A novel antigen peptide vaccine, its preparation method and application

Abstract

This invention discloses a neoantigen polypeptide vaccine, its preparation method, and its applications. The vaccine significantly promotes the maturation and antigen presentation of dendritic cells (DCs), enhances the secretion of type I interferon and IL-12, and effectively improves antigen-specific T-cell immune responses, thereby inhibiting tumor growth and metastasis, and has potential application value. The vaccine significantly inhibits tumor growth in mouse B16F10-OVA xenograft tumor and B16-OVA lung metastasis models, and significantly prolongs the survival of mice in a combined PD-L1 immunotherapy model; further, it induces long-term immune memory responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a neoantigen polypeptide vaccine, its preparation method, and its application. Background Technology

[0002] Tumor vaccines deliver tumor-specific antigens (such as neoantigen peptides) to the patient's immune system, aiming to activate a specific T-cell immune response and thereby eliminate tumor cells. The selection of immune adjuvants is crucial in the construction of tumor vaccines. Toll-like receptor (TLR) agonists, as an important class of immune adjuvants, can enhance the immune response generated by tumor vaccines by binding to corresponding Toll-like receptors and activating downstream signaling pathways.

[0003] Depending on the type of receptor activated, TLR agonists induce different types of immune responses. For example, the double-stranded RNA mimic Poly(I:C), as a TLR3 agonist (TLR3a), primarily transmits signals via the TRIF-dependent pathway, promoting the production of type I interferon. Studies have shown that TLR3 activation can enhance the expression of MHC class II molecules on the surface of dendritic cells (DCs) and promote antigen cross-presentation. On the other hand, imidazoquinoline drugs (such as ralsimod R848), as TLR7 / 8 agonists (TLR7 / 8a), primarily trigger signal transduction via the Myd88-dependent pathway, promoting the expression of co-stimulatory molecules on DCs and the production of IL-12.

[0004] Although single-type TLR agonists exhibit adjuvant activity, existing studies have shown that the combined use of different types of TLR agonists (such as TLR3a and TLR7 / 8a) may induce stronger and longer-lasting type I interferon and IL-12 secretion. However, how to efficiently co-deliver different types of TLR agonists with antigenic peptides into the same antigen-presenting cells (APCs) to maximize the synergistic effect of the agonists and protect the antigen from rapid degradation remains a critical technical challenge in the development of tumor vaccines. Summary of the Invention

[0005] To address the shortcomings of existing methods, the present invention aims to provide a peptide self-assembly-based nanoparticle vaccine, CP-7 / 8a / TLR3a. By co-incorporating TLR3a and TLR7 / 8a into the construction of a neoantigen peptide vaccine, a more potent neoantigen peptide vaccine can be developed. First, an R848 derivative is covalently bound to a DBCO-modified linker platform, and then linked to an azide-lysine-modified linker-antigen peptide-membrane-penetrating peptide via click chemistry. After dissolving in water, it self-assembles into nanoparticles through hydrophilic-hydrophobic interactions. Then, through electrostatic interactions, the positively charged membrane-penetrating peptide adsorbs negatively charged TLR3a, preparing a dual-agonist loaded CP-7 / 8a / TLR3a vaccine. The antigenic epitopes can be changed according to different tumor neoantigens, thus achieving versatility. CP-7 / 8a / TLR3a co-delivers TLR7 / 8a, TLR3a, and the antigenic peptide into APCs, enabling highly efficient initiation of T-cell immune responses. Experimental results showed that CP-7 / 8a / TLR3a can promote the maturation of dendritic cells (DCs), the expression of co-stimulatory molecules, and antigen presentation. CP-7 / 8a / TLR3a promotes the secretion of type I interferon and IL-12, enhances antigen-specific T cell immune responses, and thus inhibits tumor growth and metastasis.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] In a first aspect, the present invention protects a method for preparing a neoantigen polypeptide vaccine, the method comprising the following steps:

[0008] Step 1, Preparation of hydrophobic module: The small molecule TLR7 / 8 agonist is covalently coupled with the short peptide DBCO-EWEWE with a carboxyl side chain through an amidation reaction to form a hydrophobic module (i.e., TLR7 / 8a modified on amino acid E DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)). This structure provides the hydrophobic driving force for subsequent assembly.

[0009] Step 2, Preparation of the hydrophilic module: The antigen peptide (e.g., OVA257-264) is introduced into a sequence containing azide-modified lysine (N3-K) by solid-phase synthesis and covalently linked with the pAntp membrane-penetrating peptide to form an antigen conjugate with cell membrane-penetrating ability;

[0010] Step 3, Click Coupling Reaction: The hydrophobic module obtained in Step 1 and the hydrophilic module obtained in Step 2 undergo a click chemical reaction. After the reaction is completed, the module is dissolved in water to complete the assembly. The reaction is carried out at room temperature for 16 hours to generate an amphiphilic structural unit (i.e., CP-7 / 8a).

[0011] Step 4, Surface loading: Negatively charged poly(I:C) (i.e., TLR3 agonist) is added to CP-7 / 8a micelles at a ratio of 1:4 (CP-7 / 8a:Poly(I:C), mass ratio) to form a complete four-component vaccine based on electrostatic adsorption.

[0012] In a specific implementation scheme, the covalent coupling in step 1 is as follows: TLR7 / 8a and DBCO-EWEWE are reacted in anhydrous DMF and TEA system for 1 ± 0.5 hours, and high-purity DBCO-TLR7 / 8a conjugate (i.e., DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)) is obtained by high-performance liquid chromatography (HPLC).

[0013] In a specific implementation, the hydrophilic module sequence in step 2 is: K(n3)SLVRLESIINFEKLTRQIKIWFQNRRMKWKK-NH2, as shown in SEQ ID NO:1.

[0014] Wherein, K(n3) is azidolysine.

[0015] The antigenic peptides of this invention can be quickly replaced according to different tumor epitopes, requiring only modification of the antigenic peptide sequence without the need to reconstruct the delivery system.

[0016] In the specific implementation plan, the assembly method of step 3 is as follows: the hydrophobic module and the hydrophilic module are mixed in DMSO at a molar ratio of 1:1 and incubated at room temperature for 16 hours to form a micelle nanostructure CP-7 / 8a.

[0017] In a specific implementation plan, the surface load preparation method in step 4 is as follows: poly(I:C) (200–400kDa) is mixed in CP-7 / 8a nanoparticles at a mass ratio of 1:4 with CP-7 / 8a:Poly(I:C), and left to stand for 10±5 minutes to form a quaternary assembly structure. The whole process does not involve organic solvents, and is simple to operate and environmentally friendly.

[0018] In specific implementation schemes, other water-soluble TLR3 / 7 / 8a structures can be selected as TLR agonists to expand the range of indications.

[0019] In specific implementation schemes, the pAntp membrane-penetrating peptide sequence can be replaced with other functional membrane-penetrating peptides or cell-targeting peptides to enhance adaptability and specificity.

[0020] The preparation method of this invention does not rely on organic solvents, and the all-aqueous system has good biocompatibility and industrialization potential.

[0021] Secondly, the present invention protects the polypeptide vaccine prepared by the method described above.

[0022] This invention evaluates the enhancement of antigen-specific T cell responses by CP-7 / 8a / TLR3a through cytokine analysis and tetramer experiments; demonstrates that CP-7 / 8a / TLR3a can inhibit tumor growth in the B16F10-OVA model using a mouse tumor prevention model; shows that CP-7 / 8a / TLR3a enhances anti-tumor immune responses and inhibits tumor growth in B16F10-OVA; and demonstrates that CP-7 / 8a / TLR3a inhibits the metastasis of lung metastases in a mouse B16F10-OVA model, thus advancing research in the fields of tumor vaccines and immunotherapy and providing new strategies for tumor treatment.

[0023] Thirdly, the present invention also protects the use of the aforementioned polypeptide vaccine in the preparation of tumor vaccines.

[0024] Fourthly, the present invention also protects the use of the aforementioned polypeptide vaccine in the preparation of medicaments for the treatment or prevention of tumors.

[0025] In a specific implementation plan, the tumor is a melanoma.

[0026] Beneficial effects

[0027] The novel antigen polypeptide vaccine, its preparation method, and its application provided by this invention have the following advantages compared with the prior art:

[0028] (1) The CP-7 / 8a / TLR3a vaccine not only promotes the maturation of DCs and antigen presentation, but also significantly enhances the secretion of type I interferon and IL-12, thereby effectively improving the antigen-specific T cell immune response and demonstrating its potential application value in inhibiting tumor growth and metastasis.

[0029] (2) The CP-7 / 8a / TLR3a vaccine significantly inhibited tumor growth in mouse B16F10-OVA and TC-1, TC-1 liver metastasis, and B16-OVA lung metastasis models, and significantly prolonged the survival of mice in the combined PD-L1 immunotherapy model; and further induced long-term immune memory response. Attached Figure Description

[0030] Figure 1 Mass spectrometry detection chromatogram of DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)

[0031] Figure 2 Electron micrographs of CP-7 / 8a.

[0032] Figure 3 Detection of the adsorption ratio of Poly(I:C) for CP-7 / 8a.

[0033] Figure 4 The tumor growth curve is shown in the mouse subcutaneous xenograft model.

[0034] Figure 5 The survival curves after treatment in a mouse subcutaneous xenograft model are shown.

[0035] Figure 6 The number of lung tumor metastases in mice after treatment in a mouse lung metastasis model.

[0036] Figure 7 Safety evaluation of CP-7 / 8a / TLR3a vaccines. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0038] Reagents: DBCO-EWEWE (aza-dibenzocyclooctylenylamine-glutamic acid-tryptophan-glutamic acid-tryptophan-glutamic acid, synthesized by GenScript), TLR7 / 8 agonist (TLR7 / 8a, CAS: 210304-20-4), DMF (CAS: 68-12-2), triethylamine (CAS: 121-44-8), HATU (CAS: 148893-10-1), hydrochloric acid, hydrophilic module sequence: K(n3)SLVRLESIINFEKLTRQIKIWFQNRRMKWKK-NH2 (synthesized by GenScript).

[0039] Example 1: Construction of CP-7 / 8a

[0040] The hydrophobic module DBCO-TLR7 / 8a conjugate (DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)) was prepared as follows: First, 10 mg DBCO-EWEWE (0.0094 mmol, 1 eq) and 10.6 mg TLR7 / 8a (0.0338 mmol, 3.6 eq) were dissolved in 2 mL DMF (N,N-dimethylformamide) in a round-bottom flask. 15.7 μL of triethylamine (0.1128 mmol, 12 eq) was added. The round-bottom flask was cooled to 4°C on crushed ice. The flask and crushed ice were then placed on a magnetic stirrer, and stirring was started. 11.8 mg HATU (0.031 mmol, 3.3 eq) was added, and the reaction was carried out on ice for 1 hour. Wash twice with 1 mL of 1M hydrochloric acid (4000 g, centrifuged for 5 minutes), wash once with pure water (4000 g, centrifuged for 5 minutes), freeze-dry, and weigh. Dissolve the dried solid in DMSO and purify for 12 minutes on an Agilent C-18 column using a preparative high-performance liquid chromatography system with a 34-44% acetonitrile / H2O (0.05% TFA) gradient. Freeze-dry the obtained components and weigh. Calculate C. 108 H 121 N 23 O 13 (i.e., the MS (ESI) m / z of the DBCO-TLR7 / 8a conjugate (DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)) is 1949 (yielding 1950 [M+H]+ and 975 [M+2H]2+) Figure 1 ).

[0041] Chemical coupling and assembly: The hydrophilic module and the hydrophobic module (molar ratio 1:1) were dissolved in DMSO (20 mg / mL), mixed thoroughly, and reacted at room temperature in the dark for 16 hours. The DMSO solution was then diluted to 1 mg / mL with PBS buffer, allowed to stand, and allowed to self-assemble into nanoparticles. The nanoparticles were then stored at 4°C.

[0042] like Figure 2 As shown, the assembled nanoparticles have a particle size of approximately 170 nm, and the zeta potential decreased significantly from +26.37 ± 0.38 mV to +5 mV, indicating that poly(I:C) was successfully coated and stably exists on the particle surface.

[0043] Example 2: Preparation of CP-7 / 8a / TLR3a

[0044] Electrostatic adsorption: poly(I:C) (CAS: 24939-03-5) was mixed in different mass ratios with the CP-7 / 8a nanoparticles obtained in Example 1, and the mixture was allowed to stand for 10 minutes to form an assembled structure. The whole process does not involve organic solvents, and is simple to operate and environmentally friendly.

[0045] Further screening of specific ratios and steps are as follows: Prepare sample solutions according to mass ratios of CP-7 / 8a:Poly (I:C) of 16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, and 1:16; use Malvern particle size potential to detect the particle size potential.

[0046] The results are as follows Figure 3 As shown, different ratios of CP-7 / 8a:Poly (I:C) were added to the system, and the optimal ratio was selected based on the particle size and potential. Therefore, the final ratio of CP-7 / 8a:Poly (I:C) was determined to be 1:4 to construct CP-7 / 8a / TLR3a.

[0047] Example 3: Experimental Treatment of Subcutaneous Xenograft Tumors

[0048] The mice were subcutaneously inoculated with 5 × 10⁸ C57BL / 6 mice (8-week-old females, purchased from the Experimental Animal Center of Nanjing Medical University). 5 We established a mouse subcutaneous tumor model using B16F10-OVA cells.

[0049] The study included an untreated group, a long peptide treatment group (LP: SMLVLLPDEVSGLEQLESIINFEKLTEWTS + TLR3a / TLR7 / 8a), a transmembrane peptide-epitope peptide treatment group (CP: K(n3)SLVRLESIINFEKLTRQIKIWFQNRRMKWKK-NH2+TLR3a / TLR7 / 8a), and a CP-7 / 8a / TLR3a group, with the corresponding vaccines administered on days 4, 10, and 16.

[0050] like Figure 4 As shown, compared with the mice treated with LP+TLR3a / TLR7 / 8a and CP+TLR3a / TLR7 / 8a, the mice treated with CP-7 / 8a / TLR3a showed more significant inhibition of tumor growth.

[0051] like Figure 5As shown, compared with untreated mice (mean survival 20.25 days), the survival of mice in the LP + TLR3a / TLR7 / 8a treatment group was extended by only about 7 days (mean survival 27 days), and the survival of mice in the CP + TLR3a / TLR7 / 8a treatment group was extended by about 13 days (mean survival 33 days), but no mice achieved long-term survival. However, CP-7 / 8a / TLR3a treatment significantly prolonged the survival of B16F10-OVA tumor-bearing mice, and 1 / 7 of the mice achieved long-term survival. In summary, these results demonstrate that CP-7 / 8a / TLR3a treatment can significantly inhibit tumor growth in the B16F10-OVA tumor model and significantly prolong the survival of mice.

[0052] Example 4: Experimental Treatment of Lung Metastases

[0053] Healthy C57BL / 6 mice (8-week-old females, purchased from the Experimental Animal Center of Nanjing Medical University) were selected and injected with 5×10 mmol / L via the tail vein. 5 We established a mouse lung metastasis model using B16F10-OVA cells.

[0054] The study included an untreated group, a long peptide treatment group (LP: SMLVLLPDEVSGLEQLESIINFEKLTEWTS + TLR3a / TLR7 / 8a), a transmembrane peptide-epitope peptide treatment group (CP: K(n3)SLVRLESIINFEKLTRQIKIWFQNRRMKWKK-NH2 +TLR3a / TLR7 / 8a), and a CP-7 / 8a / TLR3a group, with the corresponding vaccines administered on days 3, 9, and 15.

[0055] like Figure 6 As shown, compared with the LP + TLR3a / TLR7 / 8a and CP + TLR3a / TLR7 / 8a treatment groups, the growth of lung metastatic tumors in the CP-7 / 8a / TLR3a treatment group was more significantly inhibited.

[0056] Example 5: Safety Evaluation

[0057] 1. Weight records and organ weight ratios:

[0058] (1) On day 0, 6-8 week old female C57BL / 6 mice (8-week-old female mice, purchased from the Experimental Animal Center of Nanjing Medical University) were injected with 100 μg of vaccine via subcutaneous injection. The weight of each mouse was measured on days 1, 3, 5, 7, 9, 11 and 14.

[0059] (2) On day 14, mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and brain were collected and weighed to calculate the organ specific gravity coefficient. The organ specific gravity coefficient was determined using the following formula: Organ specific gravity coefficient (%) = (organ weight / body weight) × 100%.

[0060] 2. Serological testing:

[0061] On day 14, peripheral blood was collected from mice. After the blood coagulated, it was centrifuged at 3000 rpm for 5 minutes, and serum was collected. The serum samples were analyzed using a fully automated biochemical analyzer BS-200 to detect the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (UREA), creatinine (CRE), and lactate dehydrogenase (LDH).

[0062] 3. Pathological examination:

[0063] (1) On the 14th day, the heart, liver, spleen, lungs and kidneys were removed and soaked in 4% paraformaldehyde for 24-48 hours for fixation.

[0064] (2) The fixed tissues were dehydrated with ethanol, cleared with xylene, and embedded in paraffin to form paraffin specimens;

[0065] (3) Use a paraffin slicer to slice the wax, then bake the slices and dewax them to remove water;

[0066] (4) Perform HE staining, observe under an optical microscope, select a suitable field of view, and take pictures to save the results.

[0067] like Figure 7 As shown, after treatment with the CP-7 / 8a / TLR3a peptide vaccine, mice showed normal body weight and serum biological indicators compared to the control group, and HE staining of various organs demonstrated no significant toxic damage; therefore, it is demonstrated that CP-7 / 8a / TLR3a has good safety.

[0068] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing a neoantigen polypeptide vaccine, characterized in that, The method includes the following steps: Step 1, Preparation of hydrophobic module: The TLR7 / 8 agonist is covalently coupled with the short peptide DBCO-EWEWE with a carboxyl side chain through an amidation reaction to form a hydrophobic module; Step 2, Preparation of the hydrophilic module: The antigen peptide is introduced into a sequence containing lysine modified by azide by solid-phase synthesis and covalently linked with pAntp membrane-penetrating peptide to form an antigen conjugate with cell membrane-penetrating ability; Step 3, Click Coupling Reaction: The hydrophobic module obtained in Step 1 and the hydrophilic module obtained in Step 2 undergo a click chemical reaction. After the reaction is completed, the module is dissolved in water to complete the assembly. The reaction is carried out at room temperature for 16 hours to generate an amphiphilic structural unit, namely CP-7 / 8a. Step 4, Surface loading: Negatively charged poly(I:C) is added to CP-7 / 8a micelles in a mass ratio of CP-7 / 8a:Poly(I:C) of 1:4, forming a complete four-component vaccine based on electrostatic adsorption.

2. The method for preparing a neoantigen polypeptide vaccine according to claim 1, characterized in that, In step 1, the specific steps of covalent coupling are as follows: TLR7 / 8a and DBCO-EWEWE are reacted in anhydrous DMF and TEA system for 1 ± 0.5 hours. The high-purity DBCO-TLR7 / 8a conjugate is obtained by high performance liquid chromatography, namely DBCO-E(TLR7 / 8a)WE(TLR7 / 8a)WE(TLR7 / 8a)).

3. The method for preparing a neoantigen polypeptide vaccine according to claim 1, characterized in that, The assembly method for step 3 is as follows: the hydrophobic module and the hydrophilic module are mixed in DMSO at a molar ratio of 1:1 and incubated at room temperature for 16 hours to form a micelle nanostructure CP-7 / 8a.

4. The method for preparing a neoantigen polypeptide vaccine according to claim 1, characterized in that, The surface loading preparation method in step 4 is as follows: poly(I:C) is mixed in CP-7 / 8a nanoparticles and left to stand for 10±5 minutes to form a quaternary assembly structure.

5. The preparation method according to claim 1, characterized in that, The hydrophilic module sequence in step 2 is: K(n3)SLVRLESIINFEKLTRQIKIWFQNRRMKWKK-NH2, as shown in SEQ ID NO:

1.

6. The polypeptide vaccine prepared by any one of the preparation methods according to claims 1-5.

7. The use of the polypeptide vaccine according to claim 6 in the preparation of a tumor vaccine.

8. The use of the polypeptide vaccine according to claim 6 in the preparation of a medicament for treating or preventing tumors.

9. The application according to claim 7 or 8, characterized in that, The tumor is melanoma.

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