CN111939251A - Preparation and application of self-assembled epitope vaccine of bonded small molecule agonist - Google Patents
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Abstract
The invention relates to a self-assembled epitope vaccine of a bonded small molecule agonist, which can prevent or treat tumors or be used as a combined preparation of an anticancer active preparation. The self-assembly epitope vaccine of the bonding small molecule stimulant comprises two parts of water-soluble epitope peptide and hydrophobic small molecule stimulant, wherein the epitope peptide and the small molecule stimulant are combined through covalent bonds to form a compound, and then the epitope vaccine with a nano structure is self-assembled in aqueous solution through thermodynamic interaction (hydrogen bonds, van der Waals force and secondary structures) among epitope polypeptides, hydrophobic interaction and pi-pi accumulation among the small molecule stimulants. The particle size of the self-assembly vaccine is 10-500 nm, the antigen epitope selected in the self-assembly epitope vaccine bonded with the small molecule stimulant is polypeptide with the length of 5-20 amino acids, has proper affinity with MHC molecules on DC cells, can effectively stimulate and induce to generate antigen-specific CTLs, and has the potential of forming good polypeptide vaccine and DC vaccine. The self-assembled epitope vaccine of the bonded small molecule agonist has good clinical transformation and practical application prospects.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a self-assembly epitope polypeptide vaccine bonded with a small molecule agonist and preparation and application of a multi-epitope co-assembly vaccine thereof.
Background
The era of tumor immunotherapy has come and will play a key role in future cancer treatments. Currently, there is still an increasing enthusiasm to find new ways to improve tumor immune responses. For this reason, small molecules that can reduce immunosuppression in the tumor environment or enhance the cytotoxic lymphocyte response to tumors are actively sought, and this new therapeutic strategy can be used as monotherapy or in combination with other cancer therapies to increase and augment its efficacy.
Agonists of pattern recognition receptors are used as potential immunotherapies or adjuvants and fall into two broad categories: nucleotide Oligomerization Domain (NOD) -like receptor (NLR) agonists and Toll-like receptor (TLR) agonists. TLR agonists are of greatest interest in this area, and there are 13 TLRs known to date that are widely expressed in immune cell profiles, including DCs, B cells, macrophages, NK cells, and T cells. TLRs can promote T cell responses against tumors, and therefore agonists thereof are widely developed and applied in immunooncology.
Imidazoquinolines, such as imiquimod and its derivatives, are a typical class of TLR7/8 agonists. The parent compound imiquimod has been approved by the FDA in the united states for the topical treatment of basal cell carcinoma, and clinical trials for metastatic melanoma and localized bladder cancer are also underway, the latter having achieved positive results. Imiquimod analogue resiquimod (resiquimod) has also been used for the topical treatment of cutaneous T cell lymphoma, and preliminary positive results have been obtained.
After the imidazoquinoline derivative is combined with TLR, the maturation of dendritic cells can be promoted, the dendritic cells are induced to secrete various cytokines and express various co-stimulatory molecules to play a role, and further, the cell-mediated innate immune response is stimulated, and the cellular and humoral immune response is regulated. However, one of the major limitations in this area is the associated serious potential toxicity following systemic administration of TLR7/8 agonists, as they can trigger cytokine storms, which can be fatal severely limiting their clinical utility.
To ensure that these drug molecules are safe and effective during use and that systemic activation reactions can be avoided, it is clear that the site of action is of great importance.
Polypeptide vaccines based on certain epitopes of tumor antigens have become an important research field for current tumor immunotherapy. However, the epitope vaccine has short half-life in vivo, is not easy to be taken by APC, and is difficult to enter the MHC class I antigen presenting pathway in APC, so that Th1 and CTL protective immune responses cannot be effectively stimulated, and the requirement of tumor immunotherapy cannot be met.
How to combine the tumor epitope vaccine with a TLR agonist for use together to further improve the anti-tumor immune response is a very challenging task.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a self-assembled epitope polypeptide vaccine of a bonded small molecule agonist capable of preventing and treating tumors.
The preparation method of the self-assembled epitope vaccine of the bonded small molecule agonist comprises the following steps:
(1) constructing an epitope polypeptide sequence by Fmoc solid phase synthesis;
(2) screening small molecule agonists with physicochemical properties matched with epitope peptides;
(3) modifying dihydroxydisulfide on the small molecule agonist;
(4) covalently bonding the modified small molecule agonist to an epitope peptide;
(5) dissolving one or more epitope polypeptides bonded with small molecule agonists in an aqueous solution, adjusting the concentration of the epitope polypeptides to be 1-10mg/ml, and forming the epitope vaccine taking the aqueous solution as a macroscopic expression form through polypeptide self-assembly.
Further, the epitope is a polypeptide with a length of 5-20 amino acids, including but not limited to LWVFFDYVS, RWPSCQKKF, SIINFEKL, QAVHAAHAEINE, CYTWNQMNL, YMLDLQPETT, KSPWFTTL, SPSYVYHQF, EQLESIINFEKLTE, YEEYYPLI, EADPTGHSY, KIWEELSML, FLKLDRERA, FLWGPRALI, REGVELCPGNKYEMRRHGTTHSLVIHD, ECRITSNFVIPSEYWVEEKEEKQKLIQ, TAVITPPTTTTKKARVSTPKPATPSTD.
Further, small molecule agonists include TLR agonists and STING agonists.
Further, selected small molecule agonists specifically include, but are not limited to, Imiquimod, Resiquimod,852A, VTX-2337, ADU-S100, MK-1454.
Further, the epitope vaccine is CD 8-inducing vaccine+Polypeptide vaccine for T cell proliferation.
Furthermore, the epitope vaccine is a polypeptide vaccine secreting IFN-gamma.
Further, epitope vaccines are polypeptide vaccines that induce innate immunity.
Further, the means of covalently bonding small molecule agonists is disulfide bonding.
Furthermore, the molar ratio of the antigen epitope to the small molecule agonist is 1-5: 1-3.
Further, small molecule agonists are associated with epitopes, one of which is hydrophilic and the other hydrophobic.
Further, the self-assembled epitope vaccine preparation of the bonded small molecule agonist is in a nanometer solution form.
Furthermore, the particle size of the self-assembled epitope vaccine bonded with the small molecule agonist is 10-500 nm.
The invention relates to application of the self-assembled epitope vaccine preparation of the bonded small molecule agonist in tumor prevention, wherein tumors comprise non-solid tumors and solid tumors, and the application is specifically that the self-assembled epitope vaccine preparation of the bonded small molecule agonist is given to a patient in a certain dosage in a subcutaneous, intramuscular, mucosal injection or intravenous injection mode.
The invention also relates to a method for combined treatment of the self-assembled epitope vaccine of the bonded small molecule agonist and other anti-tumor drugs, in particular to a method for combined use of the self-assembled epitope vaccine of the bonded small molecule agonist and other anti-tumor drugs, including chemotherapeutic drugs, monoclonal antibody drugs, cytokine drugs, chemokine drugs and the like.
The invention shows the synergistic effect of treatment by providing a novel epitope vaccine and combining with one or more effective medicines in anti-tumor treatment, thereby remarkably improving the survival rate and the cure rate.
The invention has the advantages that:
1. the epitope vaccine of the invention takes the epitope polypeptide with more definite components, stronger specificity, higher safety and efficiency as an antigen, and prepares the aqueous solution vaccine with a nano structure by a chemical bonding and self-assembly method, thereby improving the retention time of the epitope peptide in vivo, improving the cross presentation of the epitope peptide, improving the immunogenicity of the epitope, enhancing the immune response of specific cells and improving the tumor immunotherapy effect of the epitope polypeptide.
2. The epitope vaccine provided by the invention is independent of the physicochemical properties of the antigen epitope, is almost suitable for all short peptides, can be self-assembled as long as the epitope peptide is matched with the hydrophilic and hydrophobic physicochemical properties of a small molecule agonist, can be used for preparing personalized epitope vaccines aiming at different cancers and different antigens, and promotes the development of personalized precise tumor treatment.
3. The epitope vaccine of the invention does not need additional delivery carriers (such as liposome) to deliver antigen and adjuvant, can realize the purposes of self-delivery and co-delivery, eliminates unpredictable toxic and side effects of the delivery carriers, and improves the safety and the simplicity of vaccine preparation.
4. The epitope vaccine of the invention can present multiple antigens on the surface of the nano-particles to enhance immune response.
Drawings
FIG. 1 is a simple flow chart of the epitope vaccine preparation method of the present invention
FIG. 2 shows the structure of the related polypeptide components for preparing epitope vaccine according to the present invention
FIG. 3 shows Tyrosinase bound to TLR7/8 agonist368-376Polypeptide sequence synthesis roadmap for epitopes
FIG. 4 is a mass spectrum of example 1 of the present invention.
FIG. 5 is a high performance liquid chromatogram of example 1 of the present invention.
FIG. 6 is a circular dichroism map of example 1 of the present invention.
FIG. 7 is a TEM image of example 3 of the present invention.
FIG. 8 is a particle size distribution diagram of example 3 of the present invention.
FIG. 9 is a graph showing the in vitro induction of DCs maturation according to example 4 of the present invention.
FIG. 10 is a graph showing induction of DCs maturation in lymph nodes in mice in accordance with example 5 of the present invention.
FIG. 11 is a graph showing the effect of tumor therapy in a mouse tumor therapy model according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described with reference to the following embodiments, but the description of the embodiments does not set any limit to the protection of the present invention.
Example 1: and (3) synthesizing the epitope polypeptide.
The target epitope in this example is YMDGTMSQV polypeptide sequence, all the polypeptides in the invention are synthesized by using wang resin (degree of substitution 0.90mmol/g) as a solid phase carrier, and the Fmoc solid phase synthesis method is applied in all cases.
Before the step of coupling amino acid, the solid phase carrier is firstly swelled in Dichloromethane (DCM) solution for 30min, so that the solid phase carrier is naturally unfolded, and the efficiency of coupling amino acid can be improved. The method for solid-phase synthesis of polypeptides used in the present invention can be performed by coupling each amino acid according to the following four steps:
1) and (2) acetyl blocking, wherein in the detection process after the coupling of new amino acid, in order to reduce by-products, the uncoupled amino group in the resin needs to be blocked by acetyl, and the specific operation is as follows: adding a mixed solution of acetic anhydride/pyridine/DMF with the proportion of 2/1/3 into the resin system, oscillating for reaction for 30min, and washing the system with DMF after the reaction is finished.
2) Deprotection, either of the resin or amino acid, requires removal of the protecting group from the amino group to expose the active group before coupling to the next amino acid. The deprotection method used in the present invention is: and infiltrating the solid phase resin with 20% piperidine/DMF solution, after oscillation reaction for 5min, independently washing with DMF for three times, infiltrating the solid phase resin with 20% piperidine/DMF solution again, and oscillating reaction for 20min to completely remove the protecting group. Then, the solid-phase resin system is pumped and washed by DMF three times to remove impurities generated in the deprotection process in the system.
3) Coupling new amino acid, dissolving the resin of 3 times excess amino acid, HBTU, HOBt and 8 times excess DIPEA in a proper amount of DMF, adding into a solid phase system, and reacting for 1h at room temperature with shaking. After the reaction was completed, the solid phase system was subjected to suction washing with DMF three times. The cycle of "deprotection → coupling → deprotection" is followed until all amino acids are coupled.
4) Cutting off the resin, adding a TFA/TIS/H2O mixed solution with the ratio of 10/0.6/0.6 into a solid phase synthesis system, oscillating for reaction for 3 hours, filtering to remove the resin, and evaporating most of the solvent in the system under reduced pressure. In the resin cutting process, the protecting group of the amino acid side chain is cut off, the system contains impurities left by the deprotection of the side chain, and crude purification is carried out by an ether precipitation method to obtain a crude polypeptide. Separating the crude product by high performance liquid chromatography, and lyophilizing to obtain high purity product.
Example 2: preparing and characterizing the epitope polypeptide bonding matter of the bonding small molecule excitant.
Selecting different small molecule excitomotors and epitopes as adjuvant and antigen, and preparing the polypeptide sequence containing the antigen epitope by solid phase synthesis technology or chemical reaction between the small molecule excitomotors and the epitopes. FIG. 2 shows the structural formulas of epitope polypeptide and small molecule agonist involved in experiment, and FIG. 2A shows Tyrosinase368-376The structural formula is shown in figure 2B, and the TLR7/8 small molecule agonist is shown in the figure. With Tyrosinase368-376Epitopes and TLR7/8 agonists as examples, and FIG. 3 shows the preparation of Tyrosinase bound to TLR7/8a agonists368-376Polypeptide sequence synthesis scheme for epitopes. Firstly, reacting CDI with small molecule TLR7/8 agonist (TLR7/8a) with amino at room temperature to obtain activated agonist molecule, and then esterifying to obtain dihydroxy di-hydroxylAnd (3) connecting the thioether to the small molecule agonist to obtain the agonist molecule modified by the dihydroxydisulfide. Secondly, synthesizing a target epitope (YMDGTMSQV) by a solid phase synthesis technology, then reacting CDI with amino on the epitope to obtain an activated epitope, and finally carrying out esterification reaction on the activated epitope and dihydroxydisulfide modified agonist molecules to obtain a target product (YMDGTMSQV-TLR7/8 a).
The effect of adding the dihydroxydisulfide is that under GSH conditions, disulfide bonds are broken, separation of agonist molecules from epitopes is realized, and the agonist and the epitopes play roles respectively.
To avoid reaction of CDI with non-target groups, we can add excess dihydroxydisulfide-modified agonist molecules during the second reaction step.
The material structure is characterized by methods such as Mass Spectrometry (MS) (figure 4) and High Performance Liquid Chromatography (HPLC) (figure 5), and the like, and by nuclear magnetic hydrogen spectrum (M: (M))1H NMR), carbon spectrum (13C NMR), Gel Permeation Chromatography (GPC) to identify its chemical composition, and detection of polypeptide secondary structure by infrared spectroscopy (FT-IR) and Circular Dichroism (CD) (fig. 6). The results indicate successful synthesis of epitope polypeptides that bind small molecule agonists.
Example 3: self-assembly of epitope vaccines.
Dissolving a polypeptide polymer bonded with the small molecule agonist in water to prepare aqueous solutions with different concentration gradients of 1-10mg/mL, uniformly mixing the aqueous solutions through thermodynamic actions (hydrogen bonds, van der Waals force and secondary structures) among epitope polypeptides and hydrophobic interactions and pi-pi accumulation among the small molecule agonists, and incubating the mixture for 20 minutes at 4 ℃ to obtain the self-assembled nano epitope vaccine taking the aqueous solution as a macroscopic expression form.
Example 4: physical characterization of self-assembled epitope vaccines bound to small molecule agonists.
The assembly structure and size of the self-assembled epitope vaccine bonded with the small molecule agonist are observed and characterized by a Transmission Electron Microscope (TEM) (figure 7) and Dynamic Light Scattering (DLS) (figure 8). The result shows that the epitope polypeptide bonding matter bonded with the small molecule stimulant realizes self-assembly in aqueous solution to form a nano structure.
Example 5: in vitro activity studies of self-assembled epitope vaccines bound to small molecule agonists.
C57/Bl6 mouse bone marrow cells were isolated, GM-CSF and IL-4 were added to the culture medium and cultured for 6 days, immature Dendritic Cells (DCs) were harvested, PBS (Control, negative Control), naked Epitope (Epitope), Epitope vaccine with bound agonist (Epitope-aginst) and LPS (positive Control) were added to the culture medium to stimulate the immature DCs cells to differentiate into mature DCs cells, 24 hours later the expression of the major surface markers CD80, CD86 and CD40 of the mature DC cells was detected by flow cytometry (zFACS), and the secretion of cytokines (IL-6, IL-12p40, IFN-. gamma.and TNF-. alpha.) was detected by ELISA kit. FIG. 9 is a graph of in vitro induction of DCs maturation, which shows that the epitope polypeptide vaccine bonded with small molecule agonist can effectively promote induction of DCs cell maturation activation.
Example 6: the immune activity of the self-assembled epitope vaccine bound to the small molecule agonist was evaluated in vivo.
C57/BL6 mice were selected and the epitope vaccine was injected subcutaneously around the inguinal lymph node of the mice, once a week for a total of 3 injections. Separating subcutaneous tissues, lymph nodes and spleens, preparing single cell suspension, using DCs and T cell receptor specific antibody for marking, analyzing the conditions of in-situ T cell recruitment and T cell stimulation and proliferation in the spleens and the reflux lymph nodes by a flow cytometer, simultaneously detecting the activation and maturation conditions of the DCs in the reflux lymph nodes, and using a kit for detecting the expression level of related cytokines (IL-2, IL-4, TNF-alpha and IFN-gamma) in serum. FIG. 10 is a graph showing the induction of maturation of DCs in lymph nodes of a mouse, and it can be seen that the epitope polypeptide vaccine bonded with the small molecule agonist can induce the maturation of DCs cells in vivo and promote the reflux of mature DCs to lymph nodes to further activate cellular immune responses in vivo.
Example 7: and (3) investigating the in vivo anti-tumor effect of the self-assembled epitope vaccine bonded with the small molecule agonist.
Selecting C57/BL6 mice, injecting tumor cells subcutaneously to construct a tumor-bearing mouse model, and connecting the epitopeThe vaccine was injected subcutaneously around the inguinal lymph node of the mice once a week for a total of 3 times. The mice are weighed every two days, the tumor volume is measured, and the tumor inhibition rate is calculated. Detecting changes in indicators of relevant immune responses (CD 8)+The improvement of the T cell ratio and the reduction of the Treg cell ratio, the activation of NK cells and the like), and the pathway and the intrinsic mechanism of immunotherapy are determined. Figure 11A shows normal body weight growth during treatment of mice, indicating that the vaccine is biologically safe and free of significant toxic side effects. The tumor growth of the small molecule agonist-conjugated vaccine group in fig. 11B was significantly inhibited, and the therapeutic effect was superior to other naked epitope groups and the epitope and agonist physically mixed group. FIG. 11C shows infiltrating lethal CD8 in tumor tissue after treatment with the conjugated small molecule agonist vaccine+The proportion of T cells is obviously improved, and further the vaccine is proved to activate CD8 in vivo+T cells respond to the immune response and exert an anti-tumor effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A small molecule agonist bonded self-assembly epitope vaccine is characterized in that a small molecule agonist is connected to an antigen epitope peptide in a covalent bond mode, epitope polypeptide bonded with the small molecule agonist is dissolved in aqueous solution, and the self-assembly epitope vaccine is formed into the aqueous solution vaccine with a nanostructure.
2. The small molecule agonist-bonded self-assembled epitope vaccine of claim 1, wherein the small molecule agonist comprises a TLR agonist and a STING agonist.
3. The self-assembled epitope vaccine of claim 1, wherein the small molecule agonist is covalently bonded by disulfide bond.
4. The small molecule agonist-bonded self-assembling epitope vaccine of claim 1, wherein the small molecule agonist and epitope are hydrophilic and hydrophobic.
5. The small molecule agonist-bonded self-assembled epitope vaccine of claim 1, wherein the epitope peptide comprises a B cell antigen and a T cell antigen.
6. The small molecule agonist-bonded self-assembled epitope vaccine according to claim 1, wherein the molar ratio of the epitope to the small molecule agonist is 1-5: 1-3.
7. A preparation method of a self-assembled epitope vaccine bonded with a small molecule agonist is characterized by comprising the following steps:
(1) constructing an epitope polypeptide sequence by Fmoc solid phase synthesis;
(2) screening small molecule agonists with physicochemical properties matched with epitope peptides;
(3) modifying dihydroxydisulfide on the small molecule agonist;
(4) covalently bonding the modified small molecule agonist to an epitope peptide;
(5) dissolving the epitope polypeptide bonded with the small molecule agonist in water to form a solution, and forming a nano structure through self-assembly to obtain the self-assembly epitope vaccine bonded with the small molecule agonist in a macroscopic expression form of aqueous solution.
8. The method for preparing the small molecule agonist bonded self-assembled epitope vaccine according to claim 7, wherein the concentration of the small molecule agonist bonded epitope polypeptide aqueous solution is 1-10 mg/ml.
9. The method for preparing the small molecule agonist bonded self-assembled epitope vaccine according to claim 7, wherein the particle size of the small molecule agonist bonded self-assembled epitope vaccine is 10-500 nm.
10. Use of the small molecule agonist-bound self-assembled epitope vaccine of claim 1 in the field of prophylactic and therapeutic vaccines.
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| CN107157933A (en) * | 2017-05-04 | 2017-09-15 | 同济大学 | A kind of albumen self assembly novel nano vaccine and preparation method thereof |
| CN108714213A (en) * | 2018-06-04 | 2018-10-30 | 北京工业大学 | The preparation method and application of a kind of nanometer adjuvant of self assembly and the nano vaccine formed by the adjuvant |
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