Immune carrier microsphere loaded with individualized MHC-II binding polypeptide and vaccine preparation and application thereof

Abstract

The invention relates to the technical field of immune carriers, in particular to immune carrier microspheres loaded with individualized MHC-II binding polypeptide and vaccine preparation and application of the immune carrier microspheres loaded with the individualized MHC-II binding polypeptide. The core microsphere is loaded with individualized MHC-II binding polypeptide, the sequence of the MHC-II binding polypeptide is obtained by predicting and screening based on an HLA genotyping result, and each HLA allele corresponds to at least one high-affinity MHC-II binding polypeptide; and the shell is a glucan or other polymer coating layer. The preparation method has the advantages that the T epitope and the B epitope are separately subjected to immune competitive inhibition inside and outside the microspheres, so that a better immune effect is obtained. The antibody avoids cross reaction side effects; carrier molecule diversity is reduced, and side effects caused by T cell over-activation are avoided; th1 epitopes and Th2 epitopes can be contained in the microspheres, so that antibody immunity and T cell immunity functions are generated; the particle size of the microspheres is controllable, and the immunologic function can be achieved without adjuvants.

Description

Technical Field

[0001] This invention relates to the field of immune carrier technology, specifically to immune carrier microspheres loaded with personalized MHC-II binding peptides and their vaccine preparation and application. Background Technology

[0002] I. Advances in Therapeutic B-cell Vaccine Research Monoclonal antibody technology was invented in the 1970s, a milestone in medical history that plays an irreplaceable role in disease detection and treatment. In this century, human monoclonal antibody technology has matured, leading to a surge in the market launch of therapeutic antibody products, with many more in clinical trials and preclinical research. This indicates that antibodies offer even greater possibilities for disease treatment.

[0003] B-cell vaccines can generate antibodies in the human body for a long period of time, showing promising application prospects, but few products have achieved success in treatment and have been marketed. The most researched area is cancer treatment, and the only currently marketed antibody vaccine is CIMAvax-EGF from Cuba. This vaccine induces the body to produce EGF antibodies through epidermal growth factor (EGF), blocking the binding of EGF to receptors on the surface of cancer cells, thereby inhibiting the growth and proliferation of cancer cells. CIMAvax-EGF provides a new option for adjuvant therapy of non-small cell lung cancer, bringing hope to patients. Its deeper significance lies in providing a successful example for the development of therapeutic cancer vaccines, demonstrating the feasibility of fighting cancer by inducing the body to produce specific antibodies, and pointing the way for future research.

[0004] For benign diseases, there are many monoclonal antibodies available for treatment, while therapeutic vaccines are mainly used for viral infections, and most are still in clinical trials. Encouragingly, a therapeutic vaccine for hypertension, after nearly 80 years of effort, has entered Phase III clinical trials and is expected to be available domestically and internationally in a few years. This progress represents a potential new treatment option for hypertension patients and is a milestone in therapeutic vaccines.

[0005] Antibodies exert their therapeutic effects in the body in three modes: 1. The products of antigen-antibody specific binding are not directly excreted from the body, but are taken up and broken down by phagocytes. This process not only helps to clear antigen-antibody complexes, but also releases antigenic peptides, further stimulating T-cell responses and enhancing the function of the immune system (e.g., COVID-19 vaccines). 2. They prevent functional molecules from binding to receptors, inhibiting their function (e.g., EGF vaccines). 3. They bind to antigens on tumor cell membranes, killing tumor cells through the ADCC effect (e.g., Her2 vaccines).

[0006] II. Molecular Immunological Basis of B-cell Vaccines and Vectors B cells require a dual-signal activation process to produce antibodies. The first signal is the binding of the antigenic epitope (B epitope) to the B cell receptor (BCR), a process that determines the antibody's specificity. The second signal is the induction of cytokines by the antigen's Th epitope to release from T cells.

[0007] Exogenous antigens are internalized into B cells after binding to B cell receptors. Intracellularly, antigen molecules are hydrolyzed into polypeptide fragments in lysosomes and endosomes, which then bind to MHC-II molecules on B cells and are displayed on the B cell surface. These MHC-II-bound polypeptide epitopes (also known as Th epitopes or T epitopes) are presented to the T cell receptors (TCRs) of CD4+ T cells. Subsequently, cytokines are released, activating B cells and determining their fate in terms of B cell proliferation and antibody production.

[0008] MHC-II binding peptides and Th epitope peptides are the same thing. For B cells or dendritic cells, they are called MHC-II binding peptides, while for T cells, they are called T epitope peptides. Generally, exogenous proteins contain both B and Th epitopes, and antibodies can be produced when animals and humans are immunized.

[0009] Therapeutic vaccines primarily use endogenous proteins or peptides as antigens. During T cell development, T cells capable of recognizing their own (endogenous) antigens are often cloned and eliminated, leading to a lack of T cells capable of assisting in the production of antibodies against these antigens. Therefore, it is necessary to link endogenous antigen molecules to exogenous immunocarrier molecules to provide exogenous Th epitopes that can be recognized by T cells, thereby breaking immune tolerance and generating antibodies. Traditionally used exogenous molecules include OVA, BSA, KLH, bacteriophages, or adenoviruses, which can provide sufficient diversity of MHC-II binding peptides to accommodate the diversity of HLA in animals or humans.

[0010] Peptide antigens typically require conjugation with an exogenous carrier (providing an MHC-II-binding peptide as a T epitope) to achieve an effective immune response. After Th2 cell receptors recognize the peptide epitope bound to the exogenous carrier protein, Th2 cells are activated and proliferate, subsequently releasing cytokines. These cytokines act as a second signal for B cell activation, activating B cells to enter a proliferative program and acquire the ability to secrete high-affinity, high-titer antibodies through somatic mutation mechanisms. However, resting Th cells themselves cannot meet the requirements for B cell activation. Therefore, dendritic cells (DCs) are needed to phagocytose antigens and present Th epitopes to Th cells via MHC-II, thereby activating Th cells.

[0011] Therefore, it can be seen that the secretion of antibodies by B cells is actually a complex process involving two signaling molecules (antigen molecules and carrier molecules) and three types of cells (B cells, Th cells and DC cells).

[0012] Dendritic cells (DCs) tend to engulf particulate antigens, making it difficult to generate high-titer antibodies from soluble proteins. A traditional approach is to formulate soluble proteins into emulsion particles or adsorb them onto adjuvant particles to obtain higher antibody titers.

[0013] When endogenous proteins or peptides are coupled with exogenous carrier molecules, high-titer antibodies against the exogenous carrier molecules are produced. Endogenous weak antigen molecules are inhibited by immune competition from exogenous strong antigen molecules, resulting in very low antibody titers.

[0014] III. Risks and Countermeasures of Therapeutic Vaccines 1. Implications regarding the side effects of COVID-19 vaccines COVID-19 vaccines produce antibodies against the Covid-19 virus's S protein, blocking the virus's binding to the ACE2 receptor and preventing infection. In terms of antibody titers, mRNA vaccines produce the highest titers, ranging from hundreds to thousands, adenovirus expression vaccines from several hundred to over a thousand, and inactivated virus vaccines from several hundred. Literature reports that the side effects of COVID-19 vaccines exceed the common range of traditional vaccines. Traditional vaccines primarily cause local symptoms at the injection site, while COVID-19 vaccines have a relatively high rate of systemic symptoms, and a small number of individuals experience long-term side effects in the immune, nervous, and cardiovascular systems.

[0015] The most common side effects reported in the literature include fatigue, headache, myalgia, fever, pain, or redness at the injection site, accompanied by mild to moderate symptoms. On the other hand, some rare adverse events have also been reported, such as allergic reactions after mRNA vaccine administration, and thrombosis and thrombocytopenia after non-replicating viral vector vaccine administration. Other rare adverse events described include myocarditis, Bell's palsy, transient myelitis, Guillen-Barre syndrome, recurrent herpes zoster, autoimmune attacks, seizures, and orthostatic tachycardia.

[0016] The cause is speculated to be related to the use of higher doses of antigen / carrier in pursuit of higher protective efficacy, leading to an overly strong immune response. For therapeutic vaccines, the required antibody titers may be even higher, and if there are strong side effects similar to those of COVID-19 vaccines, it will seriously affect their safety and clinical application prospects.

[0017] 2. Analysis of the sources of vaccine side effects Currently, epitope peptides are commonly used in the preparation of vaccine antigens, exhibiting good specificity. However, the potential for side effects requires further investigation. Generally speaking, vaccine side effects should originate from components other than the antigen molecule: carrier molecules and vaccine adjuvants.

[0018] (1) Antibody cross-reactivity with target antigen: For example, studies have shown that antibodies against SARS-CoV-2 S1-RBD may cross-react with the ACE2 protein in the human body [Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2]. Serum autoantibody levels against ACE2 in COVID-19 patients were significantly higher than in controls and correlated with disease severity. However, the mechanism by which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection is unclear. In this study, increased antibodies against ACE2 were confirmed in COVID-19 patients, and a positive correlation was found between the amount of antibodies against ACE2 and S1-RBD. Furthermore, after pre-adsorbing serum with S1-RBD, the binding of antibodies to ACE2 in the serum of some COVID-19 patients was significantly reduced, indicating that antibodies against S1-RBD can cross-react with ACE2.

[0019] (2) B cells produce antibodies against vaccine-ineffective components: Taking the COVID-19 vaccine as an example, only antibodies that bind to the S protein and ACE2 receptor can effectively block viral transmission. Producing antibodies against other components of the viral structural proteins is not only ineffective but can also cause cross-reactions. Inactivated virus vaccines and adenovirus vaccines produce high-titer membrane protein antibodies, and even S protein vaccines can produce antibodies against irrelevant target epitopes. Antibody specificity is relative; irrelevant polyclonal antibodies can cross-react with normal tissue molecules, ADCC effects can kill irrelevant cells and tissues, and there are even antibody-mediated infections of cells without ACE2 receptors, i.e., ADE (antibody-dependent enhancement).

[0020] Current research on vaccine preparation using peptides as antigens often shows that conjugating peptides to KLH vector molecules can achieve good immunization effects. However, the antibody titers against KLH are much higher than those against the antigen molecules, and the side effects of antibody cross-reaction cannot be determined.

[0021] (3) T cell overactivation: Each person has at most 6 types of MHC-II molecules, which can bind to a limited number of polypeptides to present activated T cells to assist in antibody production. The ultra-large exogenous immune carrier macromolecules, in addition to irrelevant antibodies, can present vaccine carrier molecular components to activated T cells through the MHC of B cells and DC cells. The activated Th cells produce excessive cytokines, and the activated CD8 cytotoxic T cells damage normal tissues, producing side effects.

[0022] Taking KLH as an example, immunization can cause T cells to secrete excessive amounts of cytokines, leading to systemic side effects. KLH is considered a safe immunogenic carrier molecule for clinical use. In fact, KLH is an ideal antigen for inducing DTH. Keyhole limpethemocyanin (KLH) is a highly immunogenic protein macromolecule that enables dendritic cells to recognize Th1 cells. It is an ideal antigen for inducing late-stage DTH. KLH is injected into mice, and a few weeks later, it is re-injected locally. The DTH response is assessed by the relative swelling at the injection site.

[0023] Following KLH immunization, IL-6 secretion increases significantly, ranging from 4 to 16 times. IL-6 is an important inflammatory cytokine, and its elevation is usually associated with the activation of the immune response. IL-6 can contribute to the development of chronic inflammatory diseases (such as rheumatoid arthritis) and cytokine storms, which occur in patients undergoing CAR-T therapy and in various diseases including those infected with SARS-CoV-2.

[0024] Following KLH immunization, IFN-γ secretion increased 21-fold, ranging from 5 to 45-fold. IFN-γ is an important immunomodulatory factor. Possible side effects of interferon include: injection site pain, inflammation, fever, chills, headache, myalgia, joint pain, fatigue, dizziness, anorexia, diarrhea, weight loss, leukopenia, thrombocytopenia, mild hair loss, oral ulcers, transient rash, delusions, depression, decreased attention, insomnia, anxiety, irritability, and autoimmune diseases such as autoantibodies, thyroid disease, diabetes, blurred vision, dry eyes, eye inflammation, hearing loss, menstrual disorders, decreased libido, pharyngitis, and rhinitis. Following KLH immunization, TNF-α secretion increased significantly, ranging from 5 to 50 times. TNF-α is an important inflammatory cytokine associated with various inflammatory responses.

[0025] C-reactive protein (CRP): Although the relationship between KLH and elevated CRP is not directly mentioned, an increase in IL-6 usually induces CRP production. Therefore, KLH-induced increases in IL-6 may indirectly lead to elevated CRP levels. These cytokines and inflammatory molecules can cause systemic reactions such as fever, headache, muscle aches, joint pain, and gastrointestinal discomfort, and can produce allergic reactions. The cytokines involved include IL-4, IL-5, and IL-13, and the measurable chemicals include histamine, trypsin inhibitors, leukotrienes, and prostaglandin D.

[0026] Attenuated vaccines, such as adenovirus vector vaccines (VLP vaccines), theoretically produce results similar to KLH.

[0027] The antigens expressed by mRNA vaccines are relatively simple, but their delivery systems (such as lipid nanoparticles, LNPs) and their own immune-activating properties may also be one of the causes of side effects.

[0028] 3. Corresponding measures (1) Use short polypeptide antigens instead of large protein antigens as much as possible to improve antibody specificity and reduce cross-reaction.

[0029] (2) Avoiding the generation of high-titer antibodies from carrier molecules: The immune carrier molecules are placed inside the microspheres to prevent the carrier molecules from binding to B cell receptors and generating antibodies. Two patents have been applied for: Immunospheres for overcoming B cell immune tolerance and their applications (application number: CN201010571277.1); and Sufficiently diverse amphiphilic MHC II-binding peptides, immune carrier microspheres, their preparation methods and applications (patent number: ZL201910115133.6). This patent is an improvement on the microsphere technology process.

[0030] (3) Avoid using ultra-large exogenous molecules as immune vectors To address the HLA diversity of the population, traditionally, macromolecules such as BSA, OVA, KLH, and viruses, as well as a wide variety of random peptides, are used as carrier molecules. This invention selects high-affinity MHC-II binding peptides instead of macromolecules or viral vectors, thus avoiding systemic side effects caused by excessive T cell activation.

[0031] (4) Design of personalized MHC-II binding peptides Based on the HLA assay results of the recipients, individualized combinations of MHC-II binding peptides were prepared to meet the antibody production requirements with a small number of high-affinity peptide molecules, avoiding the side effects of irrelevant molecules.

[0032] 4. Feasibility Analysis In tumor cell vaccine therapy, based on HLA-I assays, personalized MHC-I binding antigen epitope peptides have been used. HLA assay technology is mature, widely available, and reasonably priced.

[0033] HLA-II has three heritable alleles: HLA-DR, HLA-DP, and HLA-DQ. Allele mutations and their inheritance exhibit ethnic and regional characteristics. Globally, there are thousands of HLA-II alleles, but the diversity and dominant frequencies within the Han Chinese population are as follows: HLA-DR: HLA-DRB1*09:01: Approximately 15%-25% HLA-DRB1*12:02: Approximately 10%-20% HLA-DRB1*15:01: Approximately 5%-15% HLA-DRB1*04:05: Approximately 5%-10% HLA-DRB1*07:01: Approximately 5%-10% HLA-DRB1*08:03: Approximately 3%-8% HLA-DRB1*11:01: Approximately 3%-8% HLA-DRB1*14:05: Approximately 2%-5% HLA-DRB1*01:01≤5% HLA-DRB1*03:01≤5% HLA-DRB1*13:01 ≤ 5% or more of the 11 types account for more than 95% of the population.

[0034] HLA-DPB1 exhibits high diversity, with common subtypes and their approximate proportions as follows: HLA-DPB1*05:01: Approximately 20%-30% HLA-DPB1*02:01: Approximately 15%-25% HLA-DPB1*04:01: Approximately 10%-20% HLA-DPB1*03:01: Approximately 5%-10% HLA-DPB1*09:01: Approximately 5%-10% Other subtypes (such as HLA-DPB1*13:01, HLA-DPB1*14:01, etc.): usually account for less than 5% of the HLA-DQB1 subtype abundance. The diversity of the HLA-DQB1 gene is higher. The common subtypes and their abundance in the Han Chinese population are as follows: High-frequency subtype: HLA-DQB1*03:01: Approximately 20%-30% HLA-DQB1*03:03: Approximately 15%-25% HLA-DQB1*06:01: Approximately 10%-20% Mid-frequency subtype: HLA-DQB1*02:01: Approximately 5%-10% HLA-DQB1*05:01: Approximately 5%-10% HLA-DQB1*06:02: Approximately 5%-10% Personalized carrier microspheres do not require thousands of designs; 30-50 designs are sufficient for over 95% of the population. The design of MHC-II binding peptides can be achieved using online software, with similar results across different programs. Peptide synthesis technology is mature and inexpensive. This invention is easy to operate, exhibits good reproducibility, and the microsphere preparation method is simple and reproducible.

[0035] MHC-II binding peptide prediction: With the development of sequencing technology, the known diversity of HLA genes is increasing every year. HLA-II gene mutations and corresponding peptide sequences are well known, and there is years of experience in predicting the sequences of binding peptides. Several sequence prediction software programs are available: IEDB ProPred16, MHCPred17, SVRMHC18, ARB19, SMM-align20, and NetMHCIIpan. IEDB and NetMHCPan are relatively easy to use. The prediction results for HLA-DR are generally consistent, but there are significant differences for HLA-DQ and HLA-DP. NetMHCIIpan, which has undergone several upgrades (such as version 4.3), is considered more reliable and is frequently cited in the literature.

[0036] In addition, computer-designed peptides such as AKFVAAWTLKAAA, TT830–844ILMQYIKANSKFIGI, and TT947-967 diphtheria toxin QSIAISSLMAQAIP can bind to a variety of MHC-II molecules, and this universal Th epitope can serve as a supplement. Summary of the Invention

[0037] The purpose of this invention is to overcome the immune competition inhibition between vaccine carrier molecules and antigen epitopes, improve antibody titers, avoid the side effects caused by the production of carrier molecule antibodies, avoid the side effects of excessive T cell activation, adapt to human HLA diversity, and use personalized MHC-II binding polypeptide microspheres to replace immune carrier macromolecules containing sufficient MHC-II diversity: OVA, BSA, KLH, bacteriophages, viruses or single MHC-II molecules.

[0038] To achieve the above objectives, an immune carrier microsphere loaded with personalized MHC-II binding peptides is provided. The microsphere has a core-shell structure, comprising a core and an outer shell. The core microsphere is loaded with 3-12 personalized MHC-II binding peptides. The MHC-II binding peptide sequences are obtained through computer prediction and screening based on the HLA genotyping results of the recipient, and each HLA allele corresponds to at least one high-affinity MHC-II binding peptide. The outer shell is a coating layer of dextran or other hydrophilic polymers with thiol groups, whose thiol groups serve as coupling groups to connect antigens for vaccine construction, achieving internal and external isolation of the T / B epitope microspheres.

[0039] Preferably, the immune carrier microspheres contain 3-12 high-affinity MHC-II binding peptides screened by computer and at least one universal MHC-II binding peptide for assisting B cells in antibody production. The universal MHC-II binding peptide is selected from at least one of the following: YFAVYLQETPY (SEQ ID NO: 1), AKFVAAWTLKAAA (SEQ ID NO: 2), QYIKANSKFIGITEL(SEQ ID NO:3), FNNFTVSFWLRVPKVSASHLE(SEQ ID NO: 4), AWLEAQEEEEVGF (SEQ ID NO: 5), QYIKANSKFIGITELKK (SEQ ID NO: 6), EPRAPWIEQEGPEYWDQE(SEQ ID NO:7), ADVEVYRAVTPLGPPD (SEQ ID NO: 8), DTLRSYYADWYQQKPG (SEQ ID NO: 9), FVNQHLAGSHLVEAL (SEQ ID NO: 10), LNEDLRSWTAADTAA (SEQ ID NO: 11).

[0040] Preferably, the MHC-II binding peptide is obtained by HLA typing and screening using IEDB ProPred, MHCPred, SVRMHC, ARB, SMM-align, NetMHCIIpan, IEDB and NetMHCPan prediction software, and through affinity scoring.

[0041] Preferably, the microspheres have a particle size of 0.1-5 micrometers.

[0042] This invention also provides a method for preparing immune carrier microspheres loaded with personalized MHC-II binding peptides, comprising the following three methods: MHC-II binding peptide self-assembled microspheres are coated with dextran or other hydrophilic polymers; Biospheres containing personalized MHC-II binding peptides are coated with dextran or other hydrophilic polymers. mRNA microspheres expressing MHC-II binding peptides without signal peptide sequences.

[0043] The present invention also provides a vaccine prepared from immunogenic carrier microspheres loaded with personalized MHC-II binding peptides, wherein the T / B epitopes are separately placed inside and outside the microspheres to avoid competitive inhibition of T / B epitopes, and the T epitopes are located inside the microspheres.

[0044] Preferably, the microspheres are loaded with Th2 epitope peptides, and may also be loaded with Th1 epitope peptides, for the preparation of a bifunctional T and B cell vaccine with antibody and cell-mediated immunity effects.

[0045] This invention also provides the application of a vaccine prepared from immunocarrier microspheres carrying personalized MHC-II binding peptides in the preparation of drugs for tumors, cardiovascular diseases, hyperglycemia, allergic diseases, autoimmune diseases, Alzheimer's disease, viral infections, drug addiction, weight loss, and other chronic diseases that can be treated with antibodies.

[0046] Compared with the prior art, the advantages of this invention are: 1. Vaccines prepared using carrier microspheres exhibit higher antibody titers upon inoculation. This invention is based on the separate placement of T and B cell epitopes inside and outside the microspheres, avoiding competition between exogenous carrier molecules and antigenic epitopes, which could lead to inhibition.

[0047] 2. The carrier molecules are packaged inside the microspheres, which will not produce high-titer antibodies against the exogenous carrier molecules, thus avoiding the side effects caused by antibody cross-reaction.

[0048] 3. Reduce the diversity of MHC-binding peptides to avoid over-activation of T cells and the resulting side effects.

[0049] 4. Microspheres can be equipped with Th1 and Th2 epitopes, which can generate both antibody immunity and T cell immunity.

[0050] 5. The microspheres have controllable particle size and can exert their immune function without adjuvants, making them more suitable for vaccines against benign diseases. Detailed Implementation

[0051] The present invention will be further described below with reference to the embodiments. The embodiments are for explanation only and are not intended to limit the scope of protection of the present invention.

[0052] Example 1: Design of Personalized MHC-II Binding Peptides (i.e., Th2 Epitope Peptides) 1. Personalized MHC-II binding peptide design: First, the HLA-II allelic subtype is determined by PSS-PCR in the recipient.

[0053] 2. Several computer applications for predicting MHC-binding peptides are available online: IEDB ProPred16, MHCPred17, SVRMHC18, ARB19, SMM-align20, and NetMHCIIpan. IEDB and NetMHCPan are relatively easy to use. Their predictions for HLA-DR are generally consistent, but they differ significantly for HLA-DQ and HLA-DP. NetMHpan C4.3, which has undergone several upgrades, is frequently cited in the literature, and more advanced applications are expected in the future.

[0054] Based on the gene name and number determined by HLA-II typing, the foreign protein sequence for prediction is pasted into a table for computer software to calculate and output a high-affinity MHC-II binding 15-peptide.

[0055] Typically, chicken ovalbumin, bovine serum albumin, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, HIV virus, measles virus, tetanus toxin, and KLH are used. The 15-peptide sequence with the highest score (high affinity) is selected from the results column.

[0056] In this embodiment, the NetMHCIIpan-4.2 software was used to predict MGHC-II binding peptide sequences online. Taking the HLA-IIDRB1*0901 gene as an example, more than 20 high-affinity MHC-II binding peptide sequences for DRB1*0901 expression were found by inputting TT and KLH protein sequences.

[0057] 3. Taking HLA-DRB10901 as an example, the results are shown in Table 1 below: Table 1 protein source Output MHC-II binding peptide Affinity rating HCV GPSYGFQYSPAERVD (SEQ ID NO: 12) 0.9621 KLH EDVFHPSEPFFIKVS (SEQ ID NO: 13) 0.9563 HAV MRNEFRVSTTENVVN (SEQ ID NO: 14) 0.9393 Taking HLA-DQA1*01:01 / HLA-DQB1*05:01 as an example, the results in Table 2 are obtained using NetMHCPan software: Table 2 protein source Output MHC-II binding peptide Affinity rating MVF DRINAFYKDNPHPKG(SEQ ID NO:15) 0.84 HAV VVNLSNYEDARAKMS 0.84 TT EVRQITFRDLPDKFN 0.84 The results in Table 3 below were obtained using NetMHCPan software with DPA1*01:03 / DPB1*04:01: Table 3 protein source Output MHC-II binding peptide Affinity rating HCV PDKEILYEAFDEMEE 0.97 KLH NEAIFQQTKFGEFSS 0.98 TT LYTSYLSITFLRDFW 0.84 A high score indicates high affinity; a score of 0.9 or higher indicates affinity at the nM and pM levels.

[0058] 4. To provide a reference for the mouse experimental results, MHC-II binding peptides with high affinity for both humans and mice were selected. Each selected human high-affinity binding peptide sequence was further screened using affinity for MHC-II expression in Balb / c mouse H2-IAd and H2-IEd genes, identifying eight homologous sequences with high affinity as shown in Table 4 below: Table 4

[0059] The experimental mice were purebred animals with a low number of H2 alleles. The H2 gene polymorphism is as follows: The H2 alleles in Balb / c mice are classified into two classes: Class I (H2-Kd, H-Ld, and H2-Dd) and Class II (I-Ed and I-Ad). Using OVA, TT, and KLH sequences, the following H2I-Ed and H2I-Ad binding peptides can be obtained: Table 5: Balb / c mice H2I-Ad OVA LKISQAVHAAHAEIN (SEQ ID NO: 41) 0.8638 H2I-Ad TT YDTEYYLIPVASSSK(SEQ ID NO:42) 0.7569 H2I-Ad TT GTNEYSIISSMKKHS(SEQ ID NO:43) 0.2785 H2I-Ad KLH NIDFLDKKTNRAVDD(SEQ ID NO:44) 0.3590 The H2 alleles in C57BL / 6 mice are H2-Kd and H2-Kb in type I and I-Ab in type II. Table 6: C57BL / 6 mice H2-IAb TT VPYIGPALNIVKQG(SEQ ID NO: 45) 0.9531 H2-IAb TT TEYYLIPVASSSKDV (SEQ ID NO: 46) 0.8934 H2-IAb KLH DDEYAGSFVILGGAK (SEQ ID NO: 47) 0.8686 Example 2: Oxidized dextran coating and thiol group introduction Oxidized dextran was prepared by a commissioned company, and a molecular weight of 5-20kD and an oxidation degree of 20-50% were selected based on the size of the paper microspheres for coating MHC-II microspheres.

[0060] The purpose of dextran oxidation is to oxidize the hydroxyl groups of dextran to generate aldehyde groups, which then react with the amino groups on the microspheres to encapsulate the core microspheres. Excess aldehyde groups then react with mercaptoalanine to introduce thiol groups onto the dextran molecule. This is used for linking antigens to prepare peptide vaccines. Dextran oxidation (generating aldehyde groups) introduces aldehyde groups (-CHO) through the oxidation of the vicinal diol structure of dextran, providing an active site for subsequent coupling reactions.

[0061] Materials: Dextran T40, sodium periodate (NaIO4, 0.1 M), PBS buffer (pH 6.0-7.0), dialysis bags (molecular weight cutoff selected based on dextran). step: (1) Dissolve the dextran: Dissolve the dextran in PBS buffer (pH 6.0-7.0) to prepare a 5-10 mg / mL solution.

[0062] (2) Oxidation reaction: Add NaIO4 to a final concentration of 0.05-0.1M (protect from light, ice bath conditions) and stir at room temperature for 4-6 hours (or overnight at 4°C).

[0063] (3) Termination of reaction: Add ethylene glycol (final concentration 0.1M) to quench excess NaIO4 and continue stirring for 30 minutes.

[0064] (4) Purification: Transfer the solution to a dialysis bag and dialyze with PBS (pH 7.4) for 24 hours (change the solution 3 times) to remove unreacted reagents. Freeze-dry the oxidized dextran (labeled as Dex-CHO).

[0065] (5) Since DC cells have mannose receptors, a certain proportion of glucomannan is added to the glucomannan.

[0066] Example 3: Preparation of MHC-II-binding peptide self-assembled immunoglobulins 1. Peptide self-assembling microspheres (1) Amphiphilic sequence design for personalized MHC-II binding peptide self-assembly microspheres The self-assembling peptide method is largely the same as that in our Chinese patent application ZL 201910115133.6. The difference is that in the aforementioned patent, MHC-II binding peptides are linked together with antigenic peptides to prepare self-assembled microspheres, while the improvement of this invention is that it is limited to preparing self-assembled microspheres from MHC-II binding peptides, which are then encapsulated with dextran to stabilize them and used for antigen linkage.

[0067] For the self-assembly of MHC-II binding peptides, a hydrophobic sequence with a cysteine ​​residue needs to be introduced at the N-terminus, and a charged hydrophilic sequence needs to be introduced at the C-terminus, making it an amphiphilic peptide. In addition to hydrophobic binding, the cysteine ​​residue at the N-terminus forms a disulfide bond to stabilize the microspheres. Examples include: FVFLFCLSG (SEQ ID NO: 17) and Pal-FCLSG (SEQ ID NO: 18). The positive and negative charges of the two peptides at the hydrophilic ends attract each other, and the terminal Lys amino group reacts with the oxidized dextran aldehyde group. Two examples are shown below: KSDKSDK (SEQ ID NO: 19) and DSKDSKD (SEQ ID NO: 20).

[0068] (2) Peptide self-assembly scheme and selection of globularity of amphiphilic MHC-II binding peptides There are two possible outcomes: classic self-assembly forms microspheres of 20-200 nm, while the literature more often shows fibrous structures. This invention involves dissolving an amphiphilic MHC-II-binding peptide in a 50% acetonitrile / water solution (insoluble in water), then evaporating the acetonitrile under stirring conditions using vacuum, resulting in spherical particles of 1-10 micrometers in water with good dispersibility. Particle size can be controlled by adjusting the pH and adding certain components.

[0069] Not all amphiphilic peptides can self-assemble into microspheres using the method described below. After setting the end sequences, an MHC-II binding peptide is loaded in the middle, and its solubility in water is analyzed using algorithms or software. From the above eight DRB10901 MHC-II binding peptides, the following three sequences were selected as expected to dissolve in a 50% acetonitrile aqueous solution, and after removing the acetonitrile, precipitated into microspheres in water. Examples are as follows: VFLFCL-FDNVIENITSLTIGK-DSKDSKD (SEQ ID NO: 21) FVFLFCL-HLKPFAFSSPLNNNE-SDKSDK(SEQ ID NO: 48) FVFLFCL-FYLHHSNTDRIWAIW-DSKDSK(SEQ ID NO: 49) Experimental Results: Using the first and second sequences described above, a company was commissioned to synthesize the following: 5 mg of each peptide was dissolved in 5 ml of 50% acetonitrile / PBS and placed in a 50 ml round flask. The flask was gently shaken and then aerated. After 10 minutes, 3-7 micrometer spherical precipitates were observed under a microscope, indicating good dispersion.

[0070] (3) MHC-II self-assembled microspheres encapsulated with dextran The purpose of encapsulating MHC-II binding peptide microspheres with dextran is primarily to separate the MHC-II peptide from the antigenic peptide, preventing the formation of antibodies against the MHC-binding peptide and ensuring the stability and non-lysis of the encapsulated microspheres. This also avoids side effects associated with the microsphere material. All types of MHC-II binding peptide immunocarriers are designed with free amino groups on their surface for subsequent reactions. The hydroxyl groups of dextran are oxidized to aldehydes, which react with the amino groups of the MHC-II binding peptide to achieve the function of the encapsulated microspheres.

[0071] Materials: Oxidized dextran with different molecular weights and oxidation degrees is available commercially or can be customized to meet specific needs. Selection of oxidized dextran molecular weight for encapsulation: 100-150 KD for micron-sized microspheres, 50 KD for nano-sized microspheres, and 5-10 KD for molecular-sized microspheres. The oxidation degree should be 20-30%. Oxidized dextran is soluble in water, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, and DMF.

[0072] Oxidized dextran-encapsulated microspheres: Oxidized dextran is covalently bound to the surface of MHC-II-binding polypeptide microspheres by reacting aldehyde groups with the amino groups of proteins or polypeptides.

[0073] In an aqueous solution, MHC-binding polypeptide microspheres were suspended in phosphate buffer (pH 7.8) (concentration 1-5 mg / mL), and oxidized dextran solution (final concentration 1-2 mg / mL) was added. The mixture was stirred at room temperature for 4-6 hours. Unbound Dex-CHO was removed by centrifugation (5000 rpm, 10 min). The microspheres were washed three times with pH 7.4 PBS to obtain Dex-CHO-coated protein microspheres. Introducing thiol groups: The remaining aldehyde groups on the oxidized dextran react with mercaptopropylamine (final concentration 5-10 mM) to introduce thiol groups of the coupled antigen onto the microsphere surface. Excess mercaptopropylamine is removed by centrifugation and washing in the presence of a reducing agent. NaBH3CN (final concentration 5-10 mM) is added, and the reduction reaction is carried out at 4°C for 2 hours to stabilize the Schiff base.

[0074] Thiol group determination and thiol group blocking: Reacting with Ellmans reagent at a relatively high pH (8.0) produces a yellow color, allowing for the calculation of the number of thiol groups using a colorimetric method. The microspheres are designed to have 100-5000 thiol groups. Under these conditions, nitrobenzoic acid is attached to the surface of the microspheres, making the surface negatively charged and preventing aggregation. The number of thiol groups on the microsphere surface is controlled between 300-500.

[0075] The chemical reactions involved in microsphere encapsulation and surface modification are all commonly used chemical reactions in the literature, so there is no need to provide specific details of the reaction conditions here.

[0076] 2. PLGA-PEG microspheres The PLGA-PEG microspheres with thiol groups are produced by our company, and the MHC-II peptide with N-terminal maleyl groups is linked to the surface of the microspheres, which is also completed by our company.

[0077] Traditional bio-microsphere materials include various types: polysaccharides, proteins, and liposomes. These are used to encapsulate functional drugs, functional proteins, MHC-II-donating peptides, DNA, and mRNA expression systems through emulsification and self-assembly. Traditional bio-microspheres with surface-coupled thiol groups are available for fabrication using technical services provided by companies. Achieving both material safety and a suitable microsphere fabrication process remains a challenge.

[0078] 3. Human serum albumin microspheres MHC-II binding peptides are easily coupled and anchored to bio-nanospheres or polymeric materials. PLGA-PEG bio-nanospheres have been approved by the FDA, and polyhuman serum albumin has the highest safety profile. Two MHC-II peptides are synthesized by introducing 40 thiol groups per HSA molecule using conventional methods: one with a Mal group at the N-terminus, and one with a lysine amino group at the C-terminus, and the other with a biotin group at the C-terminus.

[0079] Two MHC-II binding peptides are coupled to HSA molecules linked to thiol groups in a 7:3 ratio. Each HSA molecule can link to 15-20 large peptide molecules. Excess thiol groups in the HSA are blocked with iodoacetic acid. All procedures can be performed by the peptide synthesis company. Avidin is then linked to form a pentamer, and unbound avidin is removed using a Sephandex G100 separator.

[0080] Encapsulation with oxidized dextran: Dextran, with a molecular weight of 20,000, was prepared by a commissioned company with an oxidation ratio of 15-20%. The aldehyde group of the oxidized dextran was linked to the amino group of the avidin-HSA-MHC-II binding peptide, and then separated using Sephadex G100. The thiol groups introduced into the encapsulated dextran were the same as in Example 2. Avidin has a tetrameric structure, with a monomer of 151 amino acids. Although it is a heterologous protein, the encapsulation prevents contact with B cells to generate antibodies, and the small molecular weight prevents the production of excessive fragments that activate T cells, allowing it to be used as a universal MHC-II binding peptide.

[0081] Each HSA molecule is linked to only one MHC-II binding polypeptide. In practice, 3-6 different polypeptides can be used to link antigens based on individualized assay results.

[0082] Example 4: Coupling of microspheres with antigenic peptides Traditional vaccines used pathogens (smallpox, cowpox), and later used proteins (SARS-CoV-2 S protein). These antigens produced a variety of ineffective and irrelevant antibodies, reducing vaccine specificity. In recent years, peptide fragments of antigens have been increasingly used as antigens to avoid producing ineffective antibodies and corresponding side effects.

[0083] The surface of the immunocarrier microspheres of this invention contains thiol groups for coupling, which react with maleimide-containing peptides for linkage. The coupling procedure with antigens containing N-terminal maleimide groups is as follows: microspheres are suspended in PBS, and mercaptoethanol or mercaptothreitol is added to unblock the blocked disulfide bonds, exposing the surface thiol groups for antigen linkage. After one minute, centrifugation is performed to remove mercaptoethanol and 4-mercapto-2-nitrobenzoic acid. Antigen-microsphere coupling is carried out in a nitrogen-purged, oxygen-free solution with added EDTA. The reaction is carried out under stirring conditions and incubated overnight at 4°C. After centrifugation and washing, unreacted thiol groups on the microspheres are blocked with iodoacetic acid, resulting in a negatively charged surface on the vaccine microspheres that prevents them from sticking together.

[0084] The microspheres have multiple thiol groups, allowing them to connect to more than one polypeptide antigen. They can be reduced and lysed using dithiothreitol (DTT) and separated and semi-quantitatively identified by the different isoelectric points of the polypeptides in non-SDS electrophoresis. Lysis conditions are: concentration: 20-100 mM; incubation conditions: 95-100°C, heating for 5-10 minutes.

[0085] Example 5: Personalized MHC-II binding peptide mRNA liposome immunocarrier microspheres mRNA vaccine technology has been applied to the market launch of COVID-19 vaccines. Current mRNA vaccines express protein antigens. In this invention, the mRNA expresses MHC-II binding peptides, which play an immune-carrying role in the production of antibodies.

[0086] The expressed MHC-II polypeptide mRNA does not contain a signal peptide and functions within the cell. Even though the secreted polypeptide is a hapten, it does not produce antibodies against the MHC-binding polypeptide molecule. A company was commissioned to prepare liposomes expressing the personalized MHC-II binding polypeptide mRNA. After the liposomes were prepared, thiol-containing SH-PEG-DMG was inserted; the thiol groups are used to link the antigenic polypeptide.

[0087] Example 6: Preparation of an anti-IgE peptide vaccine from MHC-II-binding peptide self-assembled microspheres Allergic diseases have become one of the three major diseases requiring significant effort to better control in the 21st century. Immunoglobulin E (IgE) is involved in asthma, rhinitis, food allergies, and drug allergies. When allergens bind to IgE antibodies, the Fc of IgE binds to the high-affinity receptor FcεRI on the surface of effector cells, causing rapid degranulation of mast cells and basophils, releasing stored histamine, and subsequently synthesizing and releasing prostaglandins, leukotrienes, cytokines, and other inflammatory mediators. These attract and activate inflammatory cells, producing allergic symptoms and upregulating IgE biosynthesis in B cells to promote increased sensitivity.

[0088] Omarizumab is currently the world's first approved humanized anti-IgE monoclonal antibody, blocking the binding of IgE to its receptor and used to treat asthma. An IgE-Fc receptor II (FcεR1) monoclonal antibody is still under development. Theoretically, IgE-targeted vaccines could provide similar clinical benefits to omalizumab. Research on preparing vaccines using IgE and its receptor as antigens to generate endogenous antibodies is underway in various countries. It is expected that the combined use of the two should yield even better results.

[0089] IgE Fc binds to the receptor, and there is multiple pieces of evidence that IgE binds to the receptor at position 329-435 of the CH3 region of the heavy chain, and can act as an antigen.

[0090] 329-344 (ADSNPRGVSAYLSRPSPGGC (SEQ ID NO: 50)); 417--432 (QCRVTHPHLPRALMRS (SEQ ID NO: 51)) Immunogenicity of a peptide-based anti-IgE conjugate vaccine in non-human primates 345-352 (PFDLFIRK (SEQ ID NO: 52)): Inhibition of passive sensitization of human peripheral basophiles by synthetic human immunoglobulin E peptide fragments 367-376 (KGTVNLTWSR (SEQ ID NO: 53)): A monoclonal anti-IgE antibody against an epitope in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23). 413-435:Synthetic IgE peptide vaccine for immunotherapy of allergy This invention uses the human IgE antigen fragment 413-435 sequence: GETYQCRVTHHLPRALMRSTTK (SEQ ID NO: 22), and commissioned a company to synthesize a maleimide-containing polypeptide, the corresponding binding polypeptide of the human DRB1-0901 gene: FVFLFCL-FDNVIENITSLTIGK-DSKDSKD (SEQ ID NO: 21) FVFLFCL-HLKPFAFSSPLNNNE-SDKSDK Self-assembled microspheres were constructed as immune vectors. An experimental vaccine was prepared by conjugation using the method described in Example 1 above. The IgE antigen peptide was conjugated to the microspheres and used as a vaccine for experimental research. Balb / c mice were immunized four times, and blood samples were collected six weeks later to determine the antibody titers (see Table 7).

[0091] Table 7. Antibody titers of IgE antigen peptides and self-assembled MHC-II peptides

[0092] Antigen peptide synthesis, conjugation to KLH for experimental vaccination of mice, and antibody titer determination are routine technical services provided by peptide synthesis companies. The immunization results of the IgE peptide-KLH conjugate are shown in Table 8. Table 8. Antibody titer results in mice after IgE-KLH peptide vaccine inoculation.

[0093] The results of the two experiments showed that both the IgE peptide antigen conjugated to the novel immunogenic carrier microspheres and KLH produced high-titer antibodies. The microsphere vaccine was more effective than the KLH protein vaccine, with a titer approximately four times higher. The more significant difference was that the antibody titers of the carrier molecules KLH and MHC-II binding peptides differed by more than 100 times.

[0094] Several antigenic polypeptide epitopes have been identified in the extracellular region of the high-affinity IgE receptor FcεRI on the surface of eosinophils and basophils. Several linear polypeptide epitopes, QCRVTHPHLPRALMRS (SEQ ID NO: 51) and ADSNPRGVSAYLSRPSPGGC (SEQ ID NO: 50), can serve as antigens, and the resulting antibodies can effectively block the binding of IgE to the receptor. Combining these two epitopes may yield even better results.

[0095] Example 7: Therapeutic Vaccine for Hypertension Hypertension is a symptom of a disease, primarily caused by vascular factors: atherosclerosis, narrowing of blood vessels due to plaque buildup, and neuroendocrine dysfunction. Hypertension is a leading cause of death worldwide.

[0096] Hypertension can be adequately controlled with existing medications. While many chemical drugs are available for treating hypertension, long-term use can cause various side effects. Drug therapy also faces challenges related to patient adherence. It is estimated that approximately 50% of patients do not follow their doctor's medication regimen. Poor medication adherence is a common problem among hypertension patients. Furthermore, patients with Alzheimer's disease or severe debility are unable to manage their medications independently.

[0097] Therefore, vaccination has the potential to be a useful treatment for hypertension, providing lasting effects and avoiding the problem of patients not adhering to medication regimens. Therapeutic vaccines for hypertension aim to control blood pressure long-term by inducing antibodies to neutralize endogenous pressor factors.

[0098] Angiotensin and angiotensin II receptor molecules form a highly specific system for hypertension. Small molecule chemical drugs targeting this are ACE inhibitors and angiotensin II receptor blockers (ARBs). Currently, a range of ARBs are used clinically to inhibit receptor binding, but side effects include liver and kidney damage, with irreversible renal impairment. Calcium channel molecules, such as dihydropyridine calcium channel molecules, work by inhibiting calcium ion entry into cells to lower blood pressure. Dihydropyridine calcium channel drugs have the highest market share in China. The main side effects are leg edema and allergic dermatitis.

[0099] Adrenaline-blocking antihypertensive drugs mainly include two categories: alpha-receptor blockers and beta-receptor blockers. They lower blood pressure by blocking adrenaline receptors. Bisoprolol and metoprolol primarily exert their antihypertensive effect by blocking beta-adrenaline receptors, inhibiting overactive sympathetic nerve activity, suppressing myocardial contractility, and slowing heart rate.

[0100] The British company Protherics attempted to develop a vaccine, PMD-3117, targeting Ang I, aiming to lower blood pressure by inducing the body to produce antibodies against Ang I and blocking the activation of the renin-angiotensin system (RAAS). However, in clinical practice, the antihypertensive effect was not significant. This may be because the antibody titer is insufficient to significantly lower blood pressure, and Ang I antibodies are mainly present in plasma, failing to effectively prevent the conversion of Ang I to Ang II in tissues. The Swiss company Cytos' CYT006-AngQb vaccine, targeting angiotensin II, was once considered a very promising antihypertensive vaccine. Its specific mechanism lies in the fact that the vaccine itself contains antigenic epitopes with a structure similar to Ang II.

[0101] In subsequent clinical trials, compared with the placebo group, the high-dose group of the vaccine reduced the subjects' average daily ambulatory blood pressure by -9 / -4 mmHg from the baseline level. Unfortunately, this reduction in blood pressure is not enough to fully meet the actual clinical requirements.

[0102] In addition, Nakagami's team at Osaka University and AnGes, a venture capital pharmaceutical company initiated by Osaka University, have jointly developed several Ang II vaccines, each an improvement on the original. According to information on the company's official website and public channels, the Ang II vaccine appears to have completed Phase II clinical trials in Australia several years ago. China's Angiotensin Receptor and Vaccine Development: On June 6, 2025, the Center for Drug Evaluation of the National Medical Products Administration formally accepted the clinical trial application for HJY-ATRQβ-001, the world's first therapeutic antihypertensive vaccine that selectively modulates angiotensin II type 1 receptor (AT1R), independently developed by Wuhan Huajiyuan Biotechnology Development Co., Ltd. The vaccine entering clinical trials is CYT006-AngQb from the Swiss company Cytos, which directly uses Ang II (8-peptide: DRVYIHPF (SEQ ID NO: 54)) conjugated to the carrier protein KLH as the vaccine, and clinical trials have shown efficacy.

[0103] Another vaccine that has entered clinical trials is ATRQβ-001, constructed using Qβ phage as a vector and conjugated with the Ang II receptor heptapeptide fragment CAFHYESQ (SEQ ID NO: 55). Clinical trials are underway, and it has shown efficacy in lowering blood pressure in Phase II trials. The calcium ion-targeting peptide CPAEEDPS (SEQ ID NO: 56), a Cav1.2 epitope, acts as an antigen molecule for hypertension vaccines by inhibiting L-type calcium channels and reducing calcium ion entry into cells. Combined vaccines against angiotensin II receptor type 1 and alpha 1D-adrenergic receptor for hypertension.

[0104] Hypertension is a disease requiring lifelong treatment. Antibody therapy targeting the aforementioned molecules and corresponding vaccines for hypertension has shown effective results in preclinical and early clinical studies. The key factor is not efficacy but safety. Research into therapeutic vaccines for hypertension has been ongoing for over seventy years since the 1950s, with no products yet on the market; vaccine safety is the primary concern.

[0105] Side effects may stem from the specificity of antibodies produced by the vaccine, but more importantly, they may arise from the side effects produced by the immune vector molecules. The production of high titers of exogenous macromolecular or viral antibodies is theoretically unacceptable for long-term use. This invention focuses on addressing the potential side effects of immune vector molecules.

[0106] In this embodiment of the invention, the antigen is a bifunctional therapeutic vaccine produced using angiotensin 1 (DRVYIHFHL (SEQ ID NO: 57)) polypeptide and its receptor molecular polypeptide: TVAFHYESQNS (178-189) (SEQ ID NO: 58). The conjugated antigen design is as follows: synthesized Mal-EEEAK-GGGSGGGS-DRVYIHFHL (SEQ ID NO: 59) / Mal-EEEAK-GGGSGGGS-TVAFHYESQNS (SEQ ID NO: 60).

[0107] Immunotransmitter molecule: The scheme in Example 3, human serum albumin multimer microspheres loaded with individualized MHC-II binding peptides.

[0108] Initial results of animal experiments: Balb / c mice were subcutaneously injected with 1 mg of aluminum adjuvant, followed by a booster vaccination two weeks later. Blood samples were collected seven weeks later for antibody titer determination using ELISA.

[0109] Table 9. Antibody titers of angiotensin-I peptide plating assay

[0110] Table 10. Antibody titers of angiotensin receptor peptide plating assay

[0111] Table 11 Antibody titers on carrier protein coating

[0112] Example 8: Liposome antitumor vaccine packaged with MHC-II binding peptide expression gene Traditional chemotherapy drugs for cancer are highly toxic and prone to drug resistance. Cancer therapeutic vaccines, ideally a safe and effective treatment option, represent the most researched and developed area of ​​therapeutic vaccine products. For vaccines treating benign diseases, safety is paramount; for cancer treatment vaccines, efficacy is key.

[0113] mRNA vaccine technology has been used in the marketing of COVID-19 vaccines. Although the materials used to package mRNA have some side effects, their use in cancer treatment vaccines should be acceptable.

[0114] Tumor antigens are all relatively specific endogenous components, requiring loading of somatic molecules to generate antibodies. Expressing MHC-II binding peptides on mRNA completely avoids the competitive inhibition of the immune system using traditional techniques, and promises to obtain high-titer antibodies for tumor treatment.

[0115] Tumor cells produce mutated T-cell antigens. Immunotherapy using mRNA-expressed T-cell antigens is a mature technology. The tumor vaccine of this invention combines antibody therapy with various T-cell immunotherapies, and is expected to improve tumor treatment.

[0116] This invention primarily enhances antibody titers in tumor vaccines. While MHC-II expression in B cells requires specific conditions, it achieves high expression in dendritic cells. (High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA) Liposomes can package a variety of mRNA genes, express a variety of MHC-II binding peptides, and also use a variety of MHC-I binding peptides as tumor T cell antigens.

[0117] In this embodiment, Her2 epitope peptides were used as antigens. Her2 has multiple B-cell antigenic epitopes; Mal-GPYWMSPEYVQ was selected as the peptide antigen and conjugated to liposomes expressing MHC-II mRNA prepared by a contract manufacturer. For various MHC-II variants, this embodiment selected MVKLFNRIKNNVAGE and the universal YFAVYLQETPY mRNA expressing the DRB10901 gene as immunogenic vector molecules.

[0118] Balb / c mice were given a 0.2 ml subcutaneous injection in the abdomen, followed by a second immunization two weeks later. Blood samples were collected six weeks later to measure antibody titers.

[0119] Results of immunization in BaLB / C mice: Table 12 Antibody titers determined by Her2 peptide-coated ELISA Serial Number 1 / 1000 1 / 4000 1 / 16000 1 / 64000 negative blank 1 1.51 0.88 0.45 0.23 0.18 0.07 2 1.33 0.79 0.33 0.15 0.15 0.11 3 0.98 0.81 0.44 0.32 0.13 0.09 4 1.39 0.99 0.68 0.41 0.19 0.08 5 1.78 1.02 0.73 0.39 0.23 0.05 Table 13 Antibody titers determined by universal MHC-2 binding to universal peptide plating assay Serial Number 1 / 1000 1 / 4000 1 / 16000 1 / 64000 negative blank 1 0.27 0.13 0.15 0.11 0.12 0.06 2 0.17 0.15 0.11 0.12 0.09 0.07 3 0.23 0.20 0.13 0.09 0.09 0.08 4 0.31 0.15 0.10 0.11 0.09 0.06 5 0.19 0.11 0.12 0.09 0.07 0.07 The results showed that mRNA expression of MHC-II binding peptides supports the feasibility of generating antibodies against endogenous tumor peptide antigens.

[0120] Tumor cells are a heterogeneous population; even within the same cancer, individual cells exhibit differences. Drug treatments lead to drug resistance, and radiotherapy and surgery for metastatic cancer cells have significant side effects, making them difficult-to-treat diseases. Immunotherapy is a safe and effective treatment option with fewer side effects, and tumor vaccines are a key area of ​​research in immunotherapy. Each patient's tumor has different antibody-killing epitopes, as well as CD8-killing T epitopes. The table below lists the T / B antigen epitopes of lung cancer cells, which can serve as target molecules for vaccines. In this invention, liposomes can package the mRNA of MHC-2 and MHC-1 epitopes to achieve combined T / B cell immunotherapy.

[0121] Cancer treatment vaccines, employing multiple targets and combining B-cell antigens with T-cell antigens, hold promise for improving treatment efficacy. Cancer treatment vaccines involve three molecular individualizations: MHC-I binding peptides (T-cell antigens), MHC-II binding peptides, and B-cell antigens. Each molecule exhibits diversity, making the preparation of vaccines using protein immunocarrier microspheres a suitable approach. The diverse MHC binding peptides do not require conjugation within the microspheres; only the B-cell antigens are attached to the microsphere surface.

[0122] The effectiveness of cancer treatment vaccines may depend on antigen assays, and it is crucial to encapsulate the detected antigens into microspheres whenever possible. Taking lung cancer as an example, the lung cancer cell membrane contains the antigens shown in Table 14 below: Table 14

[0123] The above are some of the commonly used target molecules for lung cancer immunotherapy. In addition, there are various molecular T and B cell antigen epitopes such as EGF, CEA, MUC1, WT1, P53 mutant, and KRAS, as well as individualized point mutations, splice shifts, and other antigens, which need to be fully measured to obtain results.

[0124] Tumor therapeutic vaccines involve complex tumor molecular biology and tumor immunology. However, technically, it is possible to link the above-mentioned multiple lung cancer B cell antigen peptides to the surface of microspheres, and load the inside of the microspheres with MHC-2 and MHC-1 binding peptides (i.e. T epitope antigens) to create a T and B cell mixed vaccine with antibody and cell immunity effects.

[0125] Example 9 Virus Therapy Vaccine Many viral vaccines are available for market prevention, with the most successful being those for smallpox, polio, and rabies. Marketed vaccines for treatment purposes include those for hepatitis, HIV, HPV, and COVID-19. However, these are all in the clinical research stage and have not yet passed Phase III clinical trials before being marketed.

[0126] Similar to cancer vaccines, the effectiveness of therapeutic vaccines for viruses is crucial. Vaccines prepared using traditional methods are not suitable for therapeutic purposes.

[0127] Taking COVID-19 as an example, the novel immune carrier microspheres of this invention are applied, with S protein antigenic epitopes coupled to the surface of the microspheres and viral T cell epitopes loaded inside. The S protein is prone to mutation, allowing for the loading of multiple antigenic peptides, potentially increasing the effective antibody titer by several orders of magnitude. The T epitopes are relatively stable and can be loaded in larger quantities, activating CD8 cells to kill infected cells and control infection.

[0128] The COVID-19 receptor-binding region (residues 319-541) contains the ACE2 receptor-binding motif (RBM, residues 437-508), which is the main target of neutralizing antibodies.

[0129] Typical epitope sequences, such as residues K417, N439, G446, Y453, L455, F456, E484, and F490, bind to neutralizing antibodies (such as the Regeneron antibody combination and LY-CoV555). E484 and N501 often mutate in variant strains (such as Beta, Gamma, and Omicron), potentially escaping antibody recognition.

[0130] The N-terminal domain (residues 14-26, 140-158): such as residues W152, R246, etc., are targets of some neutralizing antibodies (such as 4A8, COVA1-16).

[0131] Linear epitopes: such as residues 19-26 (NITNLCPF(SEQ ID NO: 99)) and residues 141-156 (GVGYQPYR(SEQ ID NO: 100)).

[0132] S2 subunit conserved region: Fusion peptide (FP) (residues 816-833): such as the S2P6 epitope (residues 1148-1159), is conserved in various coronaviruses.

[0133] T-cell epitopes are short peptides presented by MHC molecules, and are divided into CD8+ (MHC-I) and CD4+ (MHC-II) epitopes.

[0134] The following is a verified example of a T-epitope: 1. CD8+ T cell epitopes (MHC-I restricted) HLA-A*02:01 Restrictive epitopes: 269-27 (YLYALVYFL (SEQ ID NO: 101)), 1291-1299 (NLDSKVGGN (SEQ ID NO: 102)) HLA-B*07:02 Restrictive Epitopes: 790-798 (SPRWYFYYL(SEQ ID NO: 103)) HLA-A*24:02 Restrictive Epitopes: 436-444 (NYNYLYRLF (SEQ ID NO: 104)) 2. CD4+ T cell epitopes (MHC-II restricted) HLA-DRB1*01:01 Restrictive epitopes: 685-694 (QIYKTPPIK (SEQ ID NO: 105)) HLA-DRB1*15:01 Restrictive epitopes: 332-347 (IKLLEQFGNNKTFR (SEQ ID NO: 106)) Broad-spectrum epitope: S816-830 (GVTQNVLYENQKLI(SEQ ID NO: 107)) Effects of mutant strains on epitopes: Omicron (BA.1 / BQ.1 / XBB, etc.): Numerous mutations in the RBD region (such as K417N, E484A, N501Y) may escape B cell immunity, but T cell epitopes (such as the conserved region of the S2 subunit) are usually more conserved.

[0135] Cross-reactive T epitopes, such as S2 subunit epitopes of the S protein (e.g., HLA-A*02:01 restricted epitopes 1167-1175), are highly conserved between SARS-CoV-1 and SARS-CoV-2.

[0136] Example 10 Combination Packaging Vaccines—Personalized Vaccine Dosage Forms Traditional Chinese medicine (TCM) offers many compound prescriptions, and even more importantly, personalized combination packaging. Western medicine includes both compound drugs and various combination packages. Combination packaging is a dosage form solution for personalized vaccines and a method for the combined application of multi-target vaccines. Antibody titers produced by vaccines change over time after vaccination. For vaccines targeting different targets, besides treating tumors and viral infections, the simultaneous production of high antibody titers from the target molecules, which are normally physiologically active components, may affect normal physiological functions. Therefore, applying vaccines at different times is more rational.

[0137] The above is a detailed description of the embodiments, which is intended to enable those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to technical solutions obtained by those skilled in the art based on the present invention and on the existing technology, without innovative effort but only through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.

Claims

1. An immunocarrier microsphere loaded with a personalized MHC-II binding polypeptide, characterized in that, The microspheres have a core-shell structure, comprising a core and an outer shell. The core microspheres are loaded with 3-12 individualized MHC-II binding peptides. The MHC-II binding peptide sequences are obtained through computer prediction and screening based on the HLA genotyping results of the vaccinated individuals, and each HLA allele corresponds to at least one high-affinity MHC-II binding peptide. The outer shell is a coating layer of dextran or other hydrophilic polymers with thiol groups, whose thiol groups serve as coupling groups to connect antigens to construct vaccines, achieving the separation of the T / B epitope microspheres inside and outside.

2. The core-shell structured immunocarrier microspheres as described in claim 1, characterized in that, The immune carrier microspheres contain 3-12 high-affinity MHC-II binding peptides screened by computer and at least one universal MHC-II binding peptide to assist B cells in producing antibodies. The universal MHC-II binding peptide is selected from at least one of the following: YFAVYLQETPY, AKFVAAWTLKAAA, QYIKANSKFIGITEL, FNNFTVSFWLRVPKVSASHLE, AWLEAQEEEEVGF, QYIKANSKFIGITELKK, EPRAPWIEQEGPEYWDQE, ADVEVYRAVTPLGPPD, DTLRSYYADWYQQKPG, FVNQHLAGSHLVEAL, LNEDLRSWTAADTAA.

3. The immunocarrier microspheres loaded with personalized MHC-II binding peptides as described in claim 1, characterized in that, The MHC-II binding peptides were obtained by HLA typing and screening using affinity scoring software such as IEDB ProPred, MHCPred, SVRMHC, ARB, SMM-align, NetMHCIIpan, IEDB, and NetMHCPan.

4. The immunocarrier microspheres carrying personalized MHC-II binding peptides as described in claim 1, characterized in that, The microspheres have a particle size of 0.1-5 micrometers.

5. A method for preparing immune carrier microspheres loaded with personalized MHC-II binding peptides as described in claim 1, characterized in that, Including the following three types: MHC-II binding peptide self-assembled microspheres are coated with dextran or other hydrophilic polymers; Biospheres containing personalized MHC-II binding peptides are coated with dextran or other hydrophilic polymers. mRNA microspheres expressing MHC-II binding peptides without signal peptide sequences.

6. A vaccine prepared based on immune carrier microspheres loaded with personalized MHC-II binding peptides as described in any one of 1-4, characterized in that, The T / B epitopes are placed separately inside and outside the microsphere to avoid competition and inhibition between the T / B epitopes, with the T epitope located inside the microsphere.

7. The vaccine prepared from immunocarrier microspheres carrying personalized MHC-II binding peptides as described in claim 6, characterized in that, The microspheres are loaded with Th2 epitope peptides and can also be loaded with Th1 epitope peptides to prepare a bifunctional T and B cell vaccine with both antibody and cellular immune effects.

8. The application of the vaccine of the immunocarrier microspheres carrying personalized MHC-II binding peptides as described in claims 6-7 in the preparation of drugs for tumors, cardiovascular diseases, hyperglycemia, allergic diseases, autoimmune diseases, Alzheimer's disease, viral infections, drug addiction, weight loss and other chronic diseases that can be treated with antibodies.

Images