CN109758575B - Fully-diversified amphipathic MHC II binding polypeptide, immune carrier microsphere, preparation method and application thereof - Google Patents

Abstract

The invention relates to fully diversified amphiphilic MHC II binding polypeptide, immune carrier microspheres and a preparation method and application thereof. The amphiphilic MHC II binding polypeptide sequence structure is fatty acyl-beta folded peptide-C-MHC II binding polypeptide-hydrophilic beta folded peptide-maleimide, the amphiphilic MHC II binding polypeptide forms an immune carrier microsphere through self-assembly, and a coupling group is positioned on the surface of the microsphere. Is particularly suitable for preparing antibody vaccines for treating benign diseases and preparing antibody vaccines for treating tumors. The antigen can be coupled on the coupling group on the surface of the immune carrier microsphere. Antibody vaccines can also be used in combination with FcR-CART cells or NK cells.

Description

Fully-diversified amphiphilic MHC II binding polypeptide, immune carrier microsphere, preparation method and application thereof

[ technical field ]

The invention relates to the field of immune carrier microspheres, in particular to fully diversified amphiphilic MHC II binding polypeptides, immune carrier microspheres and a preparation method and application thereof.

[ background art ]

A therapeutic vaccine

Vaccination is one of the effective means for humans to fight and control infectious diseases. The vaccine in the traditional sense is mainly a preventive vaccine, the application target of the vaccine is susceptible animals and people of a healthy group, the vaccine component is generally microbial protective antigen, and the vaccine can activate specific immune response in a healthy body to generate specific antibody and Cytotoxic T Lymphocyte (CTL), so that the immunoprophylaxis of the pathogen is achieved.

Therapeutic Vaccine (Therapeutic Vaccine) is a new concept of immunotherapy established and developed in recent years, and refers to a Vaccine capable of breaking immune tolerance in a chronic infected person and reconstructing or enhancing immune response. The therapeutic vaccine can induce specific immune response in the sick individual, eliminate pathogen or abnormal cell, so as to treat the disease, and is a new therapeutic means for resisting virus and tumor.

Therapeutic vaccines can be divided into two categories according to their mechanism of action, one of which is the production of effector cells, such as specific killer T cells, for tumor therapy or the elimination of virus (HBV, HCV, HIV, HPV, etc.) infected cells; the other is through inducing in vivo antibody production, which can bind to active molecule to block the function of active molecule, and can kill tumor cell via complement and ADCC effect. The present invention is a therapeutic vaccine based on antibody production.

Antibodies for the treatment of diseases

The antibody treatment of diseases is the result of the development of monoclonal antibody technology, particularly humanized monoclonal antibody technology, more than 30 monoclonal antibodies are on the market at present for clinical application, more than 00 monoclonal antibodies are on the market in clinical trials, more than 500 monoclonal antibodies for treatment are researched, and the sales of the monoclonal antibodies in 2013 are more than 500 hundred million dollars. Monoclonal antibodies are long in development cycle, high in production cost and expensive in treatment cost.

Therapeutic antibody-type vaccines have been developed with the goal of generating endogenous antibodies by antigen immunization in place of exogenous monoclonal antibodies to achieve therapeutic effects.

Third, immunological process of antibody production

There are two main mechanisms for B cell antibody secretion: the T cell dependent pathway (Td) and the T cell independent pathway (Ti). Most protein-producing antibodies are the Td pathway.

B cell Td-type expression antibodies require dual signal activation: the epitope (B epitope) binds to the B cell receptor, which is the first signal.

Antigen enters B cells through a binding pathway with a B cell receptor, is hydrolyzed into a polypeptide fragment in Lysosome (Lysosome) and Endosome (Endosome) of the cells and is bound with MHC II, and then is transferred to a cell membrane to be displayed on the surface of the B cells, and an MHC II binding polypeptide epitope (also called Th epitope or T epitope) is presented to a CD4+ Th cell receptor. Th cells recognize carrier protein epitopes, activate Th cells to release cytokines, activate B cells as a second signal of B cells to activate and secrete antibodies to enter a proliferation program, and the affinity maturation process (somatic mutation mechanism) obtains the capacity of secreting antibodies with high affinity and high titer.

The resting Th cells can not meet the requirement of B cell activation, and DC cells are required to phagocytose antigens, and Th epitopes are presented to the Th cells through MHC II to activate the Th cells.

This suggests that B cells secrete antibodies and are actually involved in the three basic processes described above.

DC cells tend to phagocytose particulate antigens, so soluble proteins are generally difficult to produce high titers of antibodies. Traditionally, soluble proteins are made into emulsion particles or adsorbed onto what is called immunoadjuvant geline particles, so that higher titer antibodies can be obtained. The particle antigen can obtain high-titer antibodies without immunological adjuvants. The easy production of high-titer antibodies by the granulated antigens is a common phenomenon in immune experiments.

Generally, exogenous proteins contain B and Th epitopes, and antibodies can be generated by immunizing animals and human beings under the condition of an adjuvant. Endogenous proteins do not contain Th epitopes.

Second, the existing therapeutic antibody type vaccine still has the technical problems

1. The problem of low titer of endogenous proteins as antigens

Immunization with polypeptides and endogenous proteins as antigens does not produce antibodies. The short peptide only has B epitope and does not have T epitope, so the short peptide immunity can not generate antibody, which is called hapten; endoproteins typically do not produce antibodies in animals or humans, and due to the constant mutation of the B cell antibody gene, it is possible to produce antibodies directed against endogenous protein receptors, whereas B cells with high affinity receptors for endogenous proteins induce apoptosis, leaving only B cells with low affinity receptors. On the other hand, T cells directed against endogenous proteins are cleared during development and cannot regenerate. Endogenous proteins are unable to induce high titers of antibodies in animals or humans, and are termed B cell immune tolerance.

The antigen used in therapeutic vaccines is usually a human or animal self protein, or endogenous protein. In order to produce antibodies from polypeptides or endogenous proteins, it is known to chemically couple exogenous strong antigenic proteins, immunologically called immunocarriers (immunocarriers), and introduce exogenous proteins for activating Th cells.

Commonly used immune carrier proteins are: BSA, OVA, KLH, tetanus exotoxin (TT), hepatitis B, hepatitis E HIV capsid protein. In addition to the chemical cross-linking method of endogenous protein and immune carrier (Immonocarrier) protein, the fusion protein containing immune carrier is constructed by using genetic engineering technology as a heterologous molecule to provide Th epitope. Th epitopes are also provided by alloproteins of xenogeneic animal origin.

This brings with it new problems: because the B cell clone with high affinity for endogenous protein is eliminated, the abundance of B cells with high affinity binding to foreign carrier protein epitopes is several orders of magnitude higher than that of B cells with endogenous protein epitopes, and thus there is a problem of immune competition for protein epitopes within the cross-linked protein (or fusion protein) molecule. When the vaccine enters a human body or an animal body, the foreign carrier protein epitope has competitive advantages, and the B cell receptor has higher probability of combining and phagocytosing the foreign carrier protein epitope to generate a high-titer antibody aiming at the foreign carrier protein. The corresponding antibody of endogenous protein is inhibited, and only low-titer antibody can be generated, so that the clinical treatment value is difficult to realize.

2. The problem of specificity: only high titers of antibody are produced to be therapeutic. If Th epitopes are provided by the foreign protein, in addition to the introduction of competitive inhibitors as described above, higher titers of foreign protein antibodies may produce unpredictable side effects due to antibody cross-reactivity.

3. Human HLA diversity

Most of the animal experiments were carried out using pure animals. In a common experimental animal, a mouse is taken as an example, and the gene H2 expressing MHC II is H2-IAb. H2-IAd H2-IEa H2-IEb.

The human HLA complex is the most complex gene system known to date in humans. It is classified into HLA-I and HLA-II. The former is associated with cellular immunity and the latter is associated with antibody immunity.

The HLA-II gene region is quite complex and mainly comprises three subregions of HLA-DP, DQ and DR and 3 subregions of newly determined DN, DO, DM and the like. The genes of the region are directly named with the peptide chains (alpha, beta) encoded by the genes, such as DRA, DRB1, DRB2 and the like. The HLA-II allele published in 2014 expresses as many as 1915 proteins, and is newly found every year.

MHC II consists of an alpha chain and a beta chain, and the alpha and beta chains with different alleles are combined, and the diversity is counted in ten thousand. Few native proteins provide so many MHC II binding polypeptides as are desirable for vaccine preparation in a broad population.

Patent application document CN102008619A discloses an immune microsphere for overcoming B cell immune tolerance and its application. The comparison document mainly aims at the problem of immune competition of epitopes in chemical cross-linked protein or fusion protein molecules in the prior art. The immune microsphere is adopted to solve the problems, vaccine antigen is coated outside a microsphere medium, and immune carrier protein for activating T cells is coated inside the microsphere medium. Furthermore, B cell epitopes of vaccine antigens are positioned on the surface of the immune microspheres, and T cell epitopes of immune carrier proteins are positioned in the immune microspheres. The method avoids the competition of antigen protein epitope and Th cell activation epitope with B cell receptor by respectively arranging the antigen protein and immune carrier protein on the surface and inside of the microsphere. The problems of the technology are as follows: proteins within the microspheres easily leak out of the microspheres, resulting in high titers of antibodies to the carrier protein. On the other hand, this application only produces antibody immune effects.

[ summary of the invention ]

The present invention aims to solve the above-mentioned problems in the prior art.

To achieve the above object, there is provided a substantially diverse amphipathic MHC II binding polypeptide characterized by a sequence structure:

fatty acyl-beta-sheet peptide-C-MHC II combined polypeptide-hydrophilic beta-sheet peptide-maleimide,

the beta sheet peptide between fatty acyl and C in the sequence structure is a sequence composed of 5-8 hydrophobic amino acids,

the hydrophilic beta-sheet peptide is formed by interlacing acidic amino acid and basic amino acid into a sequence with the length of 3-5,

the sequence of the MHC II binding polypeptide is a polypeptide sequence with affinity with the MHC II or a random substitution sequence of the MHC II, and the amino acid used for random substitution in the random substitution sequence is an amino acid with hydrophilicity similar to that of a substitution position, so that sufficient diversity of the MHCII binding polypeptide is formed.

The coupling group is used for coupling with an antigen.

C is cysteine, so that disulfide bonds are formed among polypeptides.

The invention also relates to a preparation method of the immune carrier microsphere, the amphiphilic MHC II binding polypeptide forms the immune carrier microsphere through self-assembly, the MHCII binding polypeptide is in the microsphere, and the coupling group is positioned on the surface of the microsphere.

The antigen of the vaccine is presented on the surface of the microsphere, and the MHCII binding polypeptide is in the microsphere, so that the binding competition with a B cell receptor is avoided.

Further, the assembling is realized by one of the following methods:

a. self-assembly in an organic solvent/water mixed solvent, or

b. Self-assembly by means of a template.

The preferable organic solvent/water mixed solvent is acetonitrile/water mixed solvent, the amphiphilic MHC II binding polypeptide is added into the mixed solvent and then stirred to form emulsion, the amphiphilic MHC II binding polypeptide is bound through a hydrophobic end to form microspheres, acetonitrile in the microspheres is volatilized, and the microspheres are obtained through centrifugal precipitation and separation.

The template is solid lipid, the solid lipid is dissolved in a volatile hydrophobic solvent and then stirred with water to form emulsion, the amphiphilic MHC II binding polypeptide is immersed in the solid lipid through a hydrophobic end to form microspheres, the volatile hydrophobic solvent is volatilized, and the microspheres are obtained by centrifugal precipitation and separation.

The invention also relates to an immune carrier microsphere prepared by the preparation method.

The immune carrier microsphere can be used for preparing antibody vaccines for treating benign diseases. Further antibody vaccines for the treatment of benign diseases include: IgE vaccines, TNF-alpha vaccines, VEGF vaccines, CETP vaccines, DPP4 polypeptide vaccines, and Alzheimer's disease vaccines.

The immune carrier microsphere can be used for preparing antibody vaccines for treating tumors. The antibody vaccine for treating the tumor comprises: EGF vaccine, EGFR vaccine, PDL-1 vaccine, TCR vaccine, BCR vaccine, CD19 vaccine, CD20 vaccine, VEGF vaccine, Her2 vaccine, MAGE vaccine, Mucin vaccine, PDL vaccine, β HCG vaccine.

The antigen is coupled on the coupling group of the immune carrier microsphere to form the antigen vaccine.

The invention also relates to a combined application comprising:

a. the antigen vaccine is characterized in that the antigen vaccine,

FcR-CART cells or NK cells.

Compared with the prior art, the invention has the following advantages:

1. polypeptide self-assembly documents are mostly reported, but the length of polypeptides of various different polypeptides is more than 20 amino acids, the conventional method cannot self-assemble into regular microspheres, and particularly, the self-assembly of fully diversified MHC II binding polypeptides or antigen polypeptides is not reported.

2. The MHC II binding polypeptide is self-assembled into microspheres to replace exogenous protein, and the MHC II binding polypeptide is provided for endogenous protein. Endogenous antigen and MHC II combined polypeptide are arranged inside and outside, so that competitive inhibition of the endogenous antigen and the MHC II combined polypeptide and B cell receptor can be avoided, and the immunity can obtain antibody with higher titer than that of exogenous protein used as a carrier.

3. After the microspheres are cracked in vivo, the cracking products of the microspheres are MHC II-bound soluble polypeptides, and although the exogenous polypeptides can be B cell epitopes, antibodies aiming at the carrier components cannot be generated after leakage because Th epitopes are not contained. The specificity is good. Is particularly suitable for the preparation of therapeutic vaccines for benign diseases.

4. Sufficient diversity of sufficiently diverse MHC ii binding polypeptides meets the diversity requirements of different individual MHC ii. Is not possible with any native protein carrier.

5. The particles are advantageously phagocytosed by DC or macrophages, which in turn activates resting Th cells for helper functions.

6. The immune carrier microsphere has universality, and can be provided with the capacity of connecting various antigens by changing functional groups on the surface of the microsphere, wherein the antigens can be polypeptides, proteins, polysaccharide antigens or other small molecular compounds.

7. The carrier comprises polypeptide or solid lipid and biocompatible polysaccharide, and has no other compound components, and the prepared vaccine has higher safety.

8. The production of antibodies against tumor cells in vivo is achieved primarily through the ADCC pathway. The effector cells are mainly Fc receptor-bearing NK cells. The combination with NK cells in vitro amplification culture can effectively kill tumor cells to create conditions.

9. The tumor immunotherapy mainly comprises cell immune killing. Tc cells are directed against mutated tumor cells, but some tumor cells have low or no expression of MHC I, so that the Tc cells lose the killing effect. The proportion of T cells expressing Fc receptors is very small. The vaccine constructed by the immune vector microsphere of the invention can generate high-titer antibodies, is used together with in vitro gene engineering for expressing Fc receptors and in vitro culture and amplification of T cells, and is expected to obtain better curative effect.

Besides the applications, the method has more application prospects: such as prophylactic vaccines, drug-withdrawal vaccines, contraceptive vaccines, experimental studies.

[ description of the drawings ]

FIG. 1 is a schematic of the self-assembly of amphipathic MHC II binding polypeptides into microcarrier microspheres.

FIG. 2 is an electron microscope image of microspheres.

Fig. 3 is GFP% of 293T cells 72h after four plasmid cotransfection, where (R2) ═ 98.3%.

FIG. 4 is a graph of infection rates of 293T cells by different gradiviruses.

Figure 5 is a graph of transfection efficiency of CAR genes on T cells. The upper panel shows the measurement results of day D7, and the lower panel shows the measurement results of day D14.

[ detailed description of the invention ]

The present invention will be further described with reference to the following examples and drawings, which are illustrative only and are not intended to limit the scope of the present invention.

Example 1 design and preparation of self-assembled amphiphilic Polypeptides for MHCII-binding Polypeptides for testing in mouse animals

1. MHCII binding polypeptides for Balb/C mice tetanus exotoxin fragments were selected according to literature:

TT830-844 QYIKANSKFIGITELD

2. MHCII binding polypeptides for C57/BL6 mice, egg ovalbumin fragments were selected according to the literature:

OVA 324-338 ISQAVHAAHAEINEA

3. the amphipathic polypeptide containing the two MHCII binding polypeptides is designed and synthesized for constructing immune carrier microspheres by adopting a conventional method:

the technical scheme includes that the reagent is Pal-AAAAGGG-C-QYIKANSKFIGITELD-RDKQKK-mal (Pal: palmitoyl, mal: maleimide).

⑵、Pal-AAAAGGGG-C-ISQAVHAAHAEINEA-QKDRDK-mal

EXAMPLE 2 preparation of microspheres in organic/aqueous solvent Mixed solvent

1. Polypeptide self-assembly balling principle:

as shown in figure 1, amphiphilic polypeptide belongs to a high molecular surfactant, and at a water/oil two-phase interface, a hydrophobic end is immersed into an oil phase, and a hydrophilic end is free from a water phase. If the oil phase is microspherical, the amphiphilic polypeptide can be polymerized and distributed in parallel across the microsphere interface by taking the oil phase microsphere as a mold. Because of the existence of two beta-sheet peptides, when the oil phase solvent is evaporated under the stirring condition, the amphiphilic polypeptide is not dissolved in water and becomes stable self-assembly microspheres in the water phase.

2. Preparation operation

The synthesized amphiphilic MHCII combined polypeptide 10mg is dissolved in 30-60% acetonitrile/water. The concentration of the amphiphilic MHCII binding polypeptide is 0.1-1 mg/ml.

And the organic solvent petroleum ether or cyclohexane which is immiscible with acetonitrile is adopted. Adding water containing 0.1-1% Tween-80, stirring, and emulsifying to obtain microsphere.

Mixing the three, the (1) and the (2), stirring for 10-30 minutes in a fume hood, placing in a rotary evaporator, and removing the organic solvent at 30-50 ℃ to obtain the self-assembled microspheres suspended in the water.

3. And (4) analyzing results: the size was measured by a particle size analyzer and the zeta potential was determined by a potential analyzer. The microspheres are 100-500 nanometer spherical particles. Zeta potential is related to the microsphere end groups and the ionic strength of the solution.

4. The microspheres were centrifuged, and after precipitation, 5ml of 0.05M, pH7.5 phosphate buffer was used for suspension, and the centrifugation and precipitation were repeated 2 times.

5. After centrifugation, the microspheres were freeze dried at low temperature as shown in FIG. 2.

Example 3 preparation of polypeptide self-assembled microspheres using solid lipid as template

1. Balling principle: dissolving palmitic acid in an organic solvent, and emulsifying to form microspheres as a template. Pouring the amphiphilic polypeptide solution into the reactor, mixing the solution with the amphiphilic polypeptide solution, embedding the hydrophobic end of the amphiphilic polypeptide into an organic phase containing palmitic acid under the stirring condition to be anchored, and placing the hydrophilic end in an aqueous phase. And forming solid particles after the organic solvent is volatilized to form the microspheres with stable dispersion. The particle size can be controlled by ultrasonic or homogenizer. Provided that the solvent in which the amphiphilic polypeptide is dissolved is immiscible with the organic phase in which palmitic acid is dissolved.

The invention uses acetonitrile/water to dissolve amphiphilic polypeptide, and uses petroleum ether or cyclohexane to dissolve palmitic acid. The two solvents are immiscible.

The polypeptide is stabilized by hydrophobic force and positive and negative ion force.

2. Preparation operation

Experimental table ii amphiphilic polypeptides for self-assembly are the same as in example one. 10mg was dissolved in 30-60% acetonitrile/water and centrifuged to remove insoluble polypeptide. The concentration is 0.5-1 mg/ml.

The composition is prepared by dissolving 100mg of palmitic acid and 50mg of lecithin in 3ml of dichloromethane/petroleum ether (2: 3), adding the solution into 30ml of a solution containing 1.0% of tween-80 acetonitrile/water, and ultrasonically emulsifying after magnetic stirring. The emulsified particles are 0.3-1 micron.

Pouring the polypeptide-acetonitrile/water solution into the emulsified medium palmitic acid-lecithin emulsion, and stirring by magnetic force to gradually volatilize the organic solvent.

The microspheres were centrifuged, precipitated, suspended in 5ml of 0.05M, ph7.5 phosphate buffer, and centrifuged for 2 times.

3. And (4) analyzing results: the size was measured by a particle size analyzer and the zeta potential was determined by a potential analyzer. The microsphere is 100-500 nanometer spherical particle. Zeta potential is related to the microsphere end groups and the ionic strength of the solution.

4. Centrifuging, freeze drying, and storing at low temperature.

Example 4 preparation of polypeptide self-assembled immune Carrier microsphere coated by Bio-gel microsphere

1. The principle is as follows: biological gel microspheres are used for coating medicines, proteins and nucleic acids, and research documents are many. The encapsulated polypeptide has poor effect only due to serious leakage. However, the amphiphilic polypeptide self-assembles into a polymer that overcomes the leakage problem. The method can simultaneously add cytokine and CpG nucleic acid with immune promoting function.

2. The operation is as follows:

first, the self-assembled microspheres prepared in example 1 were formed into microspheres of less than 1000nm during the volatilization of the organic solvent, and mixed with 1% alginic acid solution. Using a conventional method for preparing calcium alginate microspheres, and forming the calcium alginate microspheres with the particle size of 1-3 microns by a homogenizer. Coating chitosan according to a literature method to prepare the calcium alginate microspheres containing self-assembled polypeptides and coated by chitosan.

And modifying the microspheres with N-hydroxymaleimide propionate for coupling, and then fixing with glutaraldehyde or epichlorohydrin.

3. Detecting the size and the dispersibility of the microspheres by using an optical microscope

4. Washing with PBS, freeze drying, and storing at low temperature.

Example 5 clinical selection and design of polypeptides with sufficient diversity for MHCii binding

On-line NetMHCIIpan 3.0Server provides prediction of HLA-DR, HLA-DP, HLA-DQ binding sequences. Inputting the protein sequence from rubella virus shell, tetanus toxin and hepatitis A virus, and predicting the HLA-DR, HLA-DP and HLA-DQ binding sequence to obtain high affinity MHC II binding 15 peptide. DR, DP, DQ all taken from one subset of their allelic products, 10 in total, see Table below.

HLA	15 peptides	Core sequence	Affinity (nM)
				HLA-DRB1*01:01	ITDYMYLTNAPSYTN	YMYLTNAPS	4.43
HLA-DRB3*01:01	DLVKFISDKIKFLNP	FISDKIKFL	83.99
				HLA-DRB4*01:01	LLAIAGIRLHRAAIY	IAGIRLHRA	46.26
HLA-DRB5*01:01	NIYYRRLYNGLKFII	YRRLYNGLK	4.13
HLA-DPA1*01:03-DPB1*01:01	FNNFTVSFWLRVPKV	FTVSFWLRV	3.02
				HLA-DPA1*02:01-DPB1*09:01	KLYTSYLSITFLRDF	SYLSITFLR	9.99
				HLA-DQA1*01:01-DQB1*02:01	KTQLVDDFLDLDMAI	LVDDFLDLD	269.66
HLA-DQA1*04:01-DQB1*03:01	YINGVLMGSAEITGL	VLMGSAEIT	108.65
				HLA-DQA1*05:06-DQB1*03:04	SLNFLGGAPVCPGQN	FLGGAPVCP	20.62
HLA-DQA1*06:01-DQB1*03:05	DYDQYCADVAAEELM	YCADVAAEE	84.34

Each subclass synthesizes five polypeptides:

a novel polypeptide having a sequence of polypeptides,

the random substitution sequence of three amino acids at positions 4, 6, 8 and 10,

a sequence is randomly substituted by three amino acids of positions three, 3,5, 7, 9 and 11,

four, a sequence of random substitutions of three amino acids at positions 2, 4, 6, 8, 10, and 12,

and fifthly, randomly substituting the three amino acids at positions 3,5, 7, 9, 11 and 13.

The principle of random substitution of amino acids is that amino acids with similar hydrophilicity are used. The hydrophilicity indices are tabulated below:

the specific operation is as follows:

peptide (15) random substitution of four amino acids at positions 5, 7, 9 and 11

Using HLA-DRB1 x 01:01 as an example, binding peptide was predicted to have an affinity of ITDYMYLTNAPSYTN nM of 4.43nM

During polypeptide synthesis, M, A, W three amino acid raw materials are added in the 5 th synthesis step. P, T, Y three ammonia are added in the 7 th synthesis

And (3) an amino acid raw material. N, Q, E is added in the step 9, and P, T, Y three amino acid raw materials are added in the step 11. The theoretical calculation of diversity is 81

Random modification of the 4 th, 6 th, 8 th, 10 th and 12 th positions of 15 peptides

HLA-DPA 1A 01:03-DPB 1A 01:01 predicted to bind to FNNFTVSFWLRVPKV with 3.02nM affinity

During polypeptide synthesis, F, V, I three amino acid raw materials are added in the step 4. V, L, F three ammonia are added in the 6 th synthesis

Amino acid raw materials. F, V, I is added in the 8 th step, and L, W, V three amino acid raw materials are added in the 10 th step. V, L, F three amino acids are added in step 12. The diversity theory is 243.

Random modification of the 3 rd, 5 th, 7 th, 9 th, 11 th and 13 th positions of the three and 15 th peptides

Taking HLA-DQA 1:01-DQB 1: 02:01 as an example, the predicted binding sequence is KTQLVDDFLDLDMAI with 269.66nM affinity

During polypeptide synthesis, Q, D, N is added in the third step, and V, L, F three amino acid raw materials are added in the 5 th step. D, K, Q three amino acid materials are added in the 7 th synthesis. L, W, V is added in the step 9, and L, W, V three amino acid raw materials are added in the step 11. The theoretical calculation of diversity of M, A, W added in the step 13 is 729

Fourth, 15 peptides 2, 4, 6, 8, 10, 12, 14 th random modification

Using HLA-DQA1, 06:01-DQB1, 03:05 as an example, binding polypeptide DYDQYCADVAAEELM was predicted to have an affinity of 84.34nM

During polypeptide synthesis, Y, P, C is added in the second step of synthesis, and Q, D, N three amino acid raw materials are added in the 4 th step of synthesis. C, Y, G three amino acid materials are added in the step 6. Step 8 Synthesis addition D, K, Q step 10 addition of AGM three amino acid starting materials. E, N, H three amino acids are added in step 12. Step 14, L, W, V. The theory of diversity is calculated as species 2187.

The operation not only utilizes the computer prediction result, but also generates enough diversity. The diversity after polypeptide mixing was (1+81+243+729+2187) x10 ═ 32410

Example 6 clinical fully diverse MHCii-binding polypeptide self-assembled microspheres

The preparation method of the clinical immune carrier microspheres assembled by the MHCii binding polypeptide with sufficient diversity is the same as that used for mice, see examples 2, 3 and 4.

EXAMPLE 7 preparation of vaccine by conjugation of antigen to microspheres

The polypeptide antigen can be synthesized by adding cysteine to the terminal to introduce a sulfhydryl group, and the amino group of the protein molecule can be introduced into the sulfhydryl group by using an SPDP reagent.

The microsphere surface has maleimide group, and the maleimide group can react with sulfhydryl group specifically to form stable sulfhydryl ether bond under the condition of pH 6.5-7.5. At neutral pH, the rate of reaction of maleimide with thiol is 1000 times faster than amino.

The antigen and the microsphere are reacted and connected in PBS with neutral pH value to form the vaccine.

For specific operation, SIMA-aldrich is referred to: SPDP Crossslinkers

thermo-fisher scientific：Sulfhydryl-reactive Crosslinker Chemistry

Hereinafter, specific applications of the microcarrier microspheres will be further described.

Example 8 asthma vaccine

IgE-mediated diseases are a general term for allergic diseases caused by IgE, and are known for their susceptibility to excessive production of IgE antibodies when exposed to environmental allergens, often accompanied by more than one allergic disease. When allergen enters body for the first time, specific B cell is induced to produce IgE antibody response, and free IgE in serum can combine with surface receptor of mast cell and basophil through Fc segment to make body in sensitization state without combining antigen. After the organism contacts with the allergen again, the multivalent allergen is combined with two or more adjacent IgE antibodies on the surface of the sensitized cell, so that the receptor on the surface of the cell membrane is crosslinked, the degranulation of the sensitized cell is triggered, and the bioactive medium is synthesized and released, thereby causing local or systemic anaphylactic reaction.

IgE-mediated diseases usually include Atopic Dermatitis Atomic Dermatitis, AD, Allergic Rhinitis allergy Rhinitis, AR, bronchial Asthma Asthma, AS, etc., and severe symptoms may cause anaphylactic shock and life-threatening.

Allergic diseases have seriously interfered with the work and life of patients, and the social burden caused thereby is also considerable. Due to the important role of IgE in the occurrence and development links of allergic diseases, IgE becomes a new target and research hotspot for the treatment of the allergic diseases.

The molecular structure of IgE is clear, the site of binding to high-affinity and low-affinity receptors in human IgE antibodies is highlighted in the C epsilon 3 domain, and Omalizumab (Omalizumab) is a CHe3 domain that specifically recognizes the heavy chain of IgE and selectively binds to IgE, inhibiting the binding of IgE to the high-affinity IgE receptor (fcerei) on the surface of mast cells and basophils, thereby reducing the release of allergic mediators, and in addition, reducing the number of IgE receptors in some patients with specific reactivity.

IL-5 monoclonal antibodies have also been used in the treatment of asthma, the first being Nucala (Mepolizumab) of GSK, an inhibitor of interleukin 5(IL-5) which specifically binds IL-5 and prevents it from activating the IL-5 receptor. The medicine is the first monoclonal antibody which acts on IL-5 cytokine and inhibits the activity of eosinophil and is used for patients with asthma of 12 years old and above.

The second is Cinqair (Reslizumab) from Dewar, approved by the FDA at 23/3/2016, and approved by EMA at 18/8/2016 for marketing. Reslizumab is an interleukin-5 (IL-5) antagonist that binds IL-5 and prevents its binding to the IL-5 receptor, thereby slowing the eosinophil-related inflammatory response. The medicine can be used for treating severe asthma of 18 years old or above. Cinqair is the second IL-5 mab approved for entry into the severe asthma market. IL-5 can also be used for preparing corresponding vaccines for treating asthma.

The invention constructs a therapeutic vaccine capable of inducing specific neutralizing antibodies aiming at an IgE receptor binding site (C epsilon 3 structural domain),

through antigen-antibody combination, the generated part of the IgE Fc fragment combined with an IgE Fc receptor is blocked, and then an IgE-mediated allergic disease effect pathway is blocked, so that a new pathway is provided for treating IgE-mediated diseases.

The research uses IgEC epsilon 3 fragment polypeptide as antigen to construct microsphere vaccine, and compares the microsphere vaccine with KLH cross-linked substance.

The antigen: IgE C epsilon 3 fragment polypeptide sequence: GYGYQCIVDHPDFPKPIVRSITKTPGQR

The polypeptide specific to the C epsilon 3 structural domain of mouse IgE Fc is synthesized by a chemical synthesis method. The corresponding monoclonal antibody of the polypeptide is proved to block the combination of IgE and a mast cell receptor and to be effective in treating allergic diseases

Two, MHC2 sequences that bind: source ISISEIKGVIVHK of measles virus and ISQAVHAAHAEINEA of ovalbumin

The self-assembly polypeptide sequence is designed as follows:

Pal-AAAAGGG-C-ISISEIKGVIVHK-DKQK-mal

Pal-AAAAGGG C-ISQAVHAAHAEINEA-KQRK-mal

fourth, self-assembly

Dissolving the self-assembly polypeptide in a mixed solvent of water and acetonitrile (volume ratio is 1: 1), removing the acetonitrile by rotary evaporation, gradually self-assembling the self-assembly polypeptide into spheres in the volatilization process of the acetonitrile, finally obtaining an aqueous solution containing the self-assembly polypeptide microspheres, and freeze-drying to obtain the self-assembly polypeptide microspheres, wherein the particle size of the microspheres is about 2.8 microns. (see the attached drawing)

Fifthly, preparing a microsphere vaccine:

the sixth amino acid of the antigen polypeptide is cysteine, and is connected with maleimide on the microsphere by utilizing sulfydryl. The reaction conditions were the same as in example 7.

Sixthly, the IgE polypeptide-KLH conjugate is synthesized by Gell biotechnology company

The animals and the immune balb/c mice are from the Shanghai experimental animal center. Immunization was performed subcutaneously at 50 μ g once a week.

Blood was taken one week after the last immunization for antibody titer determination. Implemented by gill biotechnology.

Methods of ELISA and Lane-Mulilans are known in the literature. Implemented by gill biotechnology.

And (5) measuring and providing ELISA measuring results by a company.

A first group: IgE polypeptide fragment coupled self-assembly polypeptide carrier microsphere immunization 6-week ELSA test result

Dilution factor of serum

Second group: IgE polypeptide fragment coupling KLH immunity 6-week ELISA test result

Dilution factor of serum

And (ii) result analysis

(A) Polypeptides are haptens that require conjugation to an immunocarrier protein (e.g., BSA, OVA, TT, KLH) to induce antibody production. KLH is a classical immune carrier protein that, when ligated, is capable of high antibody titers. But produce KLH high titer antibodies and much higher titer levels than the antigen.

(B) The results show that the self-assembled microsphere of the heterologous MHC II binding polypeptide can replace the traditional heterologous protein to be used as an immune carrier, and the result is superior to the KLH effect. The antibody titer of the carrier component is very low.

IL-5 monoclonal antibodies have also been used in the treatment of asthma, the first being Nucala (Mepolizumab) of GSK, which is an interleukin 5(IL-5) inhibitor that specifically binds IL-5 and prevents it from activating the IL-5 receptor. The medicine is the first monoclonal antibody which acts on IL-5 cytokine and inhibits the activity of eosinophil and is used for patients with asthma of 12 years old and above. American FDA approval was obtained at 11/4/2015, EMA approval was obtained at 12/2/2015, and Japanese PMDA approval was obtained at 3/28/2016

The second is Cinqair (Reslizumab) from Tiwa, approved by the FDA at 3/23 in 2016 and approved by the EMA at 18/8 in 2016 for marketing. Reslizumab is an interleukin-5 (IL-5) antagonist that binds IL-5 and prevents its binding to the IL-5 receptor, thereby slowing the eosinophil-related inflammatory response. The medicine can be used for treating severe asthma of 18 years old and above. Cinqair is the second approval,

reference documents: vaccine 21(2003) 1580-1590

Synthetic IgE peptide vaccine for immunotherapy of allergy Chang Yi Wang a,*,Alan M.Walfield a,Xinde Fang a,Bruce Hammerberg b,John YE。

Example 9 type II diabetes vaccine DDP-4 polypeptide vaccine

The DPP-4 inhibitor can selectively inhibit the enzymatic activity of DPP-4, prevent GLP-1 from being cracked and inactivated, improve the plasma level of active GLPl, enhance the physiological action of the active GLPl, and reduce HbA, fasting blood sugar and postprandial blood sugar levels of type 2 diabetes patients. The DPP-4 inhibitor is used as a novel oral hypoglycemic medicament. The effectiveness, good safety and tolerability of glycemic control in the treatment of type 2 diabetes has been demonstrated in a number of clinical trials and practical applications.

Example 10 CETP polypeptide vaccine for cardiovascular diseases

The MHC II binding polypeptide sequences used for vaccine preparation were: FGFPEHLLVDFLQSLS

Cholesterol is mostly produced by the liver. The total amount of cholesterol in the human body is 100 to 200 g. Two thirds of these are self-synthesized in the body, and one third is from food. Cholesterol must be bound to lipoproteins in order to be transported to various parts of the body. Lipoproteins are further classified into low-density lipoproteins and high-density lipoproteins.

Cholesteryl Ester Transfer Protein (CETP) is a glycoprotein in plasma that is capable of transferring cholesteryl esters from HDL to Low Density Lipoprotein (LDL), Intermediate Density Lipoprotein (IDL) and Very Low Density Lipoprotein (VLDL) and exchanging triglycerides, thereby playing an important role in regulating plasma HDL levels and remodeling the composition of HDL particles, and highly active CETP can lower HDL levels. Thus, CETP inhibitors are considered to be one of the most therapeutically potential drugs for coronary atherosclerosis. And the results of clinical phase III are encouraging. CQYIKANSKFIGITE GFPEHLLVDFLQSLS is cholesteryl ester transfer protein polypeptide fragment, which is used as antigen to prepare vaccine to generate antibody capable of inhibiting CETP cholesterol transport activity and raising HDL/LDL ratio. Reducing the concentration of cholesterol in peripheral blood, and preventing arteriosclerosis and cardiovascular and cerebrovascular diseases. The test results were as follows:

dilution factor of serum

Reference documents: thromb Vasc biol.2000; 20:2106-2112.

Vaccine-Induced Antibodies Inhibit CETP Activity In Vivo and Reduce Aortic Lesions in a Rabbit Model of Atherosclerosis Charles W.Rittershaus,David P.Miller,Lawrence J.Thomas,Michele D.Picard,Christopher M.Honan,Constance D.Emmett,Carolyn L.Pettey,Hedy Adari,Russell A.Hammond,David T.Beattie,Allan D.Callow,Henry C.Marsh,Una S.Ryan。

Example 11EGF protein anti-tumor vaccine

EGF was originally developed in Cuba, and is a chemically coupled polypeptide vaccine developed by Baitai biological medicine industry Co. The EGF vaccine is prepared by emulsifying a conjugate formed by chemically crosslinking recombinant human EGF and recombinant Neisseria epidemic cerebrospinal meningitis outer membrane P64k carrier protein and an oily adjuvant Montanide ISA 51. After the vaccine is used for immunizing a human body, the immune tolerance of the body to the self antigen (EGF) can be broken, and high-level antibodies against the EGF are induced. The antibody is specifically combined with EGF in a human body, reduces the content of serum EGF, and inhibits the proliferation of tumor cells by down-regulating the activity of EGF/EGFR signal channels, thereby realizing the treatment of epithelial tumors. The vaccine is the first vaccine for realizing tumor treatment by active immunization successfully in the world.

Animal experiments and clinical researches show that the vaccine has good curative effects on malignant tumors excessively expressed by Epidermal Growth Factor Receptor (EGFR), including lung cancer, head and neck cancer, ovarian cancer and the like, and particularly has obvious curative effects on non-small cell lung cancer. Is currently marketed in copa, peru, paraguay for the treatment of advanced non-small cell lung cancer. The phase I clinical study of the Chinese patient with advanced non-small cell lung cancer is also finished, and the phase II/III clinical study is planned to be developed

Clinical trial results show that lung cancer patients with high serum EGF content are effective.

The EGF vaccine is coupled with encephalitis coccus outer membrane protein, and the novel immune carrier microsphere is expected to be superior to a commercial vaccine preparation

1. Antigen: mouse EGF (mEGF) is a 51 amino acid genetically engineered expression protein. Purchased from Peprotech corporation.

2. Construction of Mixed mouse MHC II-binding polypeptide microspheres the same as in example 4

3. EGF introduction of thiol groups was performed in literature references. (Leptorison et al: preparation of gastric carcinoma mAb-RTA conjugates and their cytotoxic Properties in modern immunology 1988,1988(05) 330-333).

4. 5mg of thiolated solution in 5ml of PBS pH 7.5; 2ml of packed volume, and suspending the mixed polypeptide microspheres with Mal MHC II in 5ml of PBS (pH7.5PBS); the two were mixed and placed on a magnetic stirrer in a refrigerator at 4 ℃ for reaction overnight. The next day, the supernatant was removed by centrifugation. PBS was washed once and suspended in 3ml PBS for immunization.

5. Immunization: Balb/C mice were injected subcutaneously in multiple doses of 300. mu.l, once every two weeks. One week after the fourth immunization the antibody titer was determined by conventional methods of exsanguination.

6. ELISA results

Experiment I, determining antibody titer by using mEGF coated enzyme label plate

Dilution factor of serum

OD450	1000	2000	4000	8000	16000	32000	64000	blank	negative
										1	2.35	1.55	1.05	0.787	0.810	0.550	0.489	0.060	0.109
2	1.97	1.59	0.98	0.660	0.545	0.422	0.385	0.077	0.119
										3	2.35	1.88	1.22	0.863	0.693	0.520	0.455	0.081	0.093
4	2.23	1.81	1.07	0.93	0.688	0.580	0.480	0.810	0.100
										5	2.59	1.26	1.02	0.865	0.725	0.632	0.580	0.087	0.095

Experiment two, can dissolve with blank microballoons pH9.0 carbonic acid buffer solution) coated ELISA plate to determine antibody titer

OD450	1000	2000	4000	8000	16000	32000	64000	blank	negative
										1	0.59	0.350	0.095	0.107	0.210	0.150	0.109	0.090	0.129
2	0.85	0.390	0.280	0.060	0.204	0.122	0.115	0.077	0.110
										3	0.75	0.280	0.120	0.163	0.133	0.120	0.155	0.091	0.122
4	0.53	0.11	0.072	0.193	0.108	0.200	0.115	0.100	0.110
										5	0.89	0.56	0.150	0.060	0.125	0.125	0.180	0.087	0.091

Reference documents: therapeutic vaccine against DPP4improves glucose in microorganism PNAS | Published online March 17,2014| E1257

Example 12 preparation of epidermal growth factor receptor type III mutant (EGFRvIII) microsphere vaccine and immunization experiment

EGFRv III is the deletion of genes in the membrane outer part of the epidermal growth factor receptor. Approximately 30% to 50% of tumors (non-small cell lung cancer cells, breast cancer and gliomas) display a protein called epithelial growth factor mutant iii (egfrviii), which is absent from normal cells. Researchers believe that EGFRvIII can drive abnormal proliferation and spread of glial cells, and is one of the causes of high malignancy of this tumor. The EGFRvIII is used as an antigen to prepare the vaccine, which can induce an organism to generate antibody immune response to cells containing the EGFRvIII and can also induce cell immune response. The novel vaccine comprises a synthetic EGFRvIII fragment and an adjuvant capable of stimulating dendritic cells.

(1) Antigen sequence: the EGFRv III epitope polypeptide is synthesized by a solid phase polypeptide synthesis method, the method is the same as the example 1, and the sequence is as follows: LEEKKANYVVTDHC

(2) The EGFRv III epitope polypeptide is coupled with the self-assembly polypeptide carrier microsphere by the same coupling method as the example 1.

(3) The immunization strategy was the same as in example 2.

(4) And (3) detection: after one week of non-completion, orbital bleeding was performed, serum was collected, antibody titer was detected by Elisa, and serum was lyophilized. The results are as follows,

dilution factor of serum

Reference documents: the literature: BMC Biotechnology 2010,10:72

Development of an EGFRvIII specific recombinant antibody Puja Gupta1,Shuang-Yin Han1,2,Marina Holgado-Madruga1,Siddhartha S Mitra1,Gordon Li1,Ryan T Nitta1,Albert J Wong

Molecular Cancer Therapeutics》,2015,14(5):1141

Characterization of ABT-806,a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody

Molecular Cancer Therapeutics

Clinical Oncology,2017,44(18):931-934.

New development of glioma immunotherapy

Li YI,Zhennan TAO,Xuejun YANG.Advances in the immunotherapy of glioma[J].Chinese Journal of

China Cancer No.8 China Cancer No. 013 Vol.22, 2013, No.8

The current situation of research and development of global antitumor drugs in about 5 years

(382–410)and HER-1(418–435)sequences were the best candidates for the production of a vaccine and a therapeutic peptide mimic

Example 13 EGFR polypeptide vaccine (EGFR418-435, EGFR382-410, EGFR 347-374)

Epidermal Growth Factor Receptor (EGFR) is the expression product of the proto-oncogene c-erb-B1. Its overexpression and mutant are closely related to the development of tumors, and in most tumors (such as bladder cancer, non-small cell lung cancer, ovarian cancer, head and neck squamous cancer, breast cancer, glioma, pancreatic cancer, gastric cancer, prostate cancer, esophageal cancer, etc.), the overexpression and/or mutation can lead to the uncontrolled and malignant cell growth by means of signal transduction [3 ]. The expression rate is different in various tumor tissues, and is closely related to the differentiation degree, the malignancy degree and the infiltration degree of tumors.

The monoclonal antibody produced by using EGFR1 as the outer membrane antigen is 90% effective in promoting tumor cell growth. The polypeptides at the extramembranous part constitute antibody type vaccines, and the EGFR418-435 and EGFR382-410 and EGFR347-374 polypeptides are confirmed to be effective.

This example uses the EGFR418-435 polypeptide: SLNITSLGLRSLKEISDG

Balb/c mice were immunized by coupling with the microspheres of example 1, and the results of antibody measurements for 8 weeks after 8 immunizations were as follows:

reference: j Immunol published online 22May 2013

Peptide Vaccines and Peptidomimetics of EGFR(HER-1)Ligand Binding Domain Inhibit Cancer Cell Growth In Vitro and In Vivo a Kevin Chu Foy,Ruthie M.Wygle,Megan J.Miller,Jay P.

Example 14 construction and immunization experiment of Her2 (human epidermal growth factor receptor 2) polypeptide immune vector microsphere vaccine

HER2 is a protooncogene, and a small amount of HER2 protein is present on the surface of a normal cell membrane in each human body, and HER2 protein can conduct signal transduction and regulate the growth and division of cells. When the HER2 gene in the cancer cell is highly expressed, excessive HER2 protein can be produced on the cell membrane, so that the wild growth of the cancer cell is stimulated, the cancer cell is subjected to uncontrolled division and growth, and the invasiveness of the cancer cell is increased; in HER2 positive breast cancers, the HER2 gene is greatly amplified, resulting in 10-100 fold overexpression of the HER2 protein, leading to uncontrolled cell division and tumor invasive growth. Statistically, approximately 20-30% of breast cancers are HER2 positive. HER2 positive breast cancer patients may have a poor prognosis and are more prone to recurrent metastases.

Trastuzumab is a monoclonal antibody against HER2 that blocks the growth of cancer cells by attaching itself to HER2 to prevent the attachment of human epidermal growth factor to HER2, and it also stimulates the body's own immune cells to destroy cancer cells. Compared with the traditional chemotherapy drugs, trastuzumab (herceptin) has more accurate targeting and low toxicity. The adverse reaction is relatively slight, and the tolerance is good.

2. Polypeptide antigen: her2 polypeptide antigen literature reports that the spatial structure formed by His245, Val286, Ser288, Leu295, His296 and Lys311 in the amino acid sequence of the Her2 dimeric region is critical for the binding of pertuzumab to Her 2. Allen et al [8] designed a mimic polypeptide of amino acids 266-296 in this region, and experiments confirmed that the effect was comparable to that of pertuzumab. Her266-296 was chosen for this study. The sequence is as follows: GCKKIFGSLAFLPES FDGPASNTAPLQPE

4. Antibody detection results: seven-week after completion of immunization, orbital bleeding was performed, serum was collected, antibody titer was detected by Elisa, and serum was lyophilized.

The results are as follows,

reference documents: vaccines 2015,3, 519-;

Anti-Tumor Effects of Peptide Therapeutic and Peptide Vaccine Antibody Co-targeting HER-1and HER-2in Esophageal Cancer(EC)and HER-1and IGF-1R in Triple-Negative Breast Cancer(TNBC)Jay Overholser 1,Kristen Henkins Ambegaokar 1,Siobhan M.Eze 2,Eduardo Sanabria-Figueroa 3,Rita Nahta 2,3,Tanios Bekaii-Saab 4and Pravin T.P.Kaumaya

example 15Mucin polypeptide vaccine

1. Study background: MUC1 is a mucin family member, exists on the surface of normal glandular epithelial cells and tumor cells derived from the normal glandular epithelial cells, and MUC1 is highly expressed in the tumor cells and is 100 times different from the normal cells. The related cancer types comprise ovarian cancer, lung cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer and multiple myeloma, and the vaccine taking MUC1 as an antigen FDA is approved as the first therapeutic vaccine entering clinical trials.

MUC1-C comprises an extracellular domain of 58 amino acids (MUC1-C/ED), a transmembrane domain of 28 amino acids and an intracellular domain of 72 amino acids (MUC1-CD), the extracellular domain being composed of a polypeptide core and a flanking sugar chain. The core peptide extracellular segment contains a plurality of repeated tandem sequences of 20 amino groups. Tandem repeats are extensively glycosylated with low glycosylation at the tumor cell surface. The extracellular polypeptide repeat sequence can be used as a vaccine antigen: CSAPDTRTPAPGSTAPPAHGVT, i.e., identical to BLP-25, which has been subjected to clinical trials, Jiang added a cysteine at the N-terminus for coupling to microspheres.

2. Preparing a vaccine: preparation of microspheres according to the scheme of example 4

3. Immunization: Balb/C mice for immunization

4. Results of antibody assay

Reference documents: PNAS | January 3,2012| vol.109| No.1| 261-

Immune recognition of tumor-associated mucin MUC1is achieved by a fully synthetic aberrantly glycosylated MUC1tripartite vaccine

Vani Lakshminarayanana,1,Pamela Thompsonb,1,Margreet A.Wolfertb,Therese Buskasb,Judy M.Bradleyc,Latha B.Pathangeyc,Cathy S.Madsena,Peter A.Cohenc,Sandra J.Gendlera,2,and Geert-Jan Boonsb,2

Example 16 Combined application of microsphere vaccine prepared by tumor cell surface antigen EGFR and NK cells

1. Background of the study

The tumor vaccine is used for immunizing and generating antibodies aiming at tumor cells, and a humoral system kills the tumor cells through complement. More important ADCC effect. The IgG antibody can mediate ADCC action by these cells, wherein NK cells are the primary cells capable of ADCC action. In the process of ADCC action mediated by the antibody, the antibody can only be specifically combined with corresponding epitope on a target cell, effector cells such as NK cells can kill any target cell combined with the antibody, so that the combination of the antibody and the antigen on the target cell is specific, and the killing action of the NK cells on the target cell is nonspecific by killing tumor cells through immune cells mediated by Fc receptors of the antibody. NK cell surface antibody Fc receptor

And the NK cells can release soluble NK cytotoxic factors (NKCF), the surface of the target cells has NKCF receptors, and the NKCF can selectively kill and lyse the target cells after being combined with the target cells.

NK cells can also release TNF-alpha and TNF-beta, which cause leakage of various hydrolases by altering the stability of target cell lysosomes; affecting cell membrane phospholipid metabolism; a decrease in PH upon alteration of carbohydrate metabolism in the target cell; activation of the target cell endonuclease degrades the DNA, causing programmed cell death.

The tumor vaccine generates antibodies, the antibodies are recruited to the tumor cell part through the Fc receptor, the NK cell non-antibody-dependent natural killing function can be exerted, and the bystander killing effect on the tumor cells without antibody combination is realized.

Cancer Cell 27,489–5012015

Synergistic Innate and Adaptive Immune Response toCombinationImmunotherapywithAnti-TumorAntigen Antibodies and Extended Serum Half-Life IL-2

Eric F.Zhu,1,5,12Shuning A.Gai,1,5,12Cary F.Opel,1,5,12Byron H.Kwan,2,5Rishi Surana,6Martin C.Mihm,

Fourth, NK cells can be amplified and cultured in vitro, and feedback improves ADCC (advanced cell-mediated isothermal amplification) tumor killing effect

2. Tumor vaccine EGFR polypeptide vaccine, see example 13

3. Animal tumor model- -mouse Lewis lung cancer cell high expression EGFR

Results of NK cell combination with tumor antibody

The NK cell surface has Fc receptors that bind mainly to the Fc segments of human IgG1 and IgG3 (cy2, cy3 functional regions). The antigen of origin, produces high titers (high titers) of antibodies in humans. Wherein the Fc portion of the antibody binds to NK cell Fc receptor (ADCC effect) to directly kill the target cell.

Comparison of NK cell group with anti-microsphere EGFR antibody + NK group in vitro killing experiment

1, in the above example, blood is taken from microsphere EGFR after four times of immunization in the last week and is measured by ELISA antibody titer, NK cells are selected and added with antiserum to be diluted by 1:10000 to a 96-well plate, target cells are A549, and the specific optical density value of each effective target is read by a 450nm test (CCK8KIT) of an enzyme labeling instrument.

2, NK cell killing target cell A549, 96-hole plate effect target specific optical density value/hole

The experimental results show that: in an in vitro killing experiment of an NK cell group, an anti-microsphere EGFR antibody and an NK group, when the target cell A549 has an effective target ratio of 5:1, the killing efficiency of the anti-microsphere EGFR antibody, the anti-NK group and the NK group is improved by about 60%, and a better killing effect result can be generated after optimized combination.

Example 17 assay of endogenous antibodies generated by immunization with EGFR polypeptide-microsphere vaccines in combination with in vitro amplification of FcR-CAR-T cells

Antibodies generated by immunization with the EGFR polypeptide-microsphere vaccine are shown in example 13.

Construction and synthesis of FcR-CAR gene, the gene sequence of mouse FcR-CAR is shown as SEQ ID No.23, and the gene of humanized FcR-CAR is shown as SEQ ID N0.24.

Lentiviral packaging of FcR-CAR

The FcR-CAR gene was ligated to the viral vector PCDH using Nhe1(gctagc) and Not1(gcggccgc) as cleavage sites.

Count of 293T cells approximately 90 x10 in 293T cell culture Collection plates ⁶ Approximately 21 cells were plated 24h before transfection in 10cm dishes to set a cotransfection group PCDH-gfp&PLI1 PLP 2P-VSVG (hereinafter referred to as GFP group) 10 plates, cotransfection group PCDH-pour&PLI1 PLP 2P-VSVG (hereinafter referred to as GFP group) 10 plates, blank control 1 plate.

The two virus supernatants were harvested together at 100ml each, and concentrated with PEG8000 to obtain virus concentrates at 300ul each, one for infecting CD8T cells, and one for subpackaging and storing at-80 deg.C. Viral titers before and after concentration were determined from infected 293T cells.

This data includes infection rate, virus titer assay of 293T cells co-transfected with four plasmids.

The transfection rate of the shuttle vector carrying the target gene to 293T cells can reach more than 90% per plate through detection, and the four-plasmid transfection 293T cells basically meet the virus packaging premise, as shown in FIG. 3.

The titer of the virus stock solution is determined to be about 2X 106T before and after the virus concentration, and the titer of the virus after the concentration is about 500X 106T. The virus titer is improved by 250 times, and the virus loss rate reaches 25 percent. The infection rates of the different gradient viruses on 293T cells are shown in FIG. 4.

FIG. 4 shows infection of 293T cells following gradient dilution of virus concentrate. The fluorescence detection results of GFP at 96h after infection at each dilution with the uninfected virus group 293T cells as a control are shown.

Preparation of FcR-CAR-T cells

1) Antibody coating: 24-well plates were coated with 1ug/ml anti CD3 and 5ug/ml anti CD28 (MACS cell activating reagent to CD8 cytosol volume ratio 1:17.5 mixed well).

2) The isolated CD8 cells were cultured in pre-coated cell plates with 30U/ml IL-2 added at 0.5X 106 cells/0.5 ml PRMI + 10% FBS medium per well.

3) Transferring into a 5% C02, 37 ℃ incubator for culture, activating for about 24h, observing the activation state of cells, and adding virus solution when the cells are obviously aggregated.

4) Recovering virus on ice, adding virus concentrated solution dropwise into T cell culture solution according to MOI of 20, and adding without blowing.

5) After infection of about 24-48, the complete cell culture medium was changed to 60U/ml IL-2 and 30U/ml IL-7.

6) Observing the cell culture state, adding or replacing a cell complete culture medium, and detecting the expression of the CAR on the surface of the T cell by using a flow cytometer after infecting the T cell for about 5 days or so. Negative and isotype controls were set.

7) The CART cells are continuously cultured, and the cell culture state is observed. Cells were harvested at about day 14 of cell culture and tested for transfection efficiency. GFP single stained T cells, expression of GFP corresponded to the transfection rate of CAR molecules.

Results of FcR-CAR-T cell use with tumor antibodies:

example 9. microsphere EGFR was bled for the last week of four immunizations for ELISA antibody titer determination. (1) CAR-T cells were diluted in 96-well plates at a 1:2 ratio, and target cells a549 were added to give an effective target ratio of 2.5: 1,5.0: 1,10.0: 1,20.0: 1; (2) the CAR-T cells are diluted in a 96-well plate according to the proportion of 1:2, EGFR antiserum diluted according to the proportion of 1:10000 is added firstly, the incubation is carried out for 60 minutes at room temperature, and then target cells A549 are added so that the effective target ratio is 2.5: 1,5.0: 1,10.0: 1,20.0: 1; and (4) performing 450nm test (CCK8KIT) by using a microplate reader, and reading specific optical density values/hole of each effect target.

FcR-CAR-T cell killing target cell A549

FcR-CAR-T cell and tumor antibody combined killing target cell A549

Sequence listing

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gaccccgtgc agctggaggt gcacatgggc tggctgctgc tgcagaccac caagtggctg 360

ttccaggagg gcgaccccat ccacctgaga tgccacagct ggcagaacag acccgtgaga 420

aaggtgacct acctgcagaa cggcaagggc aagaagtact tccacgagaa cagcgagctg 480

cccatcccca aggccaccca caacgacagc ggcagctact tctgcagagg cctgatcggc 540

cacaacaaca agagcagcgc cagcttcaga atcagcctgg gcgaccccgg cagccccagc 600

atgttccccc cctggcacag ccccaagctg ttctgggccc tggtggtggt ggccggcgtg 660

ctgttctgct acggcctgct ggtgaccgtg gccctgtgcg tgatctggac caacagcaga 720

agaaacagac tgctgcagag cgactacatg aacatgaccc ccagaagacc cggcctgacc 780

agaaagccct accagcccta cgcccccgcc agagacttcg ccgcctacag acccggcggc 840

ggcaagtgga tcagaaagaa gttcccccac atcttcaagc agcccttcaa gaagaccacc 900

ggcgccgccc aggaggagga cgcctgcagc tgcagatgcc cccaggagga ggagggcggc 960

ggcggcggct acgagctggg cggcggcaga gccaagttca gcagaagcgc cgagaccgcc 1020

gccaacctgc aggaccccaa ccagctgtac aacgagctga acctgggcag aagagaggag 1080

tacgacgtgc tggagaagaa gagagccaga gaccccgaga tgggcggcaa gcagcagaga 1140

agaagaaacc cccaggaggg cgtgtacaac gccctgcaga aggacaagat ggccgaggcc 1200

tacagcgaga tcggcaccaa gggcgagaga agaagaggca agggccacga cggcctgtac 1260

cagggcctga gcaccgccac caaggacacc tacgacgccc tgcacatgca gaccctggcc 1320

cccagatgag cggccgc 1337

<210> 24

<211> 1340

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gctagcgcca ccatgtggca gctgctgctg cccaccgccc tgctgctgct ggtgagcgcc 60

ggcatgagaa ccgaggacct gcccaaggcc gtggtgttcc tggagcccca gtggtacagc 120

gtgctggaga aggacagcgt gaccctgaag tgccagggcg cctacagccc cgaggacaac 180

agcacccagt ggttccacaa cgagagcctg atcagcagcc aggccagcag ctacttcatc 240

gacgccgcca ccgtgaacga cagcggcgag tacagatgcc agaccaacct gagcaccctg 300

agcgaccccg tgcagctgga ggtgcacatc ggctggctgc tgctgcaggc ccccagatgg 360

gtgttcaagg aggaggaccc catccacctg agatgccaca gctggaagaa caccgccctg 420

cacaaggtga cctacctgca gaacggcaag gacagaaagt acttccacca caacagcgac 480

ttccacatcc ccaaggccac cctgaaggac agcggcagct acttctgcag aggcctggtg 540

ggcagcaaga acgtgagcag cgagaccgtg aacatcacca tcacccaggg cctggccgtg 600

agcaccatca gcagcttcag cccccccggc agcagaaagc ccttctgggt gctggtggtg 660

gtgggcggcg tgctggcctg ctacagcctg ctggtgaccg tggccttcat catcttctgg 720

gtgagaagca agagaagcag actgctgcac agcgactaca tgaacatgac ccccagaaga 780

cccggcccca ccagaaagca ctaccagccc tacgcccccc ccagagactt cgccgcctac 840

agaagcggcg gcggcaagag aggcagaaag aagctgctgt acatcttcaa gcagcccttc 900

atgagacccg tgcagaccac ccaggaggag gacggctgca gctgcagatt ccccgaggag 960

gaggagggcg gctgcgagct gggcggcggc agagtgaagt tcagcagaag cgccgacgcc 1020

cccgcctacc agcagggcca gaaccagctg tacaacgagc tgaacctggg cagaagagag 1080

gagtacgacg tgctggacaa gagaagaggc agagaccccg agatgggcgg caagccccag 1140

agaagaaaga acccccagga gggcctgtac aacgagctgc agaaggacaa gatggccgag 1200

gcctacagcg agatcggcat gaagggcgag agaagaagag gcaagggcca cgacggcctg 1260

taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1320

ccccccagat gagcggccgc 1340

Claims (5)

1. The fully diversified amphipathic MHC II combined polypeptide self-assembles an immune carrier microsphere vaccine, which is characterized by comprising an immune carrier microsphere and an antigen, wherein the antigen is coupled on the surface of the microsphere through a coupling group; the immune carrier microsphere is formed by self-assembly of amphipathic MHC II binding polypeptide, the MHC II binding polypeptide is in the microsphere, and a coupling group is positioned on the surface of the microsphere; the self-assembly sequence structure of the amphipathic MHC II binding polypeptide is as follows: sequence set 1: consists of Pal-AAAAGGG-C-QYIKANSKFIGITELD-RDKQKK-mal and Pal-AAAAGGGG-C-ISQAVHAAHAEINEA-QKDRDK-mal or sequence group 2: consists of Pal-AAAAGGG-C-ISISEIKGVIVHK-DKQK-mal and sequence: Pal-AAAAGGG C-ISQAVHAAHAEINEA-KQRK-mal, wherein C in the sequence is cysteine, Pal is palmitoyl, and mal is maleimide.

2. The method for preparing an microcarrier microsphere vaccine according to claim 1, wherein the self-assembly is carried out in a mixed solvent of an organic solvent and water.

3. The method for preparing an microcarrier microsphere vaccine according to claim 2, wherein the mixed solvent of organic solvent and water is acetonitrile-water mixed solvent, the amphiphilic MHC II binding polypeptide is added to the mixed solvent and then stirred to form emulsion, the amphiphilic MHC II binding polypeptide is bound through a hydrophobic end to form microspheres, acetonitrile in the microspheres is volatilized, and the microspheres are obtained by centrifugal precipitation and separation.

4. Use of an immunovector microsphere vaccine according to claim 1 for the preparation of an antibody vaccine for the treatment of benign disease.

5. Use according to claim 4, characterized in that the antibody vaccine for the treatment of benign diseases is an IgE vaccine.

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