosorbent assay (ELISA) similar to the above method. After adding serum to a 96-well plate and incubating, goat anti-mouse IgG1, IgG2b, IgG2c, IgG3, IgM, and IgA subtype antibodies were added at a dilution of 1:1000, incubated at 37°C for 1.5 hours, washed three times, and incubated with alkaline phosphatase-labeled donkey anti-goat IgG. Other steps were the same as the above ELISA experiment ( Figure 7 d).

[0057] The results are as follows Figure 7 As shown, all glycopeptide vaccines can induce elevated antibody levels in mice, and the immune response presents a Th2 type immune response, among which the Mosaic-MUC1 glycopeptide vaccine induced the highest antibody titer level.

[0058] VI) Evaluation of the ability of vaccine-induced antiserum to bind to tumor cells

[0059] Tumor cells that highly express MUC1, such as B16-MUC1 and PANC-1 cells, were cultured and harvested when the cells reached the growth phase. The cells were washed with 1% FBS / PBS and centrifuged at 1000 rpm at 4°C for 5 minutes, and the supernatant was removed. After resuspending the cells, 100 μL of serum diluted 1:50 was added, and the cells were incubated at 4°C for 1 hour. After incubation, the cells were washed twice with 1% FBS / PBS at 1000 rpm at 4°C for 5 minutes, and the supernatant was removed. A 1:1000 dilution of the fluorescent antibody AF488 Goat Anti-Mouse IgG (H+L) was added, and the cells were incubated at 4°C for 1 hour. After incubation, the cells were washed twice with 1% FBS / PBS at 1000 rpm at 4°C for 5 minutes, and the supernatant was removed. The cells were resuspended in 600 μL of 1% FBS / PBS, passed through a 200-mesh sieve, and analyzed by flow cytometry. At the same time, B16F10 cells that do not express MUC1 were used as negative controls. By comparing the fluorescence intensity of different groups, the specific binding ability of vaccine-induced antibodies to MUC1 antigens on the surface of tumor cells can be quantitatively evaluated ( Figure 8 ).

[0060] The results are as follows Figure 8 As shown, since the Mosaic-MUC1 glycopeptide vaccine carries glycopeptides of various glycosylation types, it can more effectively induce the production of antibodies specific to various antigens. Therefore, it has the strongest binding ability to tumor cells that highly express MUC1, and no binding to B16F10 cells that do not express MUC1, which indicates the antigen specificity of the antiserum antibodies induced by the MUC1 glycopeptide vaccine.

[0061] 7) Effects of vaccines on antigen-presenting cells and T cells

[0062] Multi-parameter flow cytometry was used to perform systematic functional analysis of spleen cells from vaccine-immunized mice, mainly including three key parts: antigen-presenting cell activation detection, T cell proliferation experiment and antigen-specific T cell function analysis, to comprehensively evaluate the vaccine-induced cellular immune response.

[0063] First, a single-cell suspension of spleen was prepared. The spleen of mice immunized three times was gently ground using a 70 μm cell sieve in a clean bench. After treatment with red blood cell lysis buffer, a high-purity single-cell suspension was obtained. The cells were divided into three groups for subsequent experiments: the first group was used for antigen presenting cell analysis (2×10 6 cells), and the second group was used for T cell proliferation experiments (5×10 6 cells), and the third group was used for cytokine detection (the remaining cells).

[0064] The detection of antigen presenting cell activation is one of the focuses of this study. A multicolor flow cytometry solution was used to mark B cells (CD19 + MHCⅡ +CD80 + CD86 + ) and dendritic cells (CD11c + CD80 + CD86 + ), while flow cytometry accurately reflects the activation state of antigen-presenting cells ( Figure 9 ).

[0065] The results are as follows Figure 9 As shown, Mosaic-MUC1 glycopeptide vaccine can stimulate antigen-presenting cells to the greatest extent and promote the maturation and activation of antigen-presenting cells B cells and DC cells.

[0066] T cell proliferation assay was performed using carboxyfluorescein diacetate succinimidyl ester (CFSE) staining. Splenocytes were stained with 5 μM CFSE and 1×10 6 The cells were seeded in a 6-well plate at a density of 10 cells / well. The experimental group was stimulated with 2 μg / mL vaccine antigen, and the control group was stimulated with an equal amount of PBS. After 24 hours, the CD3 + CD4 + and CD3 + CD8 + The CFSE fluorescence intensity attenuation of T cells can quantitatively analyze the proliferation capacity of antigen-specific T cells. This method has high sensitivity and can accurately reflect the expansion of T cell clones (Figure 10)

[0068] The results are as follows Figure 10 As shown, Mosaic-MUC1 glycopeptide vaccine can effectively stimulate T cells and promote CD3 + CD4 + and CD3 + CD8 + T cells proliferate in the spleen, thereby exerting effective anti-tumor activity.

[0069] In the antigen-specific T cell function analysis part, splenocytes were first pretreated with 5 μg / mL brefeldin A for 4 hours, and then stimulated with 2 μg / mL MUC1 peptide and incubated overnight. IFNγ was detected by intracellular cytokine staining + CD8 + T cells, IFNγ + CD4 + The activation status of key functional T cell subsets such as T cells ( Figure 11All experiments were performed in triplicate, and data are presented as mean ± SEM. Flow cytometry data were analyzed using FlowJo v10, and statistical processing was performed using GraphPad Prism 8. Key experimental parameters, including antibody concentration, stimulation time, and cell density, were optimized in advance. This systematic experimental design allows for a comprehensive assessment of the characteristics of vaccine-induced cellular immune responses, providing reliable data support for research on vaccine immune mechanisms.

[0070] The results are as follows Figure 11 As shown in the figure, consistent with the proliferation of T cells, Mosaic-MUC1 glycopeptide vaccine can not only effectively promote the proliferation of T cells, but also make CD8 + T cells and CD4 + An increase in the proportion of T cells can also activate T cells and promote the secretion of cytokines.

[0071] 8) Evaluation of vaccine anti-tumor activity

[0072] MUC1.Tg mice (6 to 8 weeks old) were randomly divided into several groups. Mice were immunized once every two weeks with 100 μL of a 1:1 emulsion of MUC1 glycopeptide vaccine and aluminum adjuvant (containing 10 μg of MUC1 glycopeptide vaccine) subcutaneously for a total of three immunizations. On the 14th day after the last immunization, 2.5×10 5 B16-MUC1 cells were added and tumor volume was monitored every two days to evaluate the inhibitory effect of the established immune response on tumor growth. Tumor volume was calculated as follows: V = 0.5 × L × W 2 (L is the longest diameter of the tumor and W is the shortest diameter perpendicular to the length).

[0073] The results are as follows Figure 12 All glycopeptide vaccines can effectively inhibit the growth of tumor volume, among which Mosaic-MUC1 glycopeptide vaccine has the highest anti-tumor activity.

[0074] IX) Evaluation of the efficacy of combining vaccines with the immune checkpoint inhibitor αPD-1

[0075] MUC1.Tg mice (6 to 8 weeks old) were randomly divided into several groups. Mice were immunized once every two weeks with 100 μL of a 1:1 emulsion of MUC1 glycopeptide vaccine and aluminum adjuvant (containing 10 μg of MUC1 glycopeptide vaccine) subcutaneously for a total of three immunizations. On the 7th day after the last immunization, 2.5×10 5 Mice were implanted with B16-MUC1 cells and injected intraperitoneally with 200 μg of αPD-1 on days 8, 11, and 14. Tumor volume was monitored every two days to assess the inhibitory effect of the established immune response on tumor growth.

[0076] like Figure 13 As shown, the combination of Mosaic-MUC1 vaccine and immune checkpoint inhibitor αPD-1 has a synergistic anti-tumor effect, which can more effectively inhibit tumor volume growth and prolong the survival period of mice.

[0077] Although the above describes the specific implementation method of the present invention in conjunction with the accompanying drawings, it does not limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, those skilled in the art can make various modifications or variations without creative work and still fall within the scope of protection of the present invention.

Claims

1. A Mosaic vaccine targeting multiple tumor-associated glycopeptide antigens, characterized in that: The vaccine comprises a protein carrier and at least three tumor-associated polypeptide antigens with different glycosylation or non-glycosylation modifications connected to the protein carrier through covalent bonds. The polypeptide antigens are selected from MUC1 glycopeptide antigens with different glycosylation modifications.

2. The Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 1, characterized in that: The glycopeptide antigen is connected to the protein carrier by chemical covalent coupling with diethyl squarate cross-linking agent.

3. The Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 1, characterized in that: The molar ratio of the coupled multiple antigens is 1:1:1±0.

35.

4. The Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 1, characterized in that: The protein carrier is keyhole limpet hemocyanin, bovine serum albumin, diphtheria toxoid, tetanus toxoid or virus-like particle protein carrier.

5. The Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 1, characterized in that: The amino acid sequence of the MUC1 glycopeptide antigen contains the core repeating sequence of HGVSTAPDTRPAPGSTAPPA.

6. The method for preparing a Mosaic vaccine targeting multiple tumor glycopeptide antigens according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Synthesis and purification of glycopeptides: S1.

1. Solid-Phase Peptide Synthesis: Using H-Ala-2-CT resin as a support, the peptide backbone was synthesized via microwave-assisted Fmoc-SPPS strategy. Fmoc-GalNAc-Thr monomers were introduced into the glycosylation sites, and the coupling was performed using a HATU / HOAt activation system. S1.

2. Cleavage of the resin using trifluoroacetic acid / triisopropylsilane / water to remove acid-sensitive protecting groups while retaining glycosyl protecting groups; S1.

3. Purify the crude peptide by HPLC using a C18 column, remove the glycosyl protecting group after lyophilization, and perform secondary purification to obtain the MUC1 glycopeptide containing Tn antigen; S2. Sugar chain extension: Using glycosyltransferase and sugar nucleotide donors, enzymatic sugar chain extension is performed on the basis of Tn antigen to generate T antigen, STn antigen, 2,3-ST antigen and complex tetrasaccharide DST antigen; S3. Glycopeptide functionalization modification: The glycopeptide was dissolved in an ethanol / water mixed solvent, 3,4-diethoxy-3-cyclobutene-1,2-dione was added, and the pH was adjusted to 8.0 for cyclobutene dione modification. After acidification, the active glycopeptide that could be coupled to the carrier protein was purified. S4. Preparation of Mosaic Vaccine: At least two MUC1 glycopeptides with different glycoforms are reacted with a protein carrier in a borate buffer in proportion, the uncoupled components are removed by ultrafiltration, and the Mosaic vaccine is obtained after lyophilization.

7. The method for preparing a Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 6, characterized in that: The synthesis conditions for synthesizing the MUC1 polypeptide backbone by the microwave-assisted Fmoc-SPPS strategy in S1.1 include: deprotection using 20% ​​piperidine solution, microwave power 50 W, temperature 50° C., and time 5 minutes; coupling using 5 equivalents of Fmoc-amino acid, 5 equivalents of TBTU, and 10 equivalents of DIEA, microwave power 50 W, temperature 50° C., and time 10 minutes; and glycine monomer coupling using 3 equivalents of Fmoc-GalNAc-Thr, 5 equivalents of HATU, 5 equivalents of HOAt, and 10 equivalents of DIEA, and time 45 minutes.

8. The method for preparing a Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 6, characterized in that: In the S2, MUC1 (Tn) glycopeptide is used as the basis, and β1,3-Gal extension is performed using C1GalT1 enzyme and UDP-Gal donor to generate T antigen; α2,3-Neu5Ac extension is performed using ST3Gal1 enzyme and CMP-Neu5Ac donor to generate 2,3-ST antigen; α2,6-Neu5Ac extension is performed using ST6GalNAc1 enzyme and α2,6-Neu5Ac donor to generate STn antigen; and by combining the above enzymatic reactions, a complex tetrasaccharide DST antigen is generated.

9. The method for preparing a Mosaic vaccine targeting multiple tumor glycopeptide antigens according to claim 6, characterized in that: The molar ratio of glycopeptide to protein carrier in S4 is 25:1, and the coupling reaction is carried out in 0.07MNa2B4O7 / 0.035M KHCO3 buffer for 72 hours at room temperature.

10. Use of the Mosaic vaccine targeting multiple tumor glycopeptide antigens according to any one of claims 1 to 5 in the preparation of a drug for treating MUC1-positive tumors.

Images